[go: up one dir, main page]

WO2005085478A2 - Detection and identification of mycobacterium and nocardia using primers and probes directed to the seca1 gene - Google Patents

Detection and identification of mycobacterium and nocardia using primers and probes directed to the seca1 gene Download PDF

Info

Publication number
WO2005085478A2
WO2005085478A2 PCT/US2005/006609 US2005006609W WO2005085478A2 WO 2005085478 A2 WO2005085478 A2 WO 2005085478A2 US 2005006609 W US2005006609 W US 2005006609W WO 2005085478 A2 WO2005085478 A2 WO 2005085478A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
mycobacterium
specific
species
nocardia
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2005/006609
Other languages
French (fr)
Other versions
WO2005085478A3 (en
Inventor
Steven H. Fischer
Adrian M. Zelazny
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
Original Assignee
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services filed Critical US Department of Health and Human Services
Publication of WO2005085478A2 publication Critical patent/WO2005085478A2/en
Publication of WO2005085478A3 publication Critical patent/WO2005085478A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)

Definitions

  • Nocardia in a sample using secA nucleic acid sequences Specifically, a method is disclosed to identify the Mycobacterium and Nocardia species involved in the infection of a subject.
  • the Mycobacterium genus comprises more than 70 different species, of which about 30 have been associated with human disease.
  • One prominent pathogen of the genus is Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis.
  • BCG Bacille Calmette-Guerin
  • M. tuberculosis remains a major global public health problem with an estimated eight million new cases and 2.9 million deaths per year.
  • Mycobacteria other than M. tuberculosis also called nontuberculous mycobacteria (NTM) are ubiquitous in the environment and have the potential to colonize and cause serious infection.
  • Nocardia asteroids N. asteroids
  • N brasiliensis Ncaviae
  • Nocardia is found in soil around the world and can be contracted by inhaling contaminated dust or via contamination of a wound with soil containing Nocardia. While individuals with normal immune systems can acquire this infection, immunocompro ised individuals are at greatest risk of developing a serious disease related to Nocardia infection. Thus, the main risk factors for nocardiosis are a weakened immune system or chronic lung disease.
  • bacterial protein export is an integral part of bacterial pathogenesis.
  • secA is a highly conserved, soluble protein that is essential in the bacterial protein export pathway.
  • secA is a cytoplasmic, multifunctional, 100 kDa homodimer that interacts with acidic phospholipids in the bacterial cell membrane, the SecYEG pore (a membrane channel formed by SecE (9 kDa), SecG (11 kDa), and SecY (47 kDa)) and molecular chaperones, such as SecB (17 kDa), that deliver precursor proteins to the bacterial cell membrane translocase.
  • secA is an ATPase that provides energy for protein translocation and, through cycles of ATP binding and hydrolysis, secA transports precursor proteins to the translocase and undergoes cycles of membrane insertion and deinsertion that lead to stepwise export of tlie protein.
  • Smear microscopy is still widely used as a first method to screen for the presence of Mycobacterium or Nocardia in clinical samples.
  • the reliability of smear microscopy is highly dependent on the experience of the laboratory technician and on the number of acid-fast bacilli (AFB) and/or weakly acid fast bacilli present in the specimen.
  • the overall sensitivity of the smear has been reported to range from 22 to 80%.
  • AZA acid-fast bacilli
  • molecular methods have been utilized in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium species.
  • M Mycobacterium species
  • tuberculosis complex M. gordonae, M. kansasii, and M. avium-intracellulare complex
  • M. gordonae M. gordonae
  • M. kansasii M. avium-intracellulare complex
  • 16S rDNA sequence analysis have also allowed the recognition of many new Nocardia species.
  • 16S rDNA sequences for identifying isolates has been problematic, since closely related species show high levels of 16S rDNA similarity.
  • the present disclosure relates to methods of detecting a Mycobacterium or Nocardia in a sample.
  • the method involves amplifying a Mycobacterium/Nocardia genera- specific secAl nucleic acid with at least two Mycobacterium/Nocardia genera-specific primers.
  • the Mycobacterium/Nocardia genera-specific primers bind within a region of the nucleic acid sequence encoding a secAl protein that is conserved among members of the Mycobacterium and Nocardia genera, wherein the conserved region is in the 5' half of the secAl gene and includes a substrate specificity domain.
  • Mycobacterium or Nocardia includes hybridizing the amplified genera-specific secAl nucleic acid to a detectable secAl probe oligonucleotide.
  • distinguishing a Mycobacterium from a Nocardia includes hybridizing the amplified nucleic acid with a detectable Mycobacterium or a Nocardia species-specific secAl probe oligonucleotide or sequencing the amplified nucleic acid.
  • a Mycobacterium species is identified by amplifying a Mycobacterium species-specific secAl nucleic acid from a sample utilizing at least two Mycobacterium species-specific primers.
  • the Mycobacterium species-specific primers bind within a region of the Mycobacterium species-specific secAl nucleic acid sequence containing at least one nucleic acid difference, as compared to a secAl nucleic acid sequence present in another species of Mycobacterium.
  • the nucleic acid differences allow the species-specific primers to differentially amplify target sequences of different Mycobacterium species.
  • the region containing at least one nucleotide difference may be in the 5' half of the Mycobacterium secAl gene and includes the substrate specificity domain.
  • Mycobacterium species-specific detectable secAl probes are used to detect the amplified Mycobacterium species-specific secAl nucleic acids.
  • Methods for identifying a species of Mycobacterium or Nocardia in a sample include hybridizing nucleic acids in the sample to a plurality of Mycobacterium or Nocardia species-specific probe oligonucleotides attached to a polymeric solid support surface in an array. These probes are detectable by their location.
  • Mycobacterium and Nocardia species-specific secAl polypeptide fragments are disclosed herein. Also disclosed are polynucleotides encoding the Mycobacterium and Nocardia species- specific secAl polypeptide fragments, as well as Mycobacterium and Nocardia species-specific secAl oligonucleotide probes and primers.
  • the disclosure also provides methods of treating a subject suspected of being infected with a Mycobacterium or a Nocardia, as well as devices, including arrays, and kits for detecting a Mycobacterium or a Nocardia species in a sample.
  • FIG. 1 shows the alignment of an amplified Mycobacterium genus-specific secAl nucleic acid sequence from various Mycobacterium species, including M. abscessus (ATCC.19977(T), SEQ ID NO: 130), M. abscessus (50200759, SEQ ID NO: 131), M. africanum (ATCC.25420(T), SEQ ID NO: 132), M. africanum (ATCC.35711, SEQ ID NO:133), M. asiaticum (ATCC.25276(T), SEQ ID NO:134), M.
  • asiaticum 15031086, SEQ ID NO:135), M. avium (ATCC.25291(T), SEQ ID NO:136), M. avium (52110667.ATCC.35717, SEQ ID NO: 137), M. bovis (ATCC.19210(T), SEQ ID NO:138), M. bovis (52110665.ATCC.35734, SEQ ID NO:139), M. chelonae (ATCC.35752(T), SEQ ID NO: 140), M. chelonae (52281700, SEQ ID NO: 141), M.flavescens (ATCC.14474(T), SEQ ID NO: 142), M.
  • haemophilum ATCC.29548(T), SEQ ID NO: 152
  • M. haemophilum ATCC.33206, SEQ ID NO: 153
  • M. intracellulare ATCC.13950(T), SEQ ID NO:154
  • M. intracellulare R009, SEQ ID NO:155
  • M. kansasii ATCC.12478(T), SEQ ID NO: 156
  • M. kansasii 39211279, SEQ ID NO:157
  • M. leprae AL583919.1, SEQ ID NO: 221)
  • M. malmoense ATCC.29571(T), SEQ ID NO:158
  • malmoense (18280971, SEQ ID NO: 159), M. marinum (ATCC.927(T), SEQ ID NO: 160), M. marinum (52110619, SEQ ID NO:161), M. mucogenicum (ATCC.49650(T), SEQ ID NO: 162), M. mucogenicum (49200275, SEQ ID NO: 163), M. peregrinum (ATCC.14467(T), SEQ ID NO: 164), M. peregrinum (25110648, SEQ ID NO: 165), M. scrofulaceum (ATCC.19981(T), SEQ ID NO: 166), M. scrofulaceum (52241579, SEQ ID NO: 167), M.
  • FIG. 2 shows the alignment of the amino acid translation of the amplified Mycobacterium genus-specific secAl nucleic acid sequence from various Mycobacterium species, including M. abscessus (ATCC.19977(T), SEQ ID NO:69), M. abscessus (50200759, SEQ ID NO:70), M.
  • M.flavescens (ATCC.14474(T), SEQ ID NO:81), M.flavescens (ATCC.23008, SEQ ID NO:82), M.flavescens (ATCC.23033, SEQ ID NO:83), M.flavescens (ATCC.23046, SEQ ID NO:84), M. fortuitum (ATCC.6841(T), SEQ ID NO:85), M. fortuitum (52110571, SEQ ID NO:86), M. gastri (ATCC.15754(T), SEQ ID NO:87), M. gastri (ATCC.25157, SEQ ID NO:88), M.
  • gordonae ATCC.14470(T), SEQ ID NO:89
  • M. gordonae 49290605, SEQ ID NO:90
  • M. haemophilum ATCC.29548(T), SEQ ID N0:91
  • M. haemophilum ATCC.33206, SEQ ID NO:92
  • M. intracellulare ATCC.13950(T), SEQ ID N0:93
  • M. intracellulare R009, SEQ ID N0:94
  • M. kansasii ATCC.12478(T), SEQ ID N0:95
  • M. kansasii 39211279, SEQ ID N0:96
  • leprae (AL583919.1, SEQ ID NO: 220), M. malmoense (ATCC.29571(T), SEQ ID NO:97), M. malmoense (18280971, SEQ ID NO:98), M. marinum (ATCC.927(T), SEQ ID N0:99), M. marinum (52110619, SEQ ID NO: 100), M. mucogenicum (ATCC.49650(T), SEQ ID NO:101), M. mucogenicum (49200275, SEQ ID NO: 102), M. peregrinum (ATCC.14467(T), SEQ ID NO: 103), M. peregrinum (25110648, SEQ ID NO: 104), M.
  • scrofulaceum ATCC.19981 (T), SEQ ID NO: 105
  • M. scrofulaceum 52241579, SEQ ID NO: 106
  • M. simiae ATCC.25275(T), SEQ ID NO: 107
  • M. simiae MB8146, SEQ ID NO: 108
  • M. smegmatis ATCC.19420(T), SEQ ID NO: 109
  • M. smegmatis 52110637.ATCC.14468, SEQ ID NO: 110
  • M. szulgai ATCC.35799(T), SEQ ID NO:l l l
  • szulgai 52110639, SEQ ID NO: 112), M.
  • terrae (ATCC.15755(T), SEQ ID NO:113), M. terrae (MB2345, SEQ ID NO: 114), M. triviale (ATCC.23292(T), SEQ ID NO: 115), M. triviale (ATCC.23290, SEQ ID NO: 116), M. triviale (ATCC.23291, SEQ ID NO: 117), M. tuberculosis (ATCC.27294(T), SEQ ID NO: 118), M. tuberculosis (R011 , SEQ ID NO: 119), M. tuberculosis (52110663.H37'R.ATCC.25177, SEQ ID NO: 120), M.
  • ulcerans (ATCC.19423(T), SEQ ID NO: 121), M. ulcerans (ATCC.25900, SEQ ID NO: 122), M. ulcerans (ATCC.25897, SEQ ID NO: 123), M. ulcerans (ATCC.35839, SEQ ID NO: 124), X-Cluster (52110672, SEQ ID NO: 125), X-Cluster
  • FIGS. 3A and 3B are a series of schematic drawings demonstrating the 649 amino acidN- terminal region of the secAl protein and primer design for the amplification of the secAl gene.
  • FIG. 3 A shows that the secAl protein from tuberculosis contains two nucleotide-binding domains
  • NBD1 between amino acid positions 1 and 426; NBD2, between amino acid positions 426 and 641
  • SSD is embedded in NBD1 between amino acid positions 222 and 359.
  • the thin black bar shows the region of the protein coded by the secAl gene region targeted in the assay. Numbers indicate amino acid residues.
  • FIG. 3B shows that primers Mtu.For.l (FI) and Mtu.Rev.3 (R3) were used to generate a 700 base pair (base pair) fragment from the secAl gene. Numbers indicate nucleotide position in the amplified fragment.
  • FIG. 4 shows the interspecies similarity of partial secAl gene sequences.
  • FIG. 5 is a schematic drawing of a phylogenetic tree derived from secAl sequences from 34 mycobacterial reference strains and M. leprae (GenBank accession no. AL583919.1) and two nocardial reference strains. The tree was constructed by the neighbor-joining method, using
  • Coiynebacterium efficiens as the outgroup (GenBank accession no. AP005216.1).
  • T represents type strain; three to five digit number represents an ATCC number. Numbers on the branches represent the percentage of 1000 bootstrap samples supporting the branch; only values >50% are shown.
  • SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
  • SEQ ID NOs: 1-6 are Mycobacterium species-specific secAl minor groove binder probes.
  • SEQ ID NOs: 7 and 21 are Mycobacterium genus-specific minor groove binder probes.
  • SEQ ID NOs: 8-20 and 22-25 are Mycobacterium/Nocardia genera-specific primers.
  • SEQ ID NOs: 27, 207, and 223-227 are Mycobacterium FRETprobes.
  • SEQ ID NOs: 26, 28-35, and 191-206 are Mycobacterium species-specific FRETprobes.
  • SEQ ID NOs:.36-68, 208-219, and 222 are Mycobacterium species-specific primers.
  • SEQ ID NOs: 130-189 and 221 are Mycobacterium secAl nucleic acid sequences.
  • SEQ ID NO: 129 is the Mycobacterium majority nucleic acid sequence.
  • SEQ IS NOs: 69-128 and 220 are Mycobacterium secAl amino acid sequences.
  • SEQ ID NO: 190 is the Mycobacterium majority amino acid sequence.
  • SEQ ID NO: 228 is a Nocardia FRETprobe.
  • SEQ ID NO: 229 is an M13 forward primer.
  • SEQ ID NO: 230 is an M13 reverse primer.
  • SEQ ID NO: 231-252 are Nocardia secAl nucleic acid sequences.
  • fluorescein isothiocyanate FRET fluorescence resonance energy transfer JOE 2'7'-dimethoxy-4'5 '-dichloro-6-carboxyfluorescein
  • MAC Mycobacterium avium complex MGB minor groove binder
  • Amplification Use of a technique that increases the number of copies of a nucleic acid molecule (for example, a DNA or RNA molecule) in a specimen.
  • An example of amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the amplified target nucleic acid, or amplicon may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques.
  • a specific type of PCR is real-time PCR, where the detection of the amplified target nucleic acid is monitored in real-time.
  • Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311 ; transcription-free isothermal amplification, as disclosed in U.S. Patent No.
  • the antigen is a Mycobacterium secA polypeptide, such as a Mycobacterium species-specific secAl polypeptide.
  • the antigen is a Nocardia secA polypeptide, such as a Nocardia species-specific secAl polypeptide.
  • Monoclonal, and humanized immunoglobulins are encompassed by the disclosure.
  • the disclosure also includes synthetic and genetically engineered variants of these immunoglobulins.
  • a naturally occurring antibody for example, IgG
  • IgG includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody.
  • antigen-binding fragments are also intended to be designated by the term "antibody.”
  • binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the V L , V H , C L and C H 1 domains; (ii) an Fd fragment consisting of the V H and C H 1 domains; (iii) an Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544-546, 1989) which consists of a V H domain; and (v) an F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al, Science 242-ATi ⁇ 426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988) by recombinant methods.
  • single chain Fv single chain Fv
  • dsFv disulfide stabilized Fv
  • dimeric Fvs diabodies
  • antibody fragments for use in this disclosure are those which are capable of cross-linking their target antigen, for example, bivalent fragments such as F(ab') 2 fragments.
  • an antibody fragment which does not itself cross-link its target antigen for example, a Fab fragment
  • a secondary antibody which serves to cross-link the antibody fragment, thereby cross-linking the target antigen.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described for whole antibodies.
  • An antibody is further intended to include humanized monoclonal molecules that specifically bind the target antigen.
  • Specifically binds refers to the ability of individual antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an antibody molecule and the antigen.
  • the antigen is secA. Binding specificity is typically determined from the reference point of the ability of the antibody to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes.
  • An antibody that specifically binds to a particular epitope is referred to as a "specific antibody”.
  • a variety of methods for linking effector molecules to antibodies are well known in the art.
  • Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as 32 P, fluorophores, chemiluminescent agents, and enzymes. Also encompassed in the disclosure are the chemical or biochemical modifications that incorporate toxins in the antibody.
  • the toxin is chemically conjugated to the antibody.
  • a fusion protein is genetically engineered to include the antibody and the toxin.
  • toxins are radioactive isotopes, chemotherapeutic agents, bacterial toxins, viral toxins, or venom proteins.
  • Antigen Any molecule that can bind specifically with an antibody. An antigen is also a substance that antagonizes or stimulates the immune system to produce antibodies.
  • Antigens are often foreign substances such as allergens, bacteria or viruses that invade the body.
  • an antigen is a secA polypeptide, such as a secAl polypeptide.
  • Antisense and Sense Double-stranded DNA (dsDNA) has two strands, a 5' -> 3' strand, referred to as the plus strand, and a 3' -> 5' strand, referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription.
  • the RNA formed will have a sequence complementary to the minus strand, and identical to the plus strand (except that the base uracil is substituted for thymine).
  • Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA.
  • Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Binding or stable binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding.
  • Binding can be detected by either physical or functional properties of the targetioligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including functional or physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, Southern blotting, dot blotting and light or fluorescence absorption detection procedures.
  • a method which is widely used involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at a wavelength of 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog binds its target sequence, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and the target sequence dissociate or melt.
  • FRET Fluorescence Resonance Energy Transfer
  • a biological specimen is a sample of bodily fluid or tissue used for laboratory testing or examination. As used herein, biological specimens include all clinical samples useful for detection of microbial infection in subjects.
  • a biological specimen can be a tissue biopsy, for instance from the skin, lung or bronchial tissue, a surgical specimen, or autopsy material.
  • a biological specimen also can be a biological fluid.
  • Biological fluids include saliva, blood, derivatives and fractions of blood such as serum, and fluids of the respiratory tract, including the oropharyngeal tract, such as sputum, that has been expectorated or collected during bronchoscopy or bronchoalveolar lavage.
  • appropriate specimens for use with the current disclosure for the detection of members of the Mycobacterium or Nocardia genera or individual species of Mycobacterium or Nocardia include conventional clinical samples, for instance blood or blood-fractions (for example, serum), bronchoalveolar lavage (BAL), sputum, and induced sputum samples. Techniques for acquisition of such samples are well known in the art. Blood and blood fractions (for example, serum) can be prepared in traditional ways.
  • Oropharyngeal tract fluids can be acquired through conventional techniques, including sputum induction, BAL, and oral washing. Obtaining a sample from oral washing involves having the subject gargle with an amount of normal saline for approximately 10-30 seconds and then expectorate the wash into a sample cup.
  • cDNA complementary DNA
  • cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule.
  • UTRs untranslated regions
  • cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • Degenerate variant A polynucleotide encoding a secA polypeptide, such as a secAl polypeptide, that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon.
  • nucleotide sequences are included in the disclosure as long as the amino acid sequence of the sec A polypeptide, such as a secAl polypeptide, encoded by the nucleotide sequence is unchanged.
  • Epitope An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, for instance, that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Specific, non-limiting examples of an epitope include a tetra- to penta-peptide sequence in a polypeptide, a tri- to penta- glycoside sequence in a polysaccharide.
  • the epitope is Mycobacterium species-specific. In another embodiment, the epitope is Nocardia species-specific.
  • Expression Control Sequences Nucleic acid sequences that control and regulate the expression of a nucleic acid sequence, such as a heterologous nucleic acid sequence, to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, polyA signals, a start codon (for instance, ATG) in front of a protein-encoding polynucleotide sequence, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • a promoter is a minimal sequence sufficient to direct transcription.
  • promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene.
  • constitutive and inducible promoters are included (see for example, Bitter et al, Methods in Enzymology 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage lambda, plac, pt ⁇ , ptac (ptrp-lac-hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells for example, the metallothionein promoter
  • mammalian viruses for example, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
  • the promoter is a cytomegalovirus promoter.
  • Fluorescence Resonance Energy Transfer FRET: A spectroscopic process by which energy is passed between an initially excited donor molecule to an acceptor molecule that are separated by 10-100 A.
  • the donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule.
  • the efficiency of energy transfer is proportional to the inverse sixth power of the distance (R) between the donor and acceptor (1/R 6 ) fluorophores and occurs without emission of a photon.
  • R the distance between the donor and acceptor (1/R 6 ) fluorophores and occurs without emission of a photon.
  • FRET either can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence.
  • the donor's fluorescence is quenched it indicates the donor and acceptor molecules are within the F ⁇ rster radius (the distance where FRET has 50% efficiency, approximately 20-60 A), whereas if the donor fluoresces at its characteristic wavelength, it denotes that the distance between the donor and acceptor molecules has increased beyond the F ⁇ rster radius. This occurs, for example, when a TaqMan ® probe is degraded by Taq polymerase following hybridization of the probe to a target nucleic acid sequence or when a hairpin probe is hybridized to a target nucleic acid sequence.
  • energy is transferred via FRET between two different fluorophores such that the acceptor molecule can emit light at its characteristic wavelength, which is always longer than the emission wavelength of the donor molecule.
  • oligonucleotides using FRET that can be used to detect amplicons include linear oligoprobes, such as FRETprobes (also referred to as HybProbes), 5' nuclease oligoprobes, such as TaqMan ® probes or TaqMan ® probes with a minor groove binding moiety, hairpin oligoprobes such as molecular beacons, scorpion primers and UniPrimers, minor groove binding probes, and self-fluorescing amplicons, such as sunrise primers.
  • Fluorescent marker or Fluorescent dye A chemical compound, which when excited by exposure to a particular wavelength of light, emits light (for instance, fluoresces), for example at a different wavelength than that to which it was exposed.
  • Fluorophores can be described in terms of their emission profile, or "color.” Green fluorophores, for example Cy3, fluorescein isothiocyanate (FITC), and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 ⁇ . Red fluorophores, for example LightCycler Red 640, LightCycler Red 705, Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-7 lO ⁇ . Encompassed by the term "fluorescent marker” as it is used herein are luminescent molecules, which are chemical compounds which do not require exposure to a particular wavelength of light to fluoresce; luminescent compounds naturally fluoresce.
  • luminescent signals eliminates the need for an external source of electromagnetic radiation, such as a laser.
  • An example of a luminescent molecule includes, but is not limited to, aequorin (Tsien, Ann. Rev. Biochem. 67:509, 1998).
  • FRETProbes A pair of adjacent, fluorogenic hybridization oligoprobes used for real-time
  • FRETProbes include an upstream probe labeled with a 3' donor fluorophore (for example, FITC) and a downstream probe labeled with an acceptor fluorophore at the 5' terminus.
  • a 3' donor fluorophore for example, FITC
  • an acceptor fluorophore at the 5' terminus.
  • FRETProbes include FastStart DNA Hybridization
  • Probes (Roche Molecular Biochemicals; Mannheim, Germany). With FastStart DNA Hybridization Probes, one probe is labeled at the 5' end with a LightCycler Red fluorophore, for example a LightCycler Red 640 or Red 705 fluorophore, and to avoid extension, the probe is modified at the 3' end by phosphorylation. The other probe is labeled at the 3' end with FITC. During FRET, FITC, the donor fluorophore, is excited by the light source of the LightCycler instrument, and part of the excitation energy is transferred to LightCycler Red, the acceptor fluorophore.
  • a LightCycler Red fluorophore for example a LightCycler Red 640 or Red 705 fluorophore
  • FITC the donor fluorophore
  • Host cells Cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Also includes the cells of the subject.
  • Hybridization Oligonucleotides hybridize by hydrogen bonding, which includes Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleotide units.
  • adenine and thymine are complementary nucleotides which base pair through formation of hydrogen bonds.
  • “Complementary” refers to sequence complementarity between two nucleotide units. For example, if a nucleotide unit at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide unit at the same position of a DNA or RNA molecule, then the oligonucleotides are complementary to each other at that position.
  • oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units which can hydrogen bond with each other.
  • a "hybridization complex" is formed when complementary nucleotides in a nucleic acid molecule(s) form base pairs.
  • a hybridization complex is formed when target nucleic acids, for example amplified Mycobacterium species-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium species-specific oligonucleotide probes, chemically linked to a polymeric solid support.
  • a hybridization complex is formed when target nucleic acids, for example amplified Nocardia species-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Nocardia species-specific oligonucleotide probes, chemically linked to a polymeric solid support.
  • target nucleic acids for example amplified Nocardia species-specific secAl nucleic acids
  • oligonucleotide probes for example Nocardia species-specific oligonucleotide probes, chemically linked to a polymeric solid support.
  • “Specifically hybridizable” and “complementary” are terms which indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
  • An oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable.
  • An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, or under conditions in which an assay is performed.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na + concentration) of the hybridization buffer will determine the stringency of hybridization.
  • stringent conditions encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the primer or oligonucleotide probe and the target sequence. “Stringent conditions” can be broken down into particular levels of stringency for more precise definition.
  • “moderate stringency” conditions are those under which molecules with more than 25% sequence variation (also termed “mismatch”) will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency” are those under which sequences with more than 5% mismatch will not hybridize. Stringent conditions are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the T m for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Tijssen ( Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993).
  • Nucleic acid molecules that hybridize under stringent conditions to a Mycobacterium or a Nocardia secAl sequence will typically hybridize to a selected portion of the gene under wash conditions of 2x SSC at 50°C
  • An "isolated" biological component such as a nucleic acid molecule, protein or organelle
  • nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods.
  • a biomolecule is labeled if it is capable of being detected.
  • a nucleic acid or a polypeptide may be coupled covalently or noncovalently to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent markers or dyes, haptens, and enzymes.
  • the MGB group When attached to the terminal end of a DNA probe, the MGB group forms an extremely stable duplex with single-stranded DNA targets, allowing shorter probes (for example, as short as thirteen bases) to be used for hybridization based assays.
  • MGB probes In comparison with unmodified DNA probes, MGB probes have higher melting temperature and increased specificity, especially when a mismatch is in the MGB region of the duplex. Fluorogenic MGB probes are more specific for single base mismatches and fluorescence quenching is more efficient, giving increased sensitivity using these probes.
  • a T rich duplexes are stabilized more than G/C rich duplexes. MGB probes are more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.
  • MGB probes are useful when designing assays for closely related sequences, such as species-specific assays.
  • the MGB probes can also include a non- fluorescent quencher, which virtually eliminates the background fluorescence associated with traditional quenchers, providing better sensitivity and quantitation precision.
  • MGB probes include the TaqMan ® MGB probes (Applied Biosystems: Foster City, CA) and the MGB EclipseTM Probes (Epoch Biosciences; Bothell, WA).
  • Molecular beacon probe Oligonucleotides with stem-loop structures that contain a fluorescent marker at the 5' end, such as FAM, TAMRA, TET, or ROX, and a quencher, such as DABCYL, at the 3' end. The degree of quenching via fluorescence energy resonance transfer
  • FRET Fluorescence Reduction
  • Molecular beacon probes reform a stem-loop structure that quenches the fluorescent signal from the marker. If a PCR product whose sequence is complementary to the loop sequence is present during the heating/cooling cycle, hybridization of the molecular beacon probe to one strand of the PCR product, or amplicon, will increase the distance between tlie marker and the quencher, resulting in increased fluorescence (FRET is interrupted).
  • Molecular probes allow detection of multiple DNA species (multiplexing) using different reporter dyes on different probes or beacons.
  • Multiplex PCR Polymerase chain reaction that uses multiplex primers to produce more than one PCR product that can be detected in a single reaction tube.
  • Multiplex primers More than one pair of primers that are used simultaneously to amplify more than one target and/or control nucleic acid molecule in a single reaction tube.
  • Multiplex probes More than one probe, such as a molecular beacon probe, used simultaneously to detect more than one target and or control nucleic acid molecule in a single reaction tube.
  • Mycobacterium A genus of bacteria responsible for more disease world-wide than all other bacterial genera combined. Mycobacteria are small, 0.5 x 1 ⁇ m, weakly gram-positive bacilli that have two characteristic bacteriologic features.
  • the cell walls of these organisms contain very long chain fatty acid esters known as mycolic acids (lipids typically having chain lengths of 70 to 90 carbons).
  • mycolic acids lipids typically having chain lengths of 70 to 90 carbons.
  • This unusual cell envelope allows the mycobacteria to be impervious to many antiseptic solutions and antibiotics.
  • the pathogenic mycobacteria have an unusually slow growth rate. Unlike typical bacterial pathogens such as E. coli or Staphylococcus aureus, which double in 20-30 minutes and grow to high densities overnight in culture media, Mycobacterium species, such as Mycobacterium avium (M. avium) and M. tuberculosis, double approximately every 24 hours.
  • M. kansasii is an important pulmonary pathogen, along with the M. avium complex.
  • Mycobacterium avium complex includes two species, M. avium andM. intracellulare. Though M. avium was originally identified as a pathogen of birds, it is now known that both M. avium and M.
  • MAC is an opportunistic, rather than innate, pathogen in humans; it causes disease primarily in immunocompromised patients.
  • the Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, andM microti. Each of these species, except M microti (a cause of rodent tuberculosis), causes tuberculosis in humans.
  • M tuberculosis there are no environmental reservoirs of M. tuberculosis complex organisms, and only a few animals (cattle and occasionally deer) transmit tuberculosis to humans. Of all the culturable mycobacteria, only M tuberculosis is an obligate pathogen.
  • the two laboratory techniques that have classically been used to detect a mycobacterial infection are the acid fast smear and cultivation of the organism.
  • techniques have been developed to determine the species of the organism (for example, DNA blotting techniques). Generally, these techniques are performed on a body fluid, such as sputum or blood (Heifets and Good, "Current laboratory methods for the diagnosis of tuberculosis," in: Tuberculosis: Pathogenesis.
  • Mycobacterium/Nocardia genera-specific primer A primer that permits amplification of a Mycobacterium or Nocardia genus-specific nucleic acid, for example a conserved region of the secAl gene, present in virtually all members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus.
  • a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene that is conserved among members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus (a Mycobacterium/Nocardia genera-specific nucleic acid sequence).
  • a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene that is approximately 700 base pairs in length, is in the 5' half of the secAl gene and includes the substrate specificity domain (SSD).
  • a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene (including primers) that corresponds to a region between amino acid positions 138 and 391 of the secAl protein.
  • a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene (excluding primers) that corresponds to a region between amino acid positions 148 and 380 of the secAl protein.
  • Mycobacterium/Nocardia genera-specific probe or probe oligonucleotide A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secA gene, in virtually all members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus.
  • a Mycobacterium/Nocardia genera-specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus (a Mycobacterium/Nocardia genera-specific nucleic acid sequence).
  • Mycobacterium genus-specific probe or probe oligonucleotide A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secAl gene, in virtually all members of the Mycobacterium genus, but not in bacteria of any other genus.
  • a Mycobacterium genus-specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Mycobacterium genus, but not in bacteria of any other genus (a Mycobacterium genus-specific nucleic acid sequence).
  • Mycobacterium species-specific primer A primer which permits amplification of a nucleic acid from a single species of Mycobacterium and not other species of Mycobacterium.
  • the nucleic acid primer can be used to amplify the secAl gene from clinically relevant mycobacterial pathogens.
  • a pair of Mycobacterium species-specific secAl primers amplifies a region of the secAl gene that is unique to a particular Mycobacterium species.
  • a pair of Mycobacterium species-specific secAl primers amplifies a region of the secAl gene that is in the 5' half of the secAl gene.
  • a pair of primers amplifies a region of the secAl gene that is unique to M gordonae.
  • a pair of primers amplifies a region of the secAl gene that is unique to Mycobacterium abscessus (M abscessus), M. africanum, M. asiaticum, M. aurum, M. avium, M. bovis, M. celatum, M. chelonae, M. flavescens, M. fortuitum, M. gastri, M. genavense, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. leprae, M. malmoense, M. marinum, M. mucogenicum, M. nonchr ⁇ mogenicum, M.
  • Mycobacterium species-specific probe or probe oligonucleotide A probe oligonucleotide that hybridizes to a nucleic acid, for example the secAl gene, from one Mycobacterium species, but does not bind nucleic acid from other Mycobacterium species, under a specified set of hybridization conditions.
  • the probe binds M avium nucleic acid under a specific set of hybridization conditions, but does not bind nucleic acid from any other Mycobacterium species under identical hybridization conditions.
  • Nocardia A genus of bacteria characterized as Gram-positive rods, which in old cultures or clinical specimens may appear as branching chains resembling fungal hyphae. Nocardia are weakly acid-fast following staining with the modified Ziehl-Neelsen or Kinyoun stain. Cultures may grow in a few days, but typically require two to three weeks of incubation.
  • Nocardia brasiliensis Nocardia caviae (otitidiscaviarum), and Nocardia transvalensis represent the remaining 10% of infections.
  • Nocardia brasiliensis is the most important in tropical areas; it is most often seen as a cutaneous infection that can affect individuals with normal immune function (although 70% of cases of Nocardia brasiliensis are seen in immunocompromised individuals).
  • Nocardia has been isolated from soil and organic material throughout the world.
  • Nocardiosis is usually associated with T-cell dysfunctions, immunoglobulin deficiencies, or leukocyte abnormalities. Although nocardiosis has been diagnosed in individuals with no detectable deficiency of humoral or cell-mediated immunity, it usually occurs in patients whose immune status has been compromised by post-transplant immunosuppressive therapy, leukemia, lymphoma, pancytopenia, dysgammaglobulinemia, humoral defects, chronic granulomatous disease, or steroid therapy. The male/female ratio in nocardiosis is approximately 2:1, and infections occur from infancy to old age. There is no apparent geographic clustering of cases in the United States, except for cutaneous infection with N. brasiliensis, which is more common in the south.
  • Nocardia genus-specific probe or probe oligonucleotide A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secAl gene, in virtually all members of the Nocardia genus, but not in bacteria of any other genus.
  • a Nocardia genus-specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Nocardia genus, but not in bacteria of any other genus (a Nocardia genus-specific nucleic acid sequence).
  • Nocardia species-specific primer A primer which permits amplification of a nucleic acid from a single species of Nocardia and not other species of Nocardia.
  • the nucleic acid primer can be used to amplify the secAl gene from clinically relevant nocardial pathogens.
  • a pair of Nocardia species-specific secAl primers amplifies a region of the secAl gene that is unique to a particular Nocardia species.
  • a pair of Nocardia species-specific secAl primers amplifies a region of the secAl gene that is in the 5' half of the secAl gene.
  • a pair of primers amplifies a region of the secAl gene that is unique to Nocardia abscessus (N. abscessus).
  • a pair of primers amplifies a region of the secAl gene that is unique to N. Africana, N. asteroids drug pattern type IV, N asteroides drug pattern type VI, N asteroides type strain, N. beijingensis, N. brasiliensis, N. brevicatena, N carnea, N. corynebacteroides, N. cyriacigeorgica, N. farcinica, N. ignorata, N. kruczakiae, N. nova, N. otitidiscaviarum (N. caviae), N. paucivorans, N. pseudobrasiliensis, N seriolae, N. transvalensis, N.
  • Nocardia species-specific probe or probe oligonucleotide A probe oligonucleotide that hybridizes to a nucleic acid, for example the secAl gene, from one Nocardia species, but does not bind nucleic acid from other Nocardia species, under a specified set of hybridization conditions.
  • the probe binds Nocardia brasiliensis nucleic acid under a specific set of hybridization conditions, but does not bind nucleic acid from any other Nocardia species under identical hybridization conditions.
  • Nucleic acid sequence (or polynucleotide): A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, and includes polynucleotides encoding full length proteins and/or fragments of such full length proteins which can function as a therapeutic agent.
  • a polynucleotide is generally a linear nucleotide sequence, including sequences of greater than 100 nucleotide bases in length.
  • nucleic acid is labeled (for example, biotinylated, fluorescently labeled or labeled with radioisotopes).
  • Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, as in DNA and RNA, or a base linked to an amino acid, as in a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a nucleotide is one monomer in a polynucleotide.
  • a nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • nucleotides are labeled (for example, biotinylated, fluorescently labeled, or labeled with radioisotopes).
  • Oligonucleotide A linear polynucleotide sequence of between 10 and 100 nucleotide bases in length.
  • An oligonucleotide can be labeled (for example, using radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes) either at the 3' or the 5' end, or by the incorporation of labeled nucleotides into the oligonucleotide.
  • oligonucleotides are those which are at least 15, 25, 50, 75 or 90 nucleotide bases in length. Oligonucleotides can be primers or probes.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • ORF open reading frame
  • Ortholog Two nucleic acid or amino acid sequences are orihologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences.
  • Polymerase chain reaction (PCR) A method for amplifying specific DNA segments which exploits certain features of DNA replication. For instance replication requires a primer and specificity is determined by the sequence and size of the primer.
  • One primer is complementary to the sense-strand at one end of the DNA sequence to be amplified and the other primer is complementary to the antisense-strand at the other end of the DNA sequence to be amplified.
  • the method amplifies specific DNA segments by cycles of template denaturation; primer addition; primer annealing and replication using a thermostable DNA polymerase. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.
  • Polymeric solid support The physical structural shape of a medium that forms a solid support.
  • medium can generally include, but are not limited to: polymer films (for instance, polymers having a substantially non-porous surface); polymer filaments (for example, mesh and fabrics); polymer beads; polymer foams; polymer frits; and polymer threads.
  • Polymers can include, but are not limited to, cellulosic substrates, such as nitrocellulose, nylon, silicon, metals, such as gold, Teflon, polypropylene, polyethylene, polybutylene, polyisobutylene, plybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene- propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroefhylene, polysulfornes, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof.
  • cellulosic substrates such as nitrocellulose, nylon, silicon, metals, such as gold, Teflon, polypropylene, poly
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred in nature.
  • polypeptide or protein as used herein encompasses any amino acid sequence and includes, but may not be limited to, modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • Substantially purified polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
  • a non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain.
  • substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine; or (d) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl.
  • Variant amino acid sequences may, for example, be 80, 90 or even 95, 96, 97, 98 or 99% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website.
  • Primers are short nucleic acid molecules, such as DNA oligonucleotides, 15 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for amplification of a nucleic acid sequence, for example, by PCR, real-time PCR, or other nucleic-acid amplification methods known in the art.
  • An "upstream” or “forward” primer is a primer 5' to a reference point on a nucleic acid sequence.
  • a “downstream” or “reverse” primer is a primer 3' to a reference point on a nucleic acid sequence.
  • at least one forward and one reverse primer are included in an amplification reaction. Methods for preparing and using primers are described, for example, in Sambrook et al. (In
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • the nucleic acid can be attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al.
  • the secAl gene is approximately 2849 nucleotides in length.
  • the disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the secAl gene sequences. Such molecules can comprise at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of these sequences, and can be obtained from any region of the disclosed sequences.
  • One of ordinary skill in the art will appreciate that the specificity of a particular primer increases with its length.
  • a primer comprising 20 consecutive nucleotides of the nucleic acid encoding a Mycobacterium or a Nocardia secAl protein will anneal to a target sequence, such as a Mycobacterium or a Nocardia nucleic acid, respectively, in a sputum sample, with a higher specificity than a corresponding primer of only 15 nucleotides.
  • Probes Nucleic acid probes can also be readily prepared based on the nucleic acid molecules provided herein. A probe comprises an isolated nucleic acid. Methods for preparing and using probes are described, for example, in Sambrook et al. (la Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), Ausubel et al.
  • an oligonucleotide probe comprising 20 consecutive nucleotides of the nucleic acid encoding a Mycobacterium or Nocardia secAl protein will anneal to a target sequence, such as an amplified Mycobacterium or Nocardia nucleic acid, respectively, with a higher specificity than a corresponding oligonucleotide probe of only 15 nucleotides.
  • oligonucleotide probes and primers can be selected that comprise at least 15, 20, 25, 30, 35, 40 or more consecutive nucleotides of Mycobacterium ox Nocardia secAl gene sequences.
  • the disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the Mycobacterium and Nocardia secAl gene sequences. Such molecules can comprise at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of these sequences, and can be obtained from any region of the disclosed sequences. Nucleic acid molecules can be selected as probe sequences that comprise at least 15, 20, 25, 30, 35, 40 or more consecutive nucleotides of any of portions of the Mycobacterium or Nocardia secAl gene.
  • Purified The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein is more pure than the protein in its natural environment within a cell.
  • a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment comprising a complex mixture of oligonucleotides.
  • Quencher A molecule, such as DABCYL or TAMRA, which is capable of absorbing fluorescence emitted from a fluorescent marker or dye thereby reducing the level of fluorescence detected in a sample.
  • a quencher molecule can be coupled to an oligonucleotide to generate, for example a molecular beacon probe, a minor groove binding probe, or a TaqMan ® probe.
  • Real-time PCR detects amplification products as they accumulate in a sample during the PCR amplification process. In real-time PCR, the formation of a PCR product is monitored either continuously during amplification, or during each annealing or extension step of each cycle, by means of fluorescent probes, fluorescent primers or fluorescent dyes that bind to double-stranded DNA.
  • real-time PCR is a two-step process based on 1) hybridization of the PCR product with an oligomer probe, the potential of which to emit fluorescence is inactivated by a covalently linked quencher, and 2) separation of the fluorophore from the quencher, or digestion of the hybridized probe, resulting in a fluorescent signal enhancement.
  • FRETProbes involves two probes with different (acceptor and donor) fluorophores.
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • secA An essential, highly conserved, ATP-driven protein component of the sec-dependent protein export pathway in bacteria. The sec-dependent protein export pathway is responsible for binding presecretory proteins and promoting their translocation, via a translocase, across the bacterial plasma membrane.
  • the translocase core is composed of integral membrane proteins and the peripheral membrane protein secA.
  • secA is a soluble, cytoplasmic, multifunctional, 100 kDa homodimer that interacts with acidic phospholipids in the bacterial cell membrane, the SecYEG pore (a membrane channel formed by SecE (9 kDa), SecG (11 kDa), and SecY (47 kDa)) and molecular chaperones, such as SecB (17 kDa), that deliver precursor proteins to the translocase.
  • secA is an ATPase that provides energy for protein translocation and is composed of a helicase motor domain (belonging to the DEAD family) and a translocation domain.
  • SSD specificity domain
  • the SSD is essential for protein translocation, is unique to sec A, and is absent from other members of the DEAD family of proteins (Baud et al, J. Biol Chem, 277:13724-13731, 2002).
  • Some Gram-positive bacteria have two homologous secA proteins with non-overlapping functions, secAl and secA2. Genes encoding secAl and secA2 have been identified in M. leprae, M. avium, M. tuberculosis, andM smegmatis.
  • Both mycobacterial secAl and secA2 proteins exhibit significant sequence homology to other bacterial and plant secA homologs, and both contain the conserved ATP-binding motifs.
  • the two secA proteins are not very similar to each other, although each is highly conserved in different mycobacterial species.
  • secAl is the essential housekeeping secA protein, whereas secA2 is an accessory secretion factor.
  • secAl is believed to be functionally equivalent to the very well characterized Escherichia coli secA.
  • Nocardia secA is similar to Mycobacterium secAl (see the disclosure herein) it is also referred to herein as Nocardia secAl. The M.
  • tuberculosis H37Rv secAl protein is a 949 amino acid molecule (GenBank Accession No. NP_217757) that has been crystallized and its structure elucidated (Sharma et al, Proc. Natl. Acad. Sci. USA, 100:2243-2248, 2003).
  • the motor domain of secAl is composed of residues 2-221 and 360-387, whereas the translocation domain is composed of residues 222-359 and 587-835 of the secAl protein.
  • the SSD is located between residues 222-359 of the secAl protein and is inserted in nucleotide binding domain 1 (NBD1; Sharma et al, Proc. Nat. Acad. Sci. USA, 100:2243-2248, 2003).
  • NBD1 is located between amino acids 1 and 426 and the nucleotide binding domain 2 (NBD2) is located between amino acids 426 and 641 in the 5' half of the secAl protein.
  • the nucleic acid sequence encoding secAl has also been identified for M tuberculosis H37Rv (GenBank Accession No. BX842582; nucleotides 150019 to 152868).
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
  • NCBI Basic Local Alignment Search Tool (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • sequence identity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST sequence analysis software, for instance, the NCBI BLAST 2.0 program gapped blastn set to default parameters.
  • BLAST sequence analysis software for instance, the NCBI BLAST 2.0 program gapped blastn set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
  • Proteins with even greater identity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity, when using gapped blastp with databases such as the nr or swissprot database.
  • TaqMan ® probes A linear oligonucleotide probe with a 5' reporter fluorophore and a 3' quencher fluorophore, such as TAMRA.
  • energy is transferred (via FRET) from the short- wavelength fluorophore to the long- wavelength fluorophore on the other end, quenching the short-wavelength fluorescence.
  • FRET fluorescence transfer
  • Target nucleic acid sequence A nucleic acid sequence to which an antisense or sense oligonucleotide specifically hybridizes.
  • a Mycobacterium species-specific secA primer specifically hybridizes to a target nucleic acid sequence in a Mycobacterium species secA gene.
  • a Nocardia species-specific secA primer specifically hybridizes to a target nucleic acid sequence in a Nocardia species secA gene.
  • the target nucleic acid sequence of a secAl gene can include a substrate specificity domain (SSD) responsible for binding of secAl to the signal sequence of proteins to be exported.
  • SSD substrate specificity domain
  • a specific, non-limiting example of a target nucleic acid sequence is a region of the secAl gene that is approximately 700 base pairs in length in the 5' half of the secAl gene that includes a substrate specificity domain located between amino acid positions 222 and 359 of the secAl protein.
  • a further specific, non-limiting example of a target nucleic acid sequence is a region of the secAl gene that corresponds to a region between amino acid positions 148 and 380 of the secAl protein.
  • Variant oligonucleotides and variant analogs A variation of an oligonucleotide or an oligonucleotide analog is an oligomer having one or more base substitutions, one or more base deletions, and/or one or more base insertions, so long as the oligomer substantially retains the activity of the original oligonucleotide or analog, or has sufficient complementarity to a target sequence.
  • a variant oligonucleotide or analog may also hybridize with the target DNA or RNA, under stringency conditions as described above.
  • a variant oligonulceotide or analog also exhibits sufficient complementarity with the target DNA or RNA of the original oligonucleotide or analog as described above.
  • M abscessus Mycobacterium abscessus
  • M. africanum M. asiaticum
  • M. avium M. bovis
  • M. chelonae M.flavescens
  • M. fortuitum M. gastri
  • M. gordonae M. haemophilum
  • Nocardia malmoense, M. marinum, M. mucogenicum, M. peregrinum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans, X-Cluster, and xenopi.
  • Nocardia three species are responsible for most human infections. Examples of disease-associated Nocardia include Nocardia asteroids, Nocardia brasiliensis, and Nocardia caviae.
  • This disclosure provides the nucleic acid sequences encoding secA polypeptide fragments from different Mycobacterium and Nocardia species, including disease-associated Mycobacterium and Nocardia. In specific embodiments, these sequences are used for generating oligonucleotide primers and probes for the detection of bacteria of the Mycobacterium and Nocardia genera in samples. In other specific embodiments, these sequences are used for generating oligonucleotide primers and probes for the detection of Mycobacterium and Nocardia species in samples. Nucleic acid sequences encoding secA polypeptides, such as secAl polypeptide fragments, from different species of Mycobacterium and Nocardia are disclosed herein. Specific, non-limiting examples of Mycobacterium secAl nucleic acid sequences encoding a Mycobacterium secAl polypeptide fragment include, but are not limited to, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID
  • SEQ ID NO: 132 SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO:
  • Nocardia secAl nucleic acid sequences encoding a Nocardia secAl polypeptide fragment include, but are not limited to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245,
  • polynucleotides include DNA, cDNA, and RNA sequences that encode a Mycobacterium or a Nocardia secAl polypeptide fragment. It is understood that all polynucleotides encoding a Mycobacterium or a Nocardia secA polypeptide, such as a secAl polypeptide fragment, are also included herein.
  • a Mycobacterium secAl nucleic acid sequence is at least 80% homologous to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158,
  • a Mycobacterium secAl nucleic acid sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO:
  • a Nocardia secAl nucleic acid sequence is at least 80% homologous to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252.
  • a Nocardia secAl nucleic acid sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244,
  • the Mycobacterium and Nocardia secA polynucleotides disclosed herein include a region encoding the substrate specificity domain.
  • the sequence encoding the substrate specificity domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the substrate specificity domain of M tuberculosis.
  • the Mycobacterium and Nocardia secA polynucleotides may be a recombinant DNA molecule which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences.
  • DNA sequences encoding Mycobacterium or Nocardia secA polypeptide fragments, such as secAl polypeptide fragments can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell.
  • progeny may not be identical to the parental cell since there may be mutations that occur during replication.
  • Methods of stable transfer meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • Mycobacterium and Nocardia secA polynucleotide sequences such as secAl polynucleotide sequences, can be operatively linked to expression control sequences.
  • An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
  • the expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (for instance, ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art. Alternatively, MgCl 2 or RbCl can be used.
  • Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
  • the host is a eukaryote
  • methods of transfection of DNA as calcium phosphate coprecipitates conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used.
  • Eukaryotic cells can also be cotransformed with secA polynucleotide sequences, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Mycobacterium and Nocardia secA oligonucleotides for example secAl probes and secAl primers.
  • the Mycobacterium and Nocardia secA oligonucleotides for example secAl oligonucleotides, can be genera- or species-specific.
  • Mycobacterium/Nocardia genera-specific secA oligonucleotides include those oligonucleotides encoding a secA protein present in any Mycobacterium or Nocardia species found in clinical samples.
  • Mycobacterium or Nocardia species- specific secA oligonucleotides include those oligonucleotides encoding a secA protein present in specific Mycobacterium or Nocardia species, respectively, found in clinical samples.
  • Mycobacterium species found in clinical samples can be Mycobacterium known to be associated with disease, such as M tuberculosis, M. bovis, M. africanum, M. asiaticum, M. avium, M. celatum, M. chelonae, M. fortuitum, M. genavense, M. haemophilum, M. intracellulare, M. kansasii, M. malmoense, M. marinum, M. scrofulaceum, M.
  • Nocardia species found in clinical samples can be Nocardia known to be associated with disease, such as Nocardia asteroides complex, Nocardia farcinica, Nocardia nova, Nocardia brasiliensis, Nocardia otitidiscaviarum, and Nocardia transvalensis.
  • the methods disclosed herein take advantage of the fact that under appropriate conditions oligonucleotides, such as probes and primers, form base-paired duplexes with oligonucleotides which have a complementary base sequence.
  • the stability of the duplex is dependent on a number of factors, including the length of the oligonucleotides, the base composition, and the composition of the solution in which hybridization is effected.
  • the effects of base composition on duplex stability may be reduced by carrying out the hybridization in particular solutions, for example in the presence of high concentrations of tertiary or quaternary amines.
  • the thermal stability of the duplex is also dependent on the degree of sequence similarity between the sequences.
  • each oligonucleotide sequence can be selected to optimize binding of target Mycobacterium or Nocardia nucleic acid sequences, for example secA nucleic acid sequences, such as secAl nucleic acid sequences. An optimum length for use with a particular Mycobacterium or Nocardia nucleic acid sequence under specific screening conditions can be determined empirically.
  • Oligonucleotides, for example probes or primers, of the disclosed secAl nucleic acid sequences may be comprised of at least 15 consecutive nucleic acids, which is sufficient to permit the oligonucleotide to selectively hybridize to DNA that encodes the disclosed Mycobacterium secAl polypeptide or polypeptide fragment (for example a polynucleotide set forth as SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149
  • oligonucleotides comprising at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of the Mycobacterium or Nocardia secA sequences, such as secAl sequences, can be used.
  • the oligonucleotide includes nucleotides 1-15, 16-30, or 31-45, etc., 2-16, 17-31, or 32-46, etc., or 3-17, 18-32, or 33-47, etc. of a Mycobacterium or Nocardia secAl sequence.
  • the oligonucleotide includes nucleotides 1-20, 21-40, or 41-60, etc., 2-21, 22-41, or 42-61, etc., or 3-22, 23-42, or 43-62, etc. of ' a Mycobacterium or Nocardia secAl sequence.
  • the oligonucleotide includes nucleotides 1-25, 26-50, 51-75, etc., 2-26, 27-51, or 52-76, or 2-27, 28-52, or 53-77, etc. of a Mycobacterium or Nocardia secAl sequence.
  • the Mycobacterium secAl sequence is a target sequence for amplification.
  • the Nocardia secAl sequence is a target sequence for amplification.
  • the target sequence for amplification is in the 5' half of the Mycobacterium or Nocardia secAl gene and includes the substrate specificity domain.
  • the target sequence for amplification is a region of the secAl gene that is at least about 700 base pairs in length in the 5' half of the secAl gene and includes the substrate specificity domain.
  • the target sequence for amplification is a region of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 base pairs in length in the 5' half of the secAl gene and includes the substrate specificity domain.
  • Mycobacterium/Nocardia genera-specific secA oligonucleotides such as primers and probes, selectively hybridize to a region that is present in all Mycobacterium and Nocardia secA genes, but that is not present in a sec A nucleic acid in any other genus of bacteria.
  • a Mycobacterium/Nocardia genera-specific probe hybridizes to a region of the secAl gene that is common to all Mycobacterium and Nocardia secAl nucleic acids. Mycobacterium/Nocardia genera-specific probes thus can be used to distinguish Mycobacterium and Nocardia from other genera of bacteria. Specific, non-limiting examples of
  • Mycobacterium/Nocardia genera-specific secAl probes include probes that selectively hybridize to a nucleic acid sequence within a region in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain.
  • a Mycobacterium/Nocardia genera-specific primer can be used to sequence a Mycobacterium or Nocardia secAl nucleic acid in order to identify a Mycobacterium or Nocardia secAl nucleic acid.
  • two, or more, genera-specific primers can be used to amplify a region within the secAl gene, for example by polymerase chain reaction (PCR) or more specifically by real-time PCR, that is common to all Mycobacterium and Nocardia, but that is not present in secAl in bacteria of another genus.
  • PCR polymerase chain reaction
  • Specific, non-limiting examples of Mycobacterium/Nocardia genera-specific secAl primers include primers that selectively hybridize to a nucleic acid sequence within a region in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain.
  • Mycobacterium and Nocardia species-specific secAl oligonucleotides can be used to identify a Mycobacterium or Nocardia species of interest.
  • these species-specific probes and primers specifically hybridize to a region in a secAl gene which is unique to a particular Mycobacterium or Nocardia species and not to other species.
  • Specific, non-limiting examples of Mycobacterium or Nocardia species-specific secAl probes include probes that selectively hybridize to a nucleic acid sequence within the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain.
  • a Mycobacterium species-specific primer is used to sequence a Mycobacterium secAl nucleic acid in order to identify a Mycobacterium species-specific secAl nucleic acid.
  • a Nocardia species-specific primer is used to sequence a Nocardia secAl nucleic acid in order to identify a Nocardia species-specific secAl nucleic acid.
  • two, or more, species-specific primers can be used to amplify a region, for example by PCR or more specifically by real-time PCR, within the secAl gene that is unique to a particular Mycobacterium or Nocardia species.
  • Mycobacterium or Nocardia species-specific secAl primers include primers that selectively hybridize to a nucleic acid sequence in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain.
  • the primers and probes disclosed herein can be end-labeled (for example, radiolabeled, enzymatically-Iabeled, fluorescently-labeled, or biotinylated).
  • a primer label is a fluoresceinated Mycobacterium species specific primer.
  • a primer label is a biotinylated Nocardia species specific primer.
  • the probes disclosed herein can be fluorescently-labeled, such as for use in real-time PCR.
  • the oligonucleotide probes in the sample are labeled to render them readily detectable.
  • Detectable labels may be any species or moiety that maybe detected either visually or with the aid of an instrument. Detectable labels can be radioisotopes, chemiluminescent tags, haptens, or fluorescent markers.
  • fluorescent markers include FITC, LightCycler Red 640, Light Cycler Red 705, 6-carboxy-X-rhodamine (ROX), 5-carboxyfluorescein (FAM), 2'7'-dimethoxy- 4'5'-dichloro-6-carboxyfluorescein (JOE), and 6-carboxy-2',4,7,7'-tetrachlorofluorescein (TET).
  • the fluorescent markers coupled to the oligonucleotide probes have spectrally distinct emission spectra such that the amplified DNA sequences to which they specifically hybridize can be distinguished within the same reaction tube.
  • FRETProbes include an upstream probe labeled with a 3' donor fluorophore, such as FITC, and a downstream probe labeled with an acceptor fluorophore, such as LightCycler Red 640 or Red 705, at the 5' terminus.
  • the nucleic acid sequence of a FRETProbe includes a nucleic acid sequence that detects the amplified product from the target nucleic acid sequence of interest, such as a Mycobacterium secA sequence, for example, a Mycobacterium species-specific secAl sequence.
  • the donor fluorophore is excited by a filtered light source, such as by a LightCycler's light emitting diode (LED), and a green fluorescent light is emitted at a slightly longer wavelength.
  • a filtered light source such as by a LightCycler's light emitting diode (LED)
  • a green fluorescent light is emitted at a slightly longer wavelength.
  • the pair of FRETProbes hybridize to the target sequence, the two fluorophores are in close proximity to each other, for example within 1- 10 nucleotides of each other, and the energy emitted by the excitation of the donor fluorophore excited the acceptor fluorophore, for example a LightCycler Red 640 attached to the probe.
  • the resultant energy transfer via FRET results in the emission of a red fluorescent light at an even longer wavelength.
  • the intensity of the light emitted by the acceptor fluorophore is measured by the apparatus, such as a LightCycler.
  • the increasing amount of measured fluorescence is proportional to tlie increasing amount of the amplified target nucleic acid generated during the ongoing PCR process. Since the acceptor fluorophore only emits a signal when both labeled probes are hybridized to the target nucleic acid sequence, the fluorescence measurement is performed after the annealing step in the PCR process.
  • Molecular beacon probes include probes coupled to a fluorescent marker in combination with a quencher molecule.
  • the nucleic acid sequence of a molecular beacon probe includes a nucleic acid that detects the amplified product from the DNA sequence of interest, and sequences that permit the molecular beacon probe to form a hairpin structure. Attached to opposite ends (the 5' and the 3' end of the molecular beacon) are a fluorescent reporter molecule and a quencher molecule. When the molecular beacon is in the hairpin conformation (not hybridized to product) any fluorescence emitted by the fluorescent label is absorbed (quenched) by the quencher molecule via FRET and no fluorescence is detected.
  • a molecular beacon hybridizes to an amplified target nucleic acid with a complementary nucleic acid sequence the fluorescent label and the quencher molecule are separated, and fluorescence is detected and can be measured during each PCR cycle.
  • probes are known to one of skill in the art (see the Molecular-Probes, Eugene, OR website).
  • TaqMan ® probes include linear oligonucleotide probes with a 5' reporter fluorophore and a
  • 3' quencher fluorophore such as TAMRA.
  • energy is transferred (via FRET) from the short-wavelength fluorophore to the long- wavelength fluorophore on the other end, quenching the short-wavelength fluorescence.
  • FRET fluorescence transfer
  • quencher molecules include N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA) and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL).
  • TAMRA N,N,N',N'-tetramethyl-6- carboxyrhodamine
  • DBCYL 4-(4'-dimethylaminophenylazo) benzoic acid
  • fluorescent markers and quenchers are widely available commercially with substituents on their phenyl moieties which can be used as the site for coupling or as the coupling functionality for attachment to an oligonucleotide.
  • Mycobacterium and Nocardia secA Polypeptides SecAl polypeptide fragments from different Mycobacterium and Nocardia species are disclosed herein.
  • Mycobacterium secAl polypeptide fragments include, but are not limited to, polypeptides having a sequence as set forth as one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
  • Nocardia secAl polypeptide fragment sequences can be translated from the Nocardia nucleic acid sequences as set forth as one of SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252.
  • SecAl polypeptide fragments also include, but are not limited to, an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth as one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ
  • the Mycobacterium and Nocardia secA polypolypeptides disclosed herein include a region encoding the substrate specificity domain which is between amino acids 222 and 359 of the secAl protein.
  • the sequence encoding the substrate specificity domain is at least 80%, 85%, 90%>, 95%, 96%, 97%, 98%, or 99% homologous to the substrate specificity domain of M. tuberculosis. Fragments, variants, and fusions of the Mycobacterium and Nocardia secAl polypeptide fragments identified above are disclosed herein and can readily be prepared by one of skill in the art using molecular techniques.
  • a fragment of a Mycobacterium secAl polypeptide fragment includes at least 8, 10, 15, or 20 consecutive amino acids of a Mycobacterium secAl polypeptide fragment.
  • a fragment of ' a Nocardia secAl polypeptide fragment includes at least 8, 10, 15, or 20 consecutive amino acids of a Nocardia secAl polypeptide fragment.
  • a fragment of a Mycobacterium or a Nocardia secAl polypeptide fragment includes a species-specific antigenic epitope.
  • a fragment is at least 17 amino acids, at least 23 amino acids, at least 25 amino acids, or at least 30 amino acids in length from a sec A polypeptide fragment.
  • polypeptides include polypeptides as set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, S
  • a fragment retains at least one species-specific antigenic epitope.
  • One skilled in the art can purify a Mycobacterium or a Nocardia secAl polypeptide fragment using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the Mycobacterium or Nocardia secAl polypeptide can also be determined by amino- terminal amino acid sequence analysis. Minor modifications of the Mycobacterium or Nocardia secAl polypeptide primary amino acid sequences may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein. Isolation and purification of recombinantly expressed polypeptides may be carried out by conventional means including preparative chromatography and immunological separations.
  • a secA polypeptide or a fragment or conservative variant thereof can be used to produce antibodies which are immunoreactive or bind to a Mycobacterium or Nocardia species-specific epitope of secA, such as secAl.
  • Polyclonal antibodies, antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are included. The preparation of polyclonal antibodies is well-known to those skilled in the art.
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion- exchange chromatography.
  • Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
  • suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
  • Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
  • Large-scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or
  • Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • pristane tetramethylpentadecane
  • the desired monoclonal antibody is recovered from the body fluid of the animal.
  • Antibodies can also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in WO 91/11465, 1991, and Losman et al, Int. J. Cancer 46:310, 1990.
  • an antibody that specifically binds a secA polypeptide can be derived from a humanized monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833, 1989.
  • Antibodies can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from Stragene Cloning Systems (La Jolla, CA).
  • antibodies can be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of Hie endogenous heavy and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody- secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7:13, 1994; Lonberg et al, Nature 368:856, 1994; and Taylor et al, Int. Immunol 6:579, 1994.
  • Antibodies include intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding the epitopic determinant.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab') 2 the fragment of the antibody that can be obtained by treating whole antibody with tlie enzyme pepsin without subsequent reduction
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • An epitope is any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patents No. 4,036,945 and No. 4,331,647, and references contained therein; Nisonhoff et al, Arch. Biochem. Biophys.
  • Fv fragments comprise an association of V H and V L chains. This association maybe noncovalent (Inbar et al, Proc. Nat'l Acad. Sci.
  • variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, for example, Sandhu, supra.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No.4,946,778; Pack et al. , Bio/Technology 11 : 1271 , 1993 ; and Sandhu, supra) .
  • Another form of an antibody fragment is a peptide coding for a single complementarity- determining region (CDR).
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (Larrick et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 1991). Antibodies can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal can be derived from substantially purified polypeptide produced in host cells, in vitro translated cDNA, or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • Such commonly used earners which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid The coupled peptide is then used to immunize the animal (for example., a mouse, a rat, or a rabbit).
  • Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first mono-clonal antibody.
  • Effector molecules for example, therapeutic, diagnostic, or detection moieties, can be linked to an antibody that specifically binds a secA polypeptide, such as a secAl polypeptide, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (for example, through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the rumor site may be used.
  • Detection and Identification of Mycobacterium and Nocardia Genera Using secA Nucleic Acids Probes and primers directed to a secA nucleic acid sequence can be used for the detection and/or identification of the Mycobacterium and Nocardia genera in clinical samples using a variety of detection and identification methods.
  • the disclosed methods for detecting and identifying a Mycobacterium genus or a Nocardia genus are an advantage over previous methods because they provide rapid and accurate detection and identification of both Mycobacterium and Nocardia in a clinical sample.
  • the disclosed methods may be used to rapidly and accurately identify whether the causative agent of a pulmonary disease is either Mycobacterium or Nocardia, when it is otherwise not possible to distinguish the cause of the infection.
  • the presence of a Mycobacterium and Nocardia may be detected with the use of any one of a number of techniques.
  • the presence of a Mycobacterium can be detected by amplification of a Mycobacterium nucleic acid sequence.
  • the presence of a Nocardia can be detected by amplification of a Nocardia nucleic acid sequence.
  • Any nucleic acid amplification method can be used to detect the presence of a Mycobacterium or a Nocardia in a sample.
  • PCR is used to amplify the mycobacterial nucleic acid sequences.
  • PCR is used to amplify the norcardial nucleic acid sequences.
  • real-time PCR, RT-PCR, transcription-mediated amplification (TMA), or ligase chain reaction can be used to amplify mycobacterial or nocardial nucleic acid sequences.
  • TMA transcription-mediated amplification
  • ligase chain reaction can be used to amplify mycobacterial or nocardial nucleic acid sequences.
  • Techniques for nucleic acid amplification are well-known to those of skill in the art.
  • Mycobacterium, Nocardia, or Mycobacterium and Nocardia DNA sequences are amplified.
  • Mycobacterium, Nocardia, or Mycobacterium and Nocardia RNA sequences are reverse transcribed prior to amplification using reverse transcription- polymerase chain reaction (RT-PCR).
  • amplification of a Mycobacterium secA nucleic acid sequence can be used to detect the presence of a member of the genus Mycobacterium in a sample.
  • amplification of ' a Nocardia secA nucleic acid sequence can be used to detect the presence of a member of the genus Nocardia in a sample.
  • thermal cycler apparatus for example a PTC- 100 ® Peltier Thermal Cycler (MJ Research, Inc.; San Francisco, CA), a Robocycler ® 40 Temperature Cycler (Stratagene; La Jolla, CA), or a GeneAmp ® PCR System 9700 (Applied Biosystems; Foster City, CA) can be used to amplify nucleic acid sequences.
  • PTC- 100 Peltier Thermal Cycler
  • Robocycler ® 40 Temperature Cycler Stratagene; La Jolla, CA
  • GeneAmp ® PCR System 9700 Applied Biosystems; Foster City, CA
  • any type of real-time thermocycler apparatus for example a LightCycler (Roche; Mannheim, Germany), a 7700 Sequence Detector (Perkin Elmer/Applied Biosystems; Foster City, CA), ABI7700 (Applied Biosystems; Foster City, CA), or an MX4000 (Stratagene; La Jolla, CA) can by used to amplify nucleic acid sequences in real-time. Any region of a secA gene that is common to all Mycobacterium and Nocardia, but not in bacteria in another genus, can be amplified.
  • secA Mycobacterium/Nocardia genera-specific primers can be used to amplify a region that is at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 1000, or more base pairs in length to produce amplified Mycobacterium and/or Nocardia secA nucleic acids.
  • the target region of the secAl gene can include a substrate specificity domain (responsible for binding of secAl to the signal sequence of proteins to be exported) located between amino acid positions 222 and 359 of the secAl protein.
  • Mycobacterium/Nocardia genera-specific primers which specifically bind to a conserved region in the 5' half of the secAl gene, are used to produce an amplified Mycobacterium/Nocardia genera-specific nucleic acid sequence that is approximately 700 base pairs in length and includes the substrate specificity domain.
  • Mycobacterium/Nocardia genera-specific primers which specifically bind to a conserved region in the 5' half of the secAl gene, are used to produce an amplified Mycobacterium/Nocardia genera-specific nucleic acid sequence that is approximately 700 base pairs in length and includes the substrate specificity domain.
  • Mycobacterium/Nocardia genera-specific primers which specifically bind to a conserved region in the 5' half of the secAl gene, are used to produce an amplified Mycobacterium/Nocardia genera- specific nucleic acid sequence that is approximately 200, approximately 300, approximately 400, approximately 500, or approximately 600 base pairs in length and includes the substrate specificity domain.
  • the Mycobacterium/Nocardia genera-specific nucleic acid sequence is amplified from Mycobacterium, Nocardia, or both Mycobacterium and Nocardia. At least two primers are utilized in the amplification reaction.
  • One or both of the primers can be end-labeled (for example, radiolabeled, fluorescently-labeled, enzymatically-labeled, or biotinylated).
  • the resulting amplicon is labeled.
  • the pair of primers includes an upstream primer (which binds 5' to the downstream primer) and a downstream primer (which binds 3' to the upstream primer).
  • either the upstream primer or the downstream primer is labeled.
  • One specific, non-limiting example of a primer label is a fluoresceinated downstream primer.
  • the amplified Mycobacterium/Nocardia genera-specific product for example a
  • Mycobacterium or a Nocardia secA nucleic acid sequence can be detected in real-time, for example by real-time PCR, in order to determine the presence, the identity, and/or the amount of a Mycobacterium and or Nocardia genus in a sample.
  • an amplified DNA sequence such as an amplified Mycobacterium or Nocardia secAl nucleic acid sequence, can be detected using a probe specific for the product amplified from the DNA sequence of interest. Detecting the amplified product includes the use of labeled probes that are sufficiently complementary and hybridize to the amplified nucleic acid sequence.
  • the presence, amount, and/or identity of the amplified product can be detected by hybridizing a labeled probe, such as a fluorescently labeled probe, complementary to the amplified product.
  • a labeled probe such as a fluorescently labeled probe
  • the detection of a target nucleic acid sequence of interest includes the combined use of PCR amplification and a labeled probe such that the product is measured using real-time PCR.
  • the detection of an amplified target nucleic acid sequence of interest includes the transfer of the amplified target nucleic acid to a solid support, such as a blot, for example a Northern blot, and probing the blot with a probe, for example a labeled probe, that is complementary to the amplified target nucleic acid sequence.
  • the detection of an amplified target nucleic acid sequence of interest includes the hybridization of a labeled amplified target nucleic acid to probes that are attached, for example chemically-linked, to an array and that are complementary to the amplified target nucleic acid.
  • an assay system including a primer pair complementary to a Mycobacterium/Nocardia secA nucleic acid sequence (for example, for the amplification of a
  • Mycobacterium/Nocardia genera-specific nucleic acid also includes a labeled probe that hybridizes to the amplified Mycobacterium/Nocardia genera-specific product in order to detect the amplified product.
  • the primer pair includes Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium/Nocardia genera-specific probe.
  • the primer pair includes
  • Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium or Nocardia genus-specific probe.
  • the amplified target nucleic acid can be detected using fluorescently-labeled genus-specific probes in real time.
  • the fluorescently-labeled probes rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time.
  • FRET fluorescence resonance energy transfer
  • FRET that occurs between fluorogenic labels on different probes (for example, using FRETProbes) or between a fluorophore and a non-fluorescent quencher on the same probe (for example, using a molecular beacon or a TaqMan ® probe) can identify a probe that specifically hybridizes to the DNA sequence of interest and in this way, using Mycobacterium/Nocardia genera- specific probes, Mycobacterium genus-specific probe, or Nocardia genus-specific probes can detect the presence, identity, and/or amount of Mycobacterium and/or Nocardia genera in a sample.
  • the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube.
  • real-time PCR can be performed in a microfluidics device, such as in an I-CORE ® module (Cepheid; Sunnyvale, CA). Microfluidics involves manipulating minute volumes of liquids and is used to miniaturize biological assays. Real-time PCR using microfluidic technology offers several benefits over standard real-time PCR techniques, for example reduced sample volume and increased performance.
  • a melting curve analysis of the amplified target nucleic acid can be performed subsequent to the amplification process.
  • the Tm of a nucleic acid sequence depends on the length of the sequence and its G/C content.
  • the identification of the Tm for a nucleic acid sequence can be used to identify the amplified nucleic acid and/or single nucleotide polymorphisms (SNPs) between amplified nucleic acids.
  • SNPs single nucleotide polymorphisms
  • One specific, non-limiting example of an apparatus that can perform real-time PCR and melting curve analysis is the LightCyclerTM (Roche; Mannheim, Germany).
  • devices can be used to detect the presence of Mycobacterium and/or Nocardia, using specific oligonucleotide probes.
  • the arrays herein termed "Mycobacterium/Nocardia detection arrays,” are used to detect the presence of Mycobacterium and Nocardia genera. Other arrays are specific for the detection of either the Mycobacterium genus or the Nocardia genus.
  • a pre-determined set of probes for example Mycobacterium/Nocardia genera- specific secA oligonucleotide probes, are attached to the surface of a solid support for use in detection of the amplified Mycobacterium and Nocardia nucleic acid sequences.
  • Mycobacterium genus-specific or Nocardia genus-specific probes are attached to the surface of a solid support for use in the detection of the amplified Mycobacterium or Nocardia genus-specific nucleic acid sequences, respectively.
  • an oligonucleotide probe can be included to detect the presence of this amplified control nucleic acid.
  • the oligonucleotide probes attached to the array specifically hybridize to target nucleic acids.
  • the anay includes a plurality of Mycobacterium/Nocardia genera-specific oligonucleotide probes.
  • the anay includes a plurality of Mycobacterium genus-specific and/or Nocardia genus- specific probes.
  • a hybridization complex is formed when target nucleic acids, for example amplified secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium/Nocardia genera-specific oligonucleotide probes, Mycobacterium genus-specific probes, or Nocardia genus-specific probes, chemically linked to a polymeric solid support.
  • the hybridization complex forms a hybridization pattern of the sample.
  • a sample containing a target nucleic acid is hybridized to an array.
  • the target nucleic acid sequences are first amplified in an amplification reaction before hybridizing to the array.
  • probe sequences of use with the method when detecting, for example, secAl nucleic acid are Mycobacterium/Nocardia genera- specific oligonucleotide probes, Mycobacterium genus-specific probes, or Nocardia genus-specific probes.
  • the sequences forming the array can be directly attached or linked to the support.
  • the oligonucleotide probes can be attached to the support by non-Mycobacterium or Nocardia sequences such as oligonucleotides or other molecules that serve as spacers or linkers to the solid support.
  • the solid support can be formed from an organic polymer (a polymeric support).
  • Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene-propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroethylene, polysulfornes, hydroxylated biaxially oriented polypropylene, animated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof (see U.S. Patent No.
  • suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an oligonucleotide thereto; amenability to "in situ" synthesis of biomolecules; being chemically inert, such that at the termination of biomolecular synthesis, areas on the support not occupied by the biomolecules are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the biomolecules.
  • the solid support surface is polypropylene. Polypropylene is chemically inert and hydrophobic.
  • Non-specific binding is generally avoidable, and detection sensitivity is improved.
  • Polypropylene has good chemical resistance to a variety of organic acids (for instance, formic acid), organic agents (for instance, acetone or ethanol), bases (for instance, sodium hydroxide), salts (for instance, sodium chloride), oxidizing agents (for instance, peracetic acid), and mineral acids (for instance, hydrochloric acid).
  • Organic acids for instance, formic acid
  • organic agents for instance, acetone or ethanol
  • bases for instance, sodium hydroxide
  • salts for instance, sodium chloride
  • oxidizing agents for instance, peracetic acid
  • mineral acids for instance, hydrochloric acid
  • a surface activated organic polymer can be the solid support surface.
  • the surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge.
  • amine groups on the activated organic polymers are reactive with nucleotides such that the nucleotides can be bound to the polymers.
  • other reactive groups can be used (for example, carboxylated, hydroxylated, thiolated, or active ester groups).
  • array formats can be employed in accordance with the present disclosure.
  • One example includes a linear anay of oligonucleotide bands, generally referred to in the art as a dipstick.
  • other anay formats including, but not limited to slot (rectangular) and circular anays are equally suitable for use (see U.S. Patent No. 5,981,185, herein incorporated by reference).
  • the array is formed on a polymer medium, which is a thread, membrane or film.
  • Particularly disclosed for preparation of anays at this time are biaxially oriented polypropylene (BOPP) films; in addition to their durability, BOPP films exhibit a low background fluorescence.
  • BOPP biaxially oriented polypropylene
  • the anay formats of the present disclosure can be included in a variety of different types of formats.
  • a "format” includes any format to which the solid support can be affixed, such as microtiter plates, test tubes, inorganic sheets, dipsticks, etc.
  • the solid support is a polypropylene thread
  • one or more polypropylene threads can be affixed to a plastic dipstick-type device
  • polypropylene membranes can be affixed to glass slides.
  • the particular format is, in and of itself, unimportant. All that is necessary is that the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (for example, the dipstick or slide) is stable to any materials into which the device is introduced (for example, clinical samples, hybridization solutions, etc.).
  • the arrays of the present disclosure can be prepared by a variety of approaches which are known to those working in the field. Pursuant to one type of approach, the complete oligonucleotide probe sequences are synthesized separately and then attached to a solid support (see U.S. Patent No. 6,013,789, herein incorporated by reference). In another embodiment, the sequences can be synthesized directly onto the support to provide the desired anay (see U.S. Patent No. 5,554,501, herein incorporated by reference).
  • oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (for example, see PCT applications WO 85/01051 and WO 89/10977, or U.S. Patent No. 5,554,501, herein incorporated by reference).
  • a polypropylene support for example, a BOPP
  • a polypropylene support is first surface animated by exposure to an ammonia plasma generated by radiofrequency plasma discharge.
  • the reaction of a phosphoramidite-activated nucleotide with the animated membrane followed by oxidation with, for example, iodine provides a base stable amidate bond to the support.
  • a suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern.
  • a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (conesponding in number to the number of channels in the delivery system) across the substrate.
  • the substrate can then be rotated by 90° to permit synthesis to proceed within a second (2°) set of rows that are now perpendicular to the first set.
  • This process creates a multiple-channel anay whose intersection generates a plurality of discrete cells.
  • the oligonucleotides can be bound to the polypropylene support by either the 3' end of the oligonucleotide or by the 5' end of the oligonucleotide. In one embodiment, the oligonucleotides are bound to tlie solid support by the 3' end.
  • the internal complementarity of an oligonucleotide probe in the region of the 3' end and the 5' end determines binding to the support.
  • the end of the probe with the most internal complementarity is bound to the support, thereby leaving the end with the least internal complementarity to bind to amplified Mycobacterium and/or Nocardia sequences.
  • the presence, identity and/or amount of a Mycobacterium or a Nocardia can be determined by other hybridization methods, such as Northern or Southern blot analysis, using labeled oligonucleotides specific to a nucleic acid sequence, such as a conserved portion of the secAl gene that is common only to Mycobacterium and Nocardia (a Mycobacterium/Nocardia genera-specific nucleic acid sequence).
  • oligonucleotides such as those described above, can be labeled radioactively with isotopes (such as 32 P) or non-radioactively, with tags such as fluorophores or biotin (Ward and Langer, Proc. Natl. Acad. Sci.
  • the Phosphorimager is able to analyze both DNA samples from blots and gels using autoradiographic, direct fluorescence or chemifluorescence detection. Since the Phosphorimager is more sensitive than ordinary x-ray film, exposure times can be reduced up to ten-fold and signal quantitation of both weak and strong signals on the same blot is possible. Images can be visualized and evaluated with the aid of computer programs such as ImageQuantTM.
  • the identification of an amplified Mycobacterium/Nocardia genera-specific product also can be determined by sequencing the amplified product in order to determine if the amplified product is from a Mycobacterium, a Nocardia, or both a Mycobacterium and a Nocardia. Techniques for sequencing nucleic acids are well known to those of skill in the art. Antibodies that specifically bind secAl polypeptides also can be used to detect a Mycobacterium or a Nocardia in a sample.
  • antibodies against secAl polypeptides can be attached or linked to a support medium, such as a blot or a dish, and a protein sample applied.
  • a support medium such as a blot or a dish
  • antibodies that specifically bind secAl polypeptides can be used to detect secAl polypeptides by using them to probe blots containing a protein sample.
  • the disclosed methods for detecting and identifying a Mycobacterium species or a Nocardia species are an advantage over previous methods because they provide rapid and accurate detection and identification of Mycobacterium and Nocardia species in a clinical sample.
  • the disclosed methods may be used to rapidly and accurately identify whether the causative agent of pulmonary disease is a particular Mycobacterium or Nocardia species, when it is otherwise not possible to distinguish the cause of the infection.
  • the amplification-based detection and identification methods described above are used to detect and/or identify a Mycobacterium or a Nocardia species in a sample.
  • a pair of primers used in an amplification reaction can be Mycobacterium species-specific primers which permit amplification of a nucleic acid from a clinically relevant mycobacterial pathogen.
  • secAl species-specific primers which specifically bind to a unique region of the Mycobacterium secAl gene are used to produce an amplified Mycobacterium species-specific nucleic acid in order to detect the presence of, or identify, a particular Mycobacterium species in a sample, such as M tuberculosis, M. avium, or M kansasii.
  • a pair of primers used in an amplification reaction can be Nocardia species-specific primers which permit amplification of a nucleic acid from a clinically relevant nocardial pathogen.
  • secAl species-specific primers which specifically bind to a unique region of the Nocardia secAl gene are used to produce an amplified Nocardia species- specific nucleic acid in order to detect the presence of, or identify, a particular Nocardia species in a sample, such as TV. asteroids, TV brasiliensis, or TV caviae. Any region of a secA gene, such as a secAl gene, that is unique to a Mycobacterium or a Nocardia species may be amplified.
  • secAl species-specific primers can be used to amplify a region that is at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, at least 1000, or more base pairs in length.
  • a Mycobacterium species-specific secAl nucleic acid sequence that can be amplified to detect and or identify a Mycobacterium species in a sample include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO:
  • Nocaria species-specific secAl nucleic acid sequence that can be amplified to detect and/or identify a Nocardia species in a sample
  • examples of a Nocaria species-specific secAl nucleic acid sequence that can be amplified to detect and/or identify a Nocardia species in a sample include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ
  • More than one pair of primers can be included in an amplification reaction (multiplex primers) in order to simultaneously amplify more than one target and/or control nucleic acid molecule in a single reaction tube.
  • more than one pair of primers is included in the amplification reaction in order to amplify more than one Mycobacterium species-specific secA nucleic acid sequence present in a sample.
  • more than one pair of primers is included in the amplification reaction in order to amplify more than one Nocardia species-specific secA nucleic acid sequence in a sample, or to amplify at least one Nocardia and at least one Mycobacterium species-specific secA nucleic acid sequence in a sample.
  • At least one pair of Mycobacterium/Nocardia genera-specific primers is included in an amplification reaction with at least one pair of Mycobacterium or Nocardia species-specific primers.
  • an additional pair of primers is included in the amplification reaction as an internal control.
  • Internal control primers are designed to amplify nucleic acid from all samples, and serve to provide confirmation of appropriate amplification.
  • primer pairs are designed to amplify nucleic acid from all samples, and serve to provide confirmation of appropriate amplification.
  • An amplified Mycobacterium species-, Nocardia species-, or a Mycobacterium/Nocardia genera-specific product for example a species- or genera-specific secA nucleic acid sequence
  • can be detected in real-time for example by real-time PCR (see above) using Mycobacterium and/or Nocardia species-specific probes, in order to determine the presence, the identity, and/or the amount of a Mycobacterium species or a Nocardia species in a sample.
  • an assay system including a primer pair complementary to a Mycobacterium secA nucleic acid sequence also includes a labeled probe that hybridizes to the amplified Mycobacterium species-specific nucleic acid in order to detect a Mycobacterium species.
  • an assay system includes a labeled probe that hybridizes to an amplified Mycobacterium/Nocardia genera-specific nucleic acid or to an amplified Nocardia species-specific nucleic acid in order to detect a Nocardia species.
  • the primer pair includes Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium species-specific probe or a fluorescently-labeled Nocardia species-specific probe.
  • the primer pair includes Mycobacterium species-specific primers and the probe includes a fluorescently- labeled Mycobacterium species-specific probe.
  • the primer pair includes Nocardia species-specific primers and the probe includes a fluorescently-labeled Nocardia species-specific probe.
  • the amplified target nucleic acid can be detected using fluorescently-labeled species-specific probes in real time.
  • the fluorescently-labeled probes rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time.
  • FRET fluorescence resonance energy transfer
  • FRET that occurs between fluorogenic labels on different probes (for example, using FRETProbes) or between a fluorophore and a non-fluorescent quencher on the same probe (for example, using a molecular beacon or a TaqMan ® probe) can identify a probe that specifically hybridizes to the DNA sequence of interest and in this way, using Mycobacterium or Nocardia species-specific probes, can detect the presence, identity, and/or amount of a Mycobacterium species or a Nocardia species in a sample.
  • the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube.
  • the anay-based detection and identification methods described above can be used to detect and/or identify a Mycobacterium and/or a Nocardia species in a sample.
  • the Mycobacterium or Nocardia species-specific oligonucleotide probes attached to the array specifically hybridize to target nucleic acids.
  • the anay includes a plurality of Mycobacterium and Nocardia species-specific oligonucleotide probes.
  • the array includes a plurality of Nocardia ox Mycobacterium species-specific oligonucleotide probes.
  • a hybridization complex is formed when target nucleic acids, for example amplified Mycobacterium/Nocardia genera-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium species-specific oligonucleotide probes or Nocardia species-specific probes, attached, for example chemically linked, to a polymeric solid support.
  • the hybridization complex forms a hybridization pattern of the sample.
  • a hybridization pattern of the sample is compared to a control hybridization pattern in order to identify a Mycobacterium species or a Nocardia species in a sample.
  • Probe sequences of use with the array method are species-specific oligonucleotide probes that recognize a secAl nucleic acid only from a particular Mycobacterium species or a particular Nocardia species. Such sequences can be determined by examining the sequences of the different species, and choosing probes that specifically hybridize to a particular species, but not others (see species-specific probes listed above).
  • One of skill in the art will be able to identify other Mycobacterium and Nocardia oligonucleotide probes that can be attached to the surface of a solid support for the detection of other amplified Mycobacterium and Nocardia species-specific nucleic acid sequences.
  • the presence, identity and/or amount of a Mycobacterium species or a Nocardia species can be determined by other hybridization methods, as described above. Such methods would include Northern or Southern blot analysis, using labeled oligonucleotides specific to a Mycobacterium or a Nocardia species-specific nucleic acid sequence.
  • the identification of an amplified Mycobacterium/Nocardia genera-specific product, an amplified Mycobacterium species-specific product, or a Nocardia species-specific product, in order to identify the Mycobacterium or Nocardia species in a sample also can be determined by sequencing the amplified product. Techniques for sequencing nucleic acids are well known to those of skill in the art.
  • antibodies that specifically bind secAl polypeptides also can be used to detect a Mycobacterium species or a Nocardia species in a sample.
  • antibodies against secAl polypeptides can be linked to a support medium, such as a blot or a dish, and a protein sample applied.
  • antibodies that specifically bind a species specific secAl epitope can be used to probe blots containing a Mycobacterium protein sample or a Nocardia protein sample. Methods of using antibodies to detect proteins, such as Western blot analysis and ELISA, are well known in the art.
  • Methods of Treating a Subject Suspected of Being Infected with Mycobacterium or Nocardia Disclosed herein are methods of treating subjects suspected of being infected with Mycobacterium or Nocardia. Using the detection and identification methods described above a subject can be given the appropriate treatment once it is confirmed that the subject is infected with a Mycobacterium or a Nocardia and the nature of the treatment customized to the exact species of Mycobacterium or Nocardia infecting the subject. Methods of treatment for subjects infected with various species of Mycobacterium or Nocardia are well known to those of skill in the art.
  • Appropriate specimens for use with the current disclosure in the detection and identification of Mycobacterium genus and species, or Nocardia genus and species include any conventional clinical samples, for instance abscess samples, blood or blood- fractions (for example, serum), bone manow, bronchoalveolar lavage (BAL) samples, bronchial wash or bronchial brush samples, cerebrospinal fluid, esophageal samples, tissue drainage samples, eye conjunctiva samples, gastric biopsy or washing samples, nasopharyngeal swabs, saliva, sputum, induced sputum samples, stool samples, synovial fluid, skin lesions, sinus aspirates, tissue biopsies, transtracheal aspirates, surgical specimens, or autopsy material.
  • abscess samples for example, serum
  • blood or blood- fractions for example, serum
  • BAL bronchoalveolar lavage
  • bronchial wash or bronchial brush samples cerebrospinal fluid
  • the sample can be used directly, or concentrated (for example, by centrifugation or filtration) and a hybridization reaction or an amplification reaction performed.
  • the nucleic acid can be extracted using any conventional method prior to hybridization or amplification.
  • rapid DNA preparation can be performed using a commercially available kit (for example, the InstaGene Matrix, BioRad, Hercules, CA; the NucliSens isolation kit, Organon Teknika, Netherlands).
  • the DNA preparation technique chosen yields a nucleotide preparation that is accessible to, and amenable to, nucleic acid hybridization or amplification.
  • Kits Kits are provided which contain the necessary reagents for detecting bacteria of the Mycobacterium and Nocardia genera and/or particular Mycobacterium and Nocardia species. Instructions provided in the diagnostic kits can include calibration curves, diagrams, illustrations, or charts or the like to compare with the determined (for example, experimentally measured) values or other results.
  • the nucleotide sequences disclosed herein, and fragments thereof, can be supplied in the form of a kit for use in detection of bacteria of the Mycobacterium and Nocardia genera or specific Mycobacterium and Nocardia species.
  • an appropriate amount of one or more of the Mycobacterium/Nocardia genera specific, Mycobacterium species-specific, or Nocardia species- specific oligonucleotide primers or probes is provided in one or more containers.
  • the oligonucleotide primers and probes may be provided suspended in an aqueous solution or as a freeze- dried or lyophilized powder, for instance.
  • the container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers may be provided in pre- measured single use amounts in individual, typically disposable, tubes or equivalent containers.
  • the sample to be tested for the detection of bacteria of the Mycobacterium or Nocardia genera, or a specific Mycobacterium or Nocardia species can be added to the individual tubes described above and amplification, such as by real-time PCR, carried out directly.
  • the amount of each oligonucleotide primer or probe supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several in vitro amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction.
  • kits may also include the reagents necessary to carry out PCR in vitro amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (for example, polymerase buffer), salts (for example, magnesium chloride), and deoxyribonucleotides (dNTPs).
  • appropriate buffers for example, polymerase buffer
  • salts for example, magnesium chloride
  • dNTPs deoxyribonucleotides
  • Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of a target nucleic acid, such as an amplified nucleic acid.
  • the appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the amplification reaction.
  • a pair of primers and a probe may be provided in a single tube, or equivalent container, in a pre-measured single use. The amount of each probe provided would likely be in an amount sufficient to detect a target nucleic acid, such as an amplified nucleic acid.
  • the kit in another embodiment, includes a Mycobacterium, a Nocardia, or a Mycobacterium/Nocardia detection anay, a buffer solution, a conjugating solution for developing the signal of interest, and a detection reagent for detecting the signal of interest, each in separate packaging, such as a container.
  • the kit can also optionally include an effective amount of a pre- treatment solution for application to the probe biomolecules to produce an amplified signal. It may also be advantageous to provide in the kit one or more control sequences.
  • the design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art. The invention is illustrated by the following non-limiting Examples.
  • Example 1 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid in a Sample
  • This example demonstrates how a Mycobacterium/Nocardia genera-specific nucleic acid sequence encoding a fragment of the secAl protein can be amplified from a sample, thereby identifying the presence of Mycobacterium or Nocardia in the sample.
  • DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfuls of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at
  • the Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a region of a secAl gene that is conserved in both Mycobacterium and Nocardia. For example, in one experiment genera-specific primers were designed based on the fact that the target sequence for amplification is a region within the Mycobacterium tuberculosis secAl gene (located between base pair 150019 and base pair 152868 in the Mycobacterium tuberculosis genome; Genbank Accession No. BX842582).
  • primers were designed against a target sequence located between base pair 151730 and base pair 152428 of the Mycobacterium tuberculosis genome in order to generate an amplified sequence approximately 700 base pairs in length, excluding the primers.
  • the target sequence of the secAl gene conesponded to a sequence encoding a region between amino acid 148 and 380 (or amino acid positions 138 and 391, if including the primers) of the secAl protein, which included the substrate specificity domain (located between amino acid positions 222 and 359 of the secAl protein).
  • the amplified sequence was approximately 500 base pairs in length.
  • Mycobacterium/Nocardia genera-specific forward and reverse primers can be used to generate Mycobacterium/Nocardia genera-specific secAl nucleic acid sequences (amplicons).
  • Genus-specific secAl primers used to generate amplicons that will be sequenced, are tailed with M13 sequencing primers (M13 forward tail: 5'GTA AAA CGA CGG CCA G-3' (SEQ ID NO: 229); M13 reverse tail: 5'CAG GAA ACA GC TAT GAC-3' (SEQ ID NO: 230)).
  • Mtu.Forward primer (5'GTAAAACGACGGCCAGGACAGYGAGTGGATGGGYCGSGTGCACCG-3'; SEQ ID NO: 13; Y can be T or C, and S can be G or C) and the Mtu.Reverse primer (5'CAGGAAACAGCTATGACACCACGCCCAGCTTGTAGATCTCGTGCAGCTC-3'; SEQ ID NO: 14), which were synthesized by Midland Certified Reagent Company.
  • the PCR amplification was performed using 1 pmol of forward primer (see Table 1), 1 pmol of reverse primer (see Table 1), 1 Unit UNG, ultra pure water, and 5 ⁇ l of extracted DNA.
  • the PCR thermocycling program consisted of either (i) 12 two-step cycles of 1 minute at 95 °C and 2 minutes at 72 °C, then 29 cycles of 1 minute at 96 °C, 1 minute at 65 °C and 1 minute at 72 °C, or (ii) an initial step of 10 minutes incubation at 30°C (UNG), followed by 10 minutes at 95°C, 49 cycles of 1 minute at 95°C, 1 minute at 65°C and 1 minute at 72°C, and a final step of 10 minutes incubation at 72°C.
  • the amplicons were then purified using the Microcon-100 Microconcentrator columns and eluted in 100 ⁇ l TE. The products showing strong gel bands were diluted 1 : 10 in ultra pure water. Clinical samples were then screened to detect the presence of Mycobacterium.
  • Mycobacterium genus-specific primer sets used in these experiments included: (primer set 1) Mtu.For.l (SEQ ID NO: 8) and Mtu.Rev.2 (SEQ ID NO: 10), (primer set 2) Mtu.For.l (SEQ ID NO: 8) and Mtu.Rev.3 (SEQ ID NO: 11), and (primer set 3) Mtu.For.l.
  • Example 2 Sequencing of Mycobacterium/Nocardia secAl Amplicons Once the secAl amplicons were generated, they were sequenced in order to identify Mycobacterium and Nocardia species-specific differences in the secAl sequence.
  • One forward and one reverse sequencing reaction were prepared for each product in a 0.2 ⁇ l PCR tube. These reactions contained ABI Big Dye mix (Applied Biosystems Inc., Foster City, CA), IX sequencing buffer, 3.2 pmoles of forward or reverse M13 sequencing primers, ultra pure water, and 1 to 2 ⁇ l purified PCR product.
  • the thermocychng parameters for cycle sequencing consisted of 25 cycles of 10 seconds at 96 °C, 5 seconds at 50 °C, and 4 minutes at 60 °C.
  • Example 3 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid Sequence and Detection of a Mycobacterium in a Sample This example shows how Mycobacterium genus-specific probes can be used to detect a Mycobacterium secAl amplicon.
  • DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfuls of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at
  • the Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene (see Table 1). For example, in one experiment genera-specific primers were designed based on the fact that the target sequence for amplification is a region within the Mycobacterium tuberculosis secAl gene (located between base pair 150019 and base pair 152868 in the Mycobacterium tuberculosis genome; Genbank Accession No. BX842582). More specifically, primers were designed against a target sequence located between base pair 151730 and base pair 152428 of the Mycobacterium tuberculosis genome in order to generate an amplified sequence approximately 700 base pairs in length, excluding the primers.
  • the primers amplified a target sequence approximately 500 base pairs in length.
  • the PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 8 minutes at 95 °C, 50 cycles of 10 seconds at 95 °C, 20 seconds at 56 °C and 30 seconds at 76 °C.
  • the results in Tables 6 and 7 demonstrate that the Mycobacterium secA genus-specific MGB probe consistently detected the amplification product, regardless of the Mycobacterium species from which it was amplified.
  • the genus-specific MGB probes were capable of detecting the presence of various species of Mycobacterium.
  • Mycobacteuum/Nocardia genera-specific amplicons from samples containing different species of Mycobacterium, were generated usmg the Mycobacterium/Nocardia genera-specific pnmer pair Mtu.Forl.Edit (SEQ ID NO: 9; Table 1) and Mtu.Rev490 (SEQ ID NO: 24; Table 1) and then detected with a mix of FRETProbes (see Table 7, footnotes). Table 8. Mycobacterium and Nocardia FRETProbes.
  • the PCR thermocycling program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C.
  • the results in Table 7 demonstrate that the Mycobacterium secA FRETProbe mix consistently detected the amplification product, regardless of the Mycobacterium species from which it was amplified. Thus, the Mycobacterium FRETProbes were also capable of detecting tlie presence of various species of Mycobacterium.
  • Example 4 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid Sequence in a Sample and Identification of a Mycobacterium species This example demonstrates how a Mycobacterium/Nocardia genera-specific nucleic acid sequence encoding a fragment of the secAl protein can be amplified and then used to identify the Mycobacterium species present in the sample.
  • DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfiils of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at 10,500 x g for 5 minutes. The supernatant was removed and the pellet was washed in Tris-HCL, pH 8.0.
  • alkaline wash solution 0.5 M NaOH, 0.05 M sodium citrate
  • the Mycobacterium/Nocardia genera-specific secAl PCR primers (such as those listed in Table 1) were selected from a conserved region of the secAl gene to produce an amplified sequence approximately 700 base pairs in length, excluding the primers.
  • the real-time PCR amplification was performed in a 25 ⁇ l volume containing 2.5 mM MgCl 2 , IX LightCycler-Fast Start DNA Hybridization Probes (a pair of FRETProbes, such as those listed in Table 9, including one FITC-labeled "up” probe and one Red640-labeled “down” probe; Roche; Mannheim, Germany) specific for the Mycobacterium species of interest, 1 pmol of Mtu.For.l forward primer (SEQ ID NO: 8), 1 pmol of Mtu.rev.3 reverse primer (SEQ ID NO: 11), 1 Unit UNG, ultra pure water, and 5 ⁇ l of extracted DNA.
  • IX LightCycler-Fast Start DNA Hybridization Probes a pair of FRETProbes, such as those listed in Table 9, including one FITC-labeled "up” probe and one Red640-labeled "down” probe; Roche; Mannheim, Germany
  • Mycobacterium/Nocardia genera-specific forward and reverse primers for example pairs derived from the primers listed in Table 1, above, can be used to generate Mycobacterium secAl amplicons.
  • the PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C.
  • the numbers in each probe name represent the nucleotide position within the amplified target nucleic acid sequence that is complementary to the probe sequence.
  • a positive signal using the species-specific real-time PCR probes indicated the presence of a secAl nucleic acid sequence from the conesponding Mycobacterium species.
  • Example 5 Amplification of a Shorter Mycobacterium Nocardia Genera-Specific secAl Nucleic Acid and Identification of a Mycobacterium species in a Sample
  • This example demonstrates how a shorter nucleic acid sequence encoding the genus-specific fragment of the secAl protein from Mycobacterium can be amplified from a sample, in order to identify the presence of a Mycobacterium in the sample. Once a Mycobacterium is detected, the amplified nucleic acids can be sequenced to identify the specific Mycobacterium species present. DNA extraction, PCR amplification and sequencing were performed essentially as described in Examples 1 and 2, above.
  • the Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene to produce a sequence of approximately 513 base pairs (470 base pairs, excluding the primers).
  • the genus-specific secAl primers were tailed with M13 sequencing primers and include the Mtu.For.l.editM13 primer (5'- GTAAAACGACGGCCAGGACAGYGAGTGGATGGGYCGSGT 3'; SEQ ID NO: 15) and the Myco.Rev490-467M13 primer (5'CAGGAAACAGCTATGACGCGGACGATGTARTCCTTGTCSCG-3'; SEQ ID NO: 25; Y can be T or C, S can be G or C, and R can be G or A).
  • the PCR thermocycling program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C. The results are demonstrated in Table 10. If the baseline (real-time PCR) data were not conclusive as to which species of Mycobacterium was present in the sample, melting curves were generated, according to LightCycler instructions, in order to identify the species in the sample.
  • Example 6 Identification of Mycobacterium Species in a Sample Using Mycobacterium Species-Specific secA Minor Groove Binder (MGB) Probes This example demonstrates how species-specific secAl MGB probes can be used to identify a Mycobacterium species in a sample.
  • DNA was extracted from cultures of various species of Mycobacterium and a Mycobacterium genus-specific region of the secAl gene was amplified essentially as described in Example 3, above.
  • the Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene to produce an amplified sequence either 761 base pairs or 638 base pairs in length.
  • the real-time PCR amplification was perfo ⁇ ned in a 25 ⁇ l volume containing 2.5 mM MgCl 2 , IX MGB Eclipse Probes (Epoch Biosciences, Bothell, WA) specific for the Mycobacterium species of interest (Table 9), 1 pmol of forward primer (SEQ ID NO: 8 for the 761 base pair product or SEQ ID NO: 9 for the 638 base pair product; see Table 1), 1 pmol of reverse primer (SEQ ID NO: 11 for the 761 base pair product or SEQ ID NO: 12 for the 638 base pair product; see Table 1), 1 Unit UNG, ultra pure water, and 5 ⁇ l of extracted DNA.
  • the PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 8 minutes at 95 °C, 50 cycles of 10 seconds at 95 °C, 20 seconds at 56 °C and 30 seconds at 76 °C.
  • a positive signal during the real-time amplification procedure indicated the presence of the conesponding Mycobacterium species in the sample (Table 12).
  • Example 7 Detection and Identification of Mycobacterium and Nocardia by secAl Sequences This example demonstrates how nucleic acids encoding the secAl protein from bacteria of the genus Mycobacterium and Nocardia can be specifically amplified from a clinical sample and then sequenced in order to identify the particular Mycobacterium and Nocardia species present in the sample.
  • Mycobacterial reference strains and clinical isolates The 47 reference strains and 59 clinical isolates used in this Example are listed in Table 13. Conventional identification of clinical isolates included an initial assessment of pigment production, growth rate and colony characteristics. Slowly growing mycobacteria were identified using the AccuProbe assays (Gen-Probe, San Diego, CA), which are specific for M avium complex (MAC), M avium, M. intracellulare, M. tuberculosis (MTB) complex, M gordonae, and M kansasii. Additional biochemical testing of MTB complex isolates included niacin and nitrate tests. AccuProbe-negative slowly growing isolates were identified by 16S rDNA sequencing. Rapidly growing isolates were identified by hsp65 PRA (Telenti et al., J. Clin. Microbiol., 31:175-178, 1993), and 16S rDNA sequencing when necessary.
  • MAC M avium complex
  • M. intracellulare M. tubercul
  • Table 13 List of mycobacterial reference strains and clinical isolates Species Reference strains No. of clinical isolate: and source M abscessus ATCC 19977 (T) 6 M. africanum ATCC 25420 (T) ATCC 35711 M. asiaticum ATCC 25276 (T) 1 M. avium ATCC 25291 (T) 5 M. bovis ATCC 19210 (T) ATCC 35734 M. celatum ATCC 51131 (T) ATCC 51130 M. chelonae ATCC 35752 (T) 2 M.flavescens ATCC 14474 (T) ATCC 23008 ATCC 23033 M. fortuitum ATCC 6841 (T) 5 M.
  • Non-mycobacterial reference strains Seven American Type Culture Collection (ATCC) non-mycobacterial strains were also included in this study: Gordonia bronchialis [ATCC 25592 (T)], Gordonia terrae [ATCC 25594 (T)], Nocardia brasiliensis [ATCC 19296 (T)], Nocardia farcinica [ATCC 3318 (T)], Rhodococcus equi [ATCC 6939 (T)], Rhodococcus rhodochrous [ATCC 13808 (T) ⁇ , and Tsukamurella paurometabola [ATCC 8368 (T)].
  • ATCC 8368 (T) Seven American Type Culture Collection (ATCC) non-mycobacterial strains were also included in this study: Gordonia bronchialis [ATCC 25592 (T)], Gordonia terrae [ATCC 25594 (T)], Nocardia brasiliensis [ATCC 19296 (T)], Nocardia farcinica [ATCC 3318
  • Primer design and PCR Two conserved regions were identified after aligning secAl sequences from five Mycobacterium species, which were available in the NCBI GenBank database at the time this study was initiated. These regions were used to design two primers (Mtu.For.l; SEQ ID NO: 8 and Mtu.rev.3; SEQ ID NO: 11), which correspond to tuberculosis secAl gene positions 412-440, and 1141-1172, respectively [GenBank accession no. BX842582.1, nucleotides 150019-152868].
  • a schematic representation of the primer design is shown in FIG. 3.
  • Mtu.For.l (5'GAC AGY GAG TGG ATG GGY CGS GTG CAC CG-3 '
  • Mtu.Reverse3 (5 'ACC ACG CCC AGC TTG TAG
  • ATC TCG TGC AGC TC-3' were commercially synthesized (Midland Certified Reagent Company, Midland, TX). Primers used for sequencing of the secAl gene regions were tailed with M13 sequencing primer sites: M13 forward tail (5'GTA AAA CGA CGG CCA G-3'), M13 reverse tail (5'CAG GAA ACA GC TAT GAC-3').
  • M13 forward tail (5'GTA AAA CGA CGG CCA G-3')
  • M13 reverse tail 5'CAG GAA ACA GC TAT GAC-3'.
  • the PCR amplification using Mtu.For.l (M13 tailed) and Mtu.Rev.3 (M13 tailed) generated a product of 700 base pahs (excluding the primers, FIG. 3).
  • PCR reactions were performed in a Perkin-Elmer 9600 Thermocycler with a reaction mix containing 2.5 mM MgC12, IX LightCycler-Fast Start DNA Hybridization Probes (Roche, Mannheim, Germany), 1 pmol of forward primer, 1 pmol of reverse primer, 1 Unit of Uracil TV-glycosylase (UNG) enzyme (Roche), 5 ⁇ l of extracted DNA and ultra pure water to a final volume of 25 ⁇ l.
  • UNG Uracil TV-glycosylase
  • the PCR thermocycling program consisted of an initial step of 10 minutes incubation at 30°C (UNG), followed by 10 minutes at 95°C, 49 cycles of 1 minute at 95°C, 1 minute at 65°C and 1 minute at 72°C, and a final step of 10 minutes incubation at 72°C.
  • UNG UNG
  • a negative control of ultra pure water was included with every amplification reaction.
  • DNA samples of selected strains were also amplified and sequenced for the 16S rDNA gene using the MicroSeq Full Gene 16S rRNA Bacterial Isolation Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.
  • PCR product detection and purification PCR products were visualized by UV illumination of an ethidium bromide-stained 2% agarose gel following electrophoresis.
  • the purification of the remaining PCR product was achieved with Microcon-100 microconcentrators (Millipore, Bedford, MA), following the manufacturer's instructions.
  • the ABI Prism BigDye Terminator v 1.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used for the sequencing of the PCR product.
  • the sequencing reaction contained 4 ⁇ l of Big Dye premix, 0.5X buffer, 3.2 pmol of 10 sequencing primer, and approximately 150 ng of PCR product template in a total volume of 20 ⁇ l.
  • the following M13 primers were used for sequencing: M13 Forward, 5'GTA AAA CGA CGG CCA G-3 ' (SEQ ID NO: 229) and M13 Reverse, 5'CAG GAA ACA GCT ATG AC-3 (SEQ ID NO: 230).
  • the sequencing reaction and template preparation were performed in accordance with the instructions of the manufacturer. Sequencing products were purified with CleanSEQ Sequencing Reaction Clean- Up system (Agencourt, Beverly, MA) and analyzed using the 3100 Genetic Analyzer (Applied Biosystems), following the manufacturer's instructions.
  • the resulting trees were depicted using the TreeView version 1.4 package (Page, Comput. App. Biosci. 12:357-358, 1996).
  • the stability of the grouping was assessed by the bootstrap method using SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs. A total of 1,000 bootstrapped trees were generated.
  • Assembled 16S rDNA sequences from selected isolates were compared with 16S rDNA sequences available in GenBank databases using the standard nucleotide-nucleotide Basic Local Alignment Search Tool (Blast) program (National Center for biotechnology Information, Bethesda, MD).
  • Blast Basic Local Alignment Search Tool
  • the isolate was identified as most closely related to the reference species if its 16S rDNA sequence demonstrated the highest relatedness and had over 98.5% identical bases compared to the respective reference sequence, based on the data from a previous study (Stackebrandt and Goebe, Int. J. Syst. Bacteriol, 44:846-849, 1994).
  • Nucleotide sequence accession numbers Partial sequences of mycobacterial and nocardial secAl were deposited in GenBank under accession numbers AY724701 to AY724734, and AY781799 to AY781800, respectively. 16S rDNA sequences were deposited in GenBank under accession numbers AY734991 to AY734996.
  • Amino acid sequences of the mycobacterial type strains The deduced amino acid sequences of the amplified 700 base pair fragment of secAl comprised 233 amino acid residues (F148 to A375, M tuberculosis numbering, protein Accession no. CAE55574.1). Each type strain examined displayed unique amino acid sequences in this region, except for the members of the MTB complex, which shared the same protein sequence. Interspecies similarity at the amino acid level ranged from 87.6% to 100% (100% for the members of the MTB complex). High variability was observed in the first half of the 233 deduced amino acid sequence, with three particularly hypervariable regions between residues 5-23, 46-57, and 84-118.
  • M gastri and kansasii 99.1% similarity
  • M abscessus andM chelonae 96.6% similarity
  • M marinum andM ulcerans 98.3% similarity
  • Phylogenetic tree A phylogenetic tree of 34 sequenced mycobacterial reference strains, as well as M leprae, and two sequenced nocardial reference strains, was constructed by the neighbor-joining method, using Corynebacterium efficiens as the outgroup (FIG. 5). All mycobacterial species studied showed good separation. Within the consensus tree, four major clusters of mycobacteria could be defined. Most of the slow growing species grouped together in the first cluster. The rapid growers M flavescens, M. fortuitum, M mucogenicum, M. peregrinum, andM smegmatis grouped together in the second cluster. Interestingly, M. triviale also appeared in that group.
  • a third cluster included the closely related species M terrae and M nonchromogenicum.
  • a long separate branch included M abscessus and M chelonae.
  • the members of the M tuberculosis complex were distantly separated from all the other species.
  • Slowly growing pathogenic M kansasii and nonpathogenic M gastri could be easily distinguished, although they cannot be separated by 16S rDNA.
  • Two other pairs of species closely related by 16S rDNA sequences, M abscessus - M. chelonae, andM marinum - M. ulcerans, were separated as well.
  • the two Nocardia species could be easily differentiated from one another.
  • the 700 base pair fragment of secAl was sequenced from one or more reference strains or well characterized clinical isolates (Table 13). Five or six clinical isolates were obtained and sequenced from each of 9 commonly isolated species of mycobacteria (Table 13 and separately in Table 14). Table 14. Range of similarity to the type strain of 50 clinical isolates from nine commonly isolated Mycobacterium species
  • the intraspecies percentage of similarity at the DNA level was very high (usually >99%, range 96.0%-100%). Comparison of the deduced amino acid sequences among isolates of the same species showed between 99.6 to 100% similarity for all species tested. M gordonae showed the highest degree of intraspecies variability, with percentages of similarity at the DNA level ranging from 96.0 to 99.0, however, all six clinical isolates of M gordonae tested matched most closely with the type strain. Four clinical isolates of M kansasii showed 100% similarity to the type strain, while two other isolates had identical sequences showing 97.3% similarity to the type strain. However, all six clinical isolates of M kansasii were conectly identified.
  • Example 8 Identification of Nocardia species Using Sequence Analysis of a Portion of the secA Gene
  • This example demonstrates how nucleic acids encoding the secAl protein from bacteria of the genus Nocardia can be specifically amplified from clinical samples and then sequenced in order to identify Nocardia isolates to the species level.
  • SecAl is a key component of the major pathway of protein secretion across the cytoplasmic membrane.
  • a 520-base pair region of the secAl gene of various type or reference strains of pathogenic Nocardia species was amplified using Mycobacterium/Nocardia genera-specific primers (Mtu.For.l.editM13 primer and MycoRev490-467M13 primer; see Table 1).
  • a 434-base pair fragment of the secAl gene was sequenced from 15 clinical isolates of Nocardia species and showed a range of 99.1 to 100% similarity to the secAl sequences of the type or reference strain to which their identification corresponded. Comparison of the deduced 145 amino acid sequences of the SecAl proteins showed between 99.3 and 100% similarity to the conesponding type or reference strain. Sequencing the secAl gene, and using deduced amino acid sequences of the secAl protein, provides a more discriminative method for identification of Nocardia isolates than 16S rDNA sequencing.
  • Example 9 Identification oi Mycobacterium Species in a Sample Using Mycobacterium Species-Specific secAl Primers This example demonstrates how nucleic acids encoding the secAl protein of a Mycobacterium species can be specifically amplified and identified.
  • DNA is extracted from cultures of various species of Mycobacterium and is amplified essentially as described in Example 1, above, with the exception that Mycobacterium species-specific secAl PCR primers are used to amplify the target nucleic acid sequence.
  • the Mycobacterium species-specific secAl PCR primers are selected from unique regions (species-specific) of the Mycobacterium secAl gene.
  • the species-specific secAl primers are all reverse primers (Table 16) and were designed to be used with the Mtu.For.ledit forward primer (SEQ ID NO: 9; Table 1).
  • the numbers in each probe name represent the nucleotide position within the amplified target nucleic acid sequence that is complementary to the probe sequence.
  • Real-time PCR amplification is performed as described in Example 3.
  • a positive signal due to the hybridization of a species-specific probe indicates the presence of the conesponding Mycobacterium species in the sample.
  • the secAl amplicons are sequenced in order to confirm the origin of the Mycobacterium secAl sequence. Sequencing reactions are performed essentially as described in Example 2.
  • Example 10 Isolation oi Mycobacterium and Nocardia Genera from a Clinical Sample and the Subsequent Identification of Particular Mycobacterium and Nocardia Species in the Sample
  • This example demonstrates how nucleic acids encoding the secAl protein from bacteria of Mycobacterium and Nocardia genera can be specifically amplified from a clinical sample and then sequenced in order to identify the particular Mycobacterium or Nocardia species present in the sample.
  • DNA is extracted from clinical samples, a Mycobacterium/Nocardia genera-specific secAl nucleic acid sequence is amplified, and Mycobacterium and Nocardia species-specific probes are used to identify the origin of the secAl nucleic acid (see the protocol described in Examples 3 or 4).
  • a positive signal due to the hybridization of a species-specific probe indicates the presence of the conesponding Mycobacterium or Nocardia species in the sample.
  • Mycobacterium or Nocardia species-specific secA amplicons are detected in the sample, the amplicons are sequenced to confirm the origin of the amplified secA nucleic acid generated from the sample. Sequencing reactions are performed as described in Example 2.
  • This disclosure provides methods of detecting the presence of Mycobacterium and Nocardia in a sample using secA nucleic acid sequences that are conserved in Mycobacterium and Nocardia.
  • the disclosure also provides methods of identifying a Mycobacterium or a Nocardia species involved in infection of a subject.
  • the disclosure further provides Mycobacterium and Nocardia species-specific secAl nucleic acid sequences.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Methods of detecting the presence of Mycobacterium or Nocardia in a sample using Mycobacterium and Nocardia secA nucleic acid sequences are disclosed. Methods of identifying a Mycobacterium or a Nocardia species involved in infection of a subject are also disclosed. The disclosure further provides Mycobacterium and Nocardia species-specific secA1 nucleic acid sequences.

Description

DETECTIONAND IDENTIFICATION OF MYCOBACTERIUM AND NOCARDIA USING SECA
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 60/548,371, filed February 27, 2004, which is incorporated herein by reference.
FIELD The present disclosure is related to methods of detecting the presence of Mycobacterium and
Nocardia in a sample using secA nucleic acid sequences. Specifically, a method is disclosed to identify the Mycobacterium and Nocardia species involved in the infection of a subject.
BACKGROUND The Mycobacterium genus comprises more than 70 different species, of which about 30 have been associated with human disease. One prominent pathogen of the genus is Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis. Despite the availability of the Bacille Calmette-Guerin (BCG) vaccine and effective short-course chemotherapy, M. tuberculosis remains a major global public health problem with an estimated eight million new cases and 2.9 million deaths per year. Mycobacteria other than M. tuberculosis, also called nontuberculous mycobacteria (NTM), are ubiquitous in the environment and have the potential to colonize and cause serious infection. Serious disease caused by NTM is often associated with various forms of immunosuppression, particularly HIV infection. Members of the genus Nocardia have the potential to cause serious disease, however only three species of Nocardia are known to cause nearly all human infections: Nocardia asteroids (N. asteroids), N brasiliensis, and Ncaviae. Nocardia is found in soil around the world and can be contracted by inhaling contaminated dust or via contamination of a wound with soil containing Nocardia. While individuals with normal immune systems can acquire this infection, immunocompro ised individuals are at greatest risk of developing a serious disease related to Nocardia infection. Thus, the main risk factors for nocardiosis are a weakened immune system or chronic lung disease. In addition, people on chronic steroid therapy, those with cancer, organ or bone marrow transplants, or HIV/AIDS are also at higher risk for developing a serious disease associated with Nocardia infection. It is believed that bacterial protein export is an integral part of bacterial pathogenesis. secA is a highly conserved, soluble protein that is essential in the bacterial protein export pathway. secA is a cytoplasmic, multifunctional, 100 kDa homodimer that interacts with acidic phospholipids in the bacterial cell membrane, the SecYEG pore (a membrane channel formed by SecE (9 kDa), SecG (11 kDa), and SecY (47 kDa)) and molecular chaperones, such as SecB (17 kDa), that deliver precursor proteins to the bacterial cell membrane translocase. secA is an ATPase that provides energy for protein translocation and, through cycles of ATP binding and hydrolysis, secA transports precursor proteins to the translocase and undergoes cycles of membrane insertion and deinsertion that lead to stepwise export of tlie protein. Although the Sec-dependent protein export pathway is well characterized in various bacteria, such as Escherichia coli, little is known about the protein export pathway in Mycobacterium or Nocardia. The identification of 'a Mycobacterium or Nocardia species involved in infection is essential to determining a treatment protocol. Conventional detection and isolation of Mycobacterium and Nocardia species includes microscopic detection (smear) with acid-fast (for Mycobacterium) or modified acid-fast (for Mycobacterium and Nocardia) stain procedures, or growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. Unfortunately, the whole process may take days to one or more months. Smear microscopy is still widely used as a first method to screen for the presence of Mycobacterium or Nocardia in clinical samples. However, the reliability of smear microscopy is highly dependent on the experience of the laboratory technician and on the number of acid-fast bacilli (AFB) and/or weakly acid fast bacilli present in the specimen. The overall sensitivity of the smear has been reported to range from 22 to 80%. Thus, there remains a need to replace smear microscopy and conventional identification techniques in the bacteriology laboratory. Recently, molecular methods have been utilized in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium species. However, only a limited number of Mycobacterium species (M. tuberculosis complex, M. gordonae, M. kansasii, and M. avium-intracellulare complex) can be distinguished from other members of the genus. Molecular methodologies, especially 16S rDNA sequence analysis, have also allowed the recognition of many new Nocardia species. However, the use of 16S rDNA sequences for identifying isolates has been problematic, since closely related species show high levels of 16S rDNA similarity. At present there are no commercially available assay systems capable of detecting and identifying all members of the Mycobacterium genus and the Nocardia genus, while excluding cross-reactive signals from other microorganisms commonly present in clinical samples. In addition there are no available assay systems capable of detecting and identifying all common disease- associated Mycobacterium and Nocardia species. Thus, there remains a need for the development of a rapid method for the accurate detection of the presence of Mycobacterium and Nocardia directly in a clinical sample, as well as the identification of common Mycobacterium and Nocardia species in clinical specimens. SUMMARY The present disclosure relates to methods of detecting a Mycobacterium or Nocardia in a sample. In one embodiment, the method involves amplifying a Mycobacterium/Nocardia genera- specific secAl nucleic acid with at least two Mycobacterium/Nocardia genera-specific primers. The Mycobacterium/Nocardia genera-specific primers bind within a region of the nucleic acid sequence encoding a secAl protein that is conserved among members of the Mycobacterium and Nocardia genera, wherein the conserved region is in the 5' half of the secAl gene and includes a substrate specificity domain. A particular example of the method of detecting a. Mycobacterium or Nocardia includes hybridizing the amplified genera-specific secAl nucleic acid to a detectable secAl probe oligonucleotide. In other particular examples of the method, distinguishing a Mycobacterium from a Nocardia includes hybridizing the amplified nucleic acid with a detectable Mycobacterium or a Nocardia species-specific secAl probe oligonucleotide or sequencing the amplified nucleic acid. In another embodiment, a Mycobacterium species is identified by amplifying a Mycobacterium species-specific secAl nucleic acid from a sample utilizing at least two Mycobacterium species-specific primers. The Mycobacterium species-specific primers bind within a region of the Mycobacterium species-specific secAl nucleic acid sequence containing at least one nucleic acid difference, as compared to a secAl nucleic acid sequence present in another species of Mycobacterium. The nucleic acid differences allow the species-specific primers to differentially amplify target sequences of different Mycobacterium species. The region containing at least one nucleotide difference may be in the 5' half of the Mycobacterium secAl gene and includes the substrate specificity domain. In particular examples of the method Mycobacterium species-specific detectable secAl probes are used to detect the amplified Mycobacterium species-specific secAl nucleic acids. Methods for identifying a species of Mycobacterium or Nocardia in a sample include hybridizing nucleic acids in the sample to a plurality of Mycobacterium or Nocardia species-specific probe oligonucleotides attached to a polymeric solid support surface in an array. These probes are detectable by their location. Mycobacterium and Nocardia species-specific secAl polypeptide fragments are disclosed herein. Also disclosed are polynucleotides encoding the Mycobacterium and Nocardia species- specific secAl polypeptide fragments, as well as Mycobacterium and Nocardia species-specific secAl oligonucleotide probes and primers. The disclosure also provides methods of treating a subject suspected of being infected with a Mycobacterium or a Nocardia, as well as devices, including arrays, and kits for detecting a Mycobacterium or a Nocardia species in a sample.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the alignment of an amplified Mycobacterium genus-specific secAl nucleic acid sequence from various Mycobacterium species, including M. abscessus (ATCC.19977(T), SEQ ID NO: 130), M. abscessus (50200759, SEQ ID NO: 131), M. africanum (ATCC.25420(T), SEQ ID NO: 132), M. africanum (ATCC.35711, SEQ ID NO:133), M. asiaticum (ATCC.25276(T), SEQ ID NO:134), M. asiaticum (15031086, SEQ ID NO:135), M. avium (ATCC.25291(T), SEQ ID NO:136), M. avium (52110667.ATCC.35717, SEQ ID NO: 137), M. bovis (ATCC.19210(T), SEQ ID NO:138), M. bovis (52110665.ATCC.35734, SEQ ID NO:139), M. chelonae (ATCC.35752(T), SEQ ID NO: 140), M. chelonae (52281700, SEQ ID NO: 141), M.flavescens (ATCC.14474(T), SEQ ID NO: 142), M. flavescens (ATCC.23008, SEQ ID NO: 143), M. flavescens (ATCC.23033, SEQ ID NO: 144), M. flavescens (ATCC.23046, SEQ ID NO: 145), M. fortuitum (ATCC.6841 (T), SEQ ID NO:146), M. fortuitum (52110571, SEQ ID NO:147), M. gastri (ATCC.15754(T), SEQ ID NO:148), M. gastri (ATCC.25157, SEQ ID NO:149), M. gordonae (ATCC.14470(T), SEQ ID NO:150), M. gordonae (49290605, SEQ ID NO: 151), M. haemophilum (ATCC.29548(T), SEQ ID NO: 152), M. haemophilum (ATCC.33206, SEQ ID NO: 153), M. intracellulare (ATCC.13950(T), SEQ ID NO:154), M. intracellulare (R009, SEQ ID NO:155), M. kansasii (ATCC.12478(T), SEQ ID NO: 156), M. kansasii (39211279, SEQ ID NO:157), M. leprae (AL583919.1, SEQ ID NO: 221), M. malmoense (ATCC.29571(T), SEQ ID NO:158), M. malmoense (18280971, SEQ ID NO: 159), M. marinum (ATCC.927(T), SEQ ID NO: 160), M. marinum (52110619, SEQ ID NO:161), M. mucogenicum (ATCC.49650(T), SEQ ID NO: 162), M. mucogenicum (49200275, SEQ ID NO: 163), M. peregrinum (ATCC.14467(T), SEQ ID NO: 164), M. peregrinum (25110648, SEQ ID NO: 165), M. scrofulaceum (ATCC.19981(T), SEQ ID NO: 166), M. scrofulaceum (52241579, SEQ ID NO: 167), M. simiae (ATCC.25275(T), SEQ ID NO: 168), M. simiae (MB8146, SEQ ID NO: 169), M. smegmatis (ATCC.19420(T), SEQ ID NO:170), M. smegmatis (52110637.ATCC.14468, SEQ ID NO:171), M. szulgai (ATCC.35799(T), SEQ ID NO:172), M. szulgai (52110639, SEQ ID NO:173), M. terrae (ATCC.15755(T), SEQ ID NO: 174), M. terrae (MB2345, SEQ ID NO: 175), M. triviale (ATCC.23292(T), SEQ ID NO:176), M. triviale (ATCC.23290, SEQ ID NO: 177), M. triviale (ATCC.23291, SEQ ID NO:178), M. tuberculosis (ATCC.27294(T), SEQ ID NO:179), M. tuberculosis (R011, SEQ ID NO: 180), M tuberculosis (52110663.H37R.ATCC.25177, SEQ ID NO: 181), M. ulcerans (ATCC.19423(T), SEQ ID NO: 182), M. ulcerans (ATCC.25900, SEQ ID NO: 183), M. ulcerans (ATCC.25897, SEQ ID NO: 184), M. ulcerans (ATCC.35839, SEQ ID NO:185), X-Cluster (52110672, SEQ ID NO: 186), X-Cluster (53271416, SEQ ID NO:187), M. xenopi (ATCC.19250(T), SEQ ID NO: 188), M. xenopi (42261237, SEQ ID NO:189). FIG. 2 shows the alignment of the amino acid translation of the amplified Mycobacterium genus-specific secAl nucleic acid sequence from various Mycobacterium species, including M. abscessus (ATCC.19977(T), SEQ ID NO:69), M. abscessus (50200759, SEQ ID NO:70), M. africanum (ATCC.25420(T), SEQ ID NO:71), M. africanum (ATCC.35711, SEQ ID NO:72), M. asiaticum (ATCC.25276(T), SEQ ID NO:73), M. asiaticum (15031086, SEQ ID NO:74), M. avium (ATCC.25291(T), SEQ ID NO:75), M. avium (52110667.ATCC.35717, SEQ ID NO:76), M. bovis (ATCC.19210(T), SEQ ID NO:77), M. bovis (52110665.ATCC.35734, SEQ ID NO:78), M. chelonae (ATCC.35752(T), SEQ ID NO:79), M. chelonae (52281700, SEQ ID NO:80), M.flavescens (ATCC.14474(T), SEQ ID NO:81), M.flavescens (ATCC.23008, SEQ ID NO:82), M.flavescens (ATCC.23033, SEQ ID NO:83), M.flavescens (ATCC.23046, SEQ ID NO:84), M. fortuitum (ATCC.6841(T), SEQ ID NO:85), M. fortuitum (52110571, SEQ ID NO:86), M. gastri (ATCC.15754(T), SEQ ID NO:87), M. gastri (ATCC.25157, SEQ ID NO:88), M. gordonae (ATCC.14470(T), SEQ ID NO:89), M. gordonae (49290605, SEQ ID NO:90), M. haemophilum (ATCC.29548(T), SEQ ID N0:91), M. haemophilum (ATCC.33206, SEQ ID NO:92), M. intracellulare (ATCC.13950(T), SEQ ID N0:93), M. intracellulare (R009, SEQ ID N0:94), M. kansasii (ATCC.12478(T), SEQ ID N0:95), M. kansasii (39211279, SEQ ID N0:96), M. leprae (AL583919.1, SEQ ID NO: 220), M. malmoense (ATCC.29571(T), SEQ ID NO:97), M. malmoense (18280971, SEQ ID NO:98), M. marinum (ATCC.927(T), SEQ ID N0:99), M. marinum (52110619, SEQ ID NO: 100), M. mucogenicum (ATCC.49650(T), SEQ ID NO:101), M. mucogenicum (49200275, SEQ ID NO: 102), M. peregrinum (ATCC.14467(T), SEQ ID NO: 103), M. peregrinum (25110648, SEQ ID NO: 104), M. scrofulaceum (ATCC.19981 (T), SEQ ID NO: 105), M. scrofulaceum (52241579, SEQ ID NO: 106), M. simiae (ATCC.25275(T), SEQ ID NO: 107), M. simiae (MB8146, SEQ ID NO: 108), M. smegmatis (ATCC.19420(T), SEQ ID NO: 109), M. smegmatis (52110637.ATCC.14468, SEQ ID NO: 110), M. szulgai (ATCC.35799(T), SEQ ID NO:l l l), szulgai (52110639, SEQ ID NO: 112), M. terrae (ATCC.15755(T), SEQ ID NO:113), M. terrae (MB2345, SEQ ID NO: 114), M. triviale (ATCC.23292(T), SEQ ID NO: 115), M. triviale (ATCC.23290, SEQ ID NO: 116), M. triviale (ATCC.23291, SEQ ID NO: 117), M. tuberculosis (ATCC.27294(T), SEQ ID NO: 118), M. tuberculosis (R011 , SEQ ID NO: 119), M. tuberculosis (52110663.H37'R.ATCC.25177, SEQ ID NO: 120), M. ulcerans (ATCC.19423(T), SEQ ID NO: 121), M. ulcerans (ATCC.25900, SEQ ID NO: 122), M. ulcerans (ATCC.25897, SEQ ID NO: 123), M. ulcerans (ATCC.35839, SEQ ID NO: 124), X-Cluster (52110672, SEQ ID NO: 125), X-Cluster
(53271416, SEQ ID NO: 126), M. xenopi (ATCC.19250(T), SEQ ID NO: 127), M. xenopi (42261237, SEQ ID NO: 128). FIGS. 3A and 3B are a series of schematic drawings demonstrating the 649 amino acidN- terminal region of the secAl protein and primer design for the amplification of the secAl gene. FIG. 3 A shows that the secAl protein from tuberculosis contains two nucleotide-binding domains
(NBD1, between amino acid positions 1 and 426; NBD2, between amino acid positions 426 and 641) and a substrate specificity domain (SSD). SSD is embedded in NBD1 between amino acid positions 222 and 359. The thin black bar shows the region of the protein coded by the secAl gene region targeted in the assay. Numbers indicate amino acid residues. FIG. 3B shows that primers Mtu.For.l (FI) and Mtu.Rev.3 (R3) were used to generate a 700 base pair (base pair) fragment from the secAl gene. Numbers indicate nucleotide position in the amplified fragment. FIG. 4 shows the interspecies similarity of partial secAl gene sequences. FIG. 5 is a schematic drawing of a phylogenetic tree derived from secAl sequences from 34 mycobacterial reference strains and M. leprae (GenBank accession no. AL583919.1) and two nocardial reference strains. The tree was constructed by the neighbor-joining method, using
Coiynebacterium efficiens as the outgroup (GenBank accession no. AP005216.1). T represents type strain; three to five digit number represents an ATCC number. Numbers on the branches represent the percentage of 1000 bootstrap samples supporting the branch; only values >50% are shown. SEQUENCE LISTING The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOs: 1-6 are Mycobacterium species-specific secAl minor groove binder probes. SEQ ID NOs: 7 and 21 are Mycobacterium genus-specific minor groove binder probes. SEQ ID NOs: 8-20 and 22-25 are Mycobacterium/Nocardia genera-specific primers. SEQ ID NOs: 27, 207, and 223-227 are Mycobacterium FRETprobes. SEQ ID NOs: 26, 28-35, and 191-206 are Mycobacterium species-specific FRETprobes. SEQ ID NOs:.36-68, 208-219, and 222 are Mycobacterium species-specific primers. SEQ ID NOs: 130-189 and 221 are Mycobacterium secAl nucleic acid sequences. SEQ ID NO: 129 is the Mycobacterium majority nucleic acid sequence. SEQ IS NOs: 69-128 and 220 are Mycobacterium secAl amino acid sequences. SEQ ID NO: 190 is the Mycobacterium majority amino acid sequence. SEQ ID NO: 228 is a Nocardia FRETprobe. SEQ ID NO: 229 is an M13 forward primer. SEQ ID NO: 230 is an M13 reverse primer. SEQ ID NO: 231-252 are Nocardia secAl nucleic acid sequences.
DETAILED DESCRIPTION
I. Abbreviations AFB acid-fast bacilli BAL bronchoalveolar lavage bp base pair DABCYL 4-(4'-dimethylaminophenylazo)benzoic acid FAM 5-carboxyfluorescein FITC • -. fluorescein isothiocyanate FRET fluorescence resonance energy transfer JOE 2'7'-dimethoxy-4'5 '-dichloro-6-carboxyfluorescein MAC Mycobacterium avium complex MGB minor groove binder NTM nontuberculous mycobacteria PCR polymerase chain reaction ROX 6-carboxy-X-rhodamine RT-PCR reverse-transcription polymerase chain reaction SSD substrate specificity domain TAMRA N,N,N',N'-tetramethyl-6-carboxyrhodamine TET 6-carboxy-2',4,7,7'-tetrachlorofluorescein Tm melting temperature
II. Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182- 9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:
Amplification: Use of a technique that increases the number of copies of a nucleic acid molecule (for example, a DNA or RNA molecule) in a specimen. An example of amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The amplified target nucleic acid, or amplicon, may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. A specific type of PCR is real-time PCR, where the detection of the amplified target nucleic acid is monitored in real-time. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311 ; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134. Animal: Living multicellular vertebrate organisms, a category that includes, for example, mammals and birds. Antibody: Immunoglobulin (Ig) molecules and immunologically active portions of Ig molecules, for instance, molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen. In one embodiment the antigen is a Mycobacterium secA polypeptide, such as a Mycobacterium species-specific secAl polypeptide. In another embodiment the antigen is a Nocardia secA polypeptide, such as a Nocardia species-specific secAl polypeptide. Monoclonal, and humanized immunoglobulins are encompassed by the disclosure. The disclosure also includes synthetic and genetically engineered variants of these immunoglobulins. A naturally occurring antibody (for example, IgG) includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term "antibody." Examples of binding fragments encompassed within the term antibody include (i) an Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al, Nature 341:544-546, 1989) which consists of a VH domain; and (v) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. Furthermore, although the two domains of the Fv fragment are coded for by separate genes, a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al, Science 242-ATi~426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988) by recombinant methods. Such single chain antibodies, as well as dsFv, a disulfide stabilized Fv (Bera et al, J. Mol. Biol. 281:475-483, 1998), and dimeric Fvs (diabodies), that are generated by pairing different polypeptide chains (Holliger et al, Proc. Natl. Acad. Sci. 90:6444-6448, 1993), are also included. In one embodiment, antibody fragments for use in this disclosure are those which are capable of cross-linking their target antigen, for example, bivalent fragments such as F(ab')2 fragments. Alternatively, an antibody fragment which does not itself cross-link its target antigen (for example, a Fab fragment) can be used in conjunction with a secondary antibody which serves to cross-link the antibody fragment, thereby cross-linking the target antigen. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described for whole antibodies. An antibody is further intended to include humanized monoclonal molecules that specifically bind the target antigen. "Specifically binds" refers to the ability of individual antibodies to specifically immunoreact with an antigen. This binding is a non-random binding reaction between an antibody molecule and the antigen. In one embodiment, the antigen is secA. Binding specificity is typically determined from the reference point of the ability of the antibody to differentially bind the antigen of interest and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes. An antibody that specifically binds to a particular epitope is referred to as a "specific antibody". A variety of methods for linking effector molecules to antibodies are well known in the art. Detectable labels useful for such purposes are also well known in the art, and include radioactive isotopes such as 32P, fluorophores, chemiluminescent agents, and enzymes. Also encompassed in the disclosure are the chemical or biochemical modifications that incorporate toxins in the antibody. In one embodiment, the toxin is chemically conjugated to the antibody. In another embodiment, a fusion protein is genetically engineered to include the antibody and the toxin. Specific, non-limiting examples of toxins are radioactive isotopes, chemotherapeutic agents, bacterial toxins, viral toxins, or venom proteins. Antigen: Any molecule that can bind specifically with an antibody. An antigen is also a substance that antagonizes or stimulates the immune system to produce antibodies. Antigens are often foreign substances such as allergens, bacteria or viruses that invade the body. In one embodiment, an antigen is a secA polypeptide, such as a secAl polypeptide. Antisense and Sense: Double-stranded DNA (dsDNA) has two strands, a 5' -> 3' strand, referred to as the plus strand, and a 3' -> 5' strand, referred to as the minus strand. Because RNA polymerase adds nucleic acids in a 5' -> 3' direction, the minus strand of the DNA serves as the template for the RNA during transcription. Thus, the RNA formed will have a sequence complementary to the minus strand, and identical to the plus strand (except that the base uracil is substituted for thymine). Antisense molecules are molecules that are specifically hybridizable or specifically complementary to either RNA or the plus strand of DNA. Sense molecules are molecules that are specifically hybridizable or specifically complementary to the minus strand of DNA. Binding or stable binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized to its target nucleic acid, to permit detection of that binding. Binding can be detected by either physical or functional properties of the targetioligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including functional or physical binding assays. Binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, Southern blotting, dot blotting and light or fluorescence absorption detection procedures. For example, a method which is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at a wavelength of 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog binds its target sequence, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and the target sequence dissociate or melt. In another example, Fluorescence Resonance Energy Transfer (FRET) is used to determine if an oligonucleotide binds its target nucleic acid sequence. The binding between an oligomer and its target nucleic acid is frequently characterized by the melting temperature (Tm) at which 50% of the oligomer is melted from its target. A higher Tm means a stronger or more stable complex relative to a complex with a lower Tm. Biological Specimen: A biological specimen is a sample of bodily fluid or tissue used for laboratory testing or examination. As used herein, biological specimens include all clinical samples useful for detection of microbial infection in subjects. A biological specimen can be a tissue biopsy, for instance from the skin, lung or bronchial tissue, a surgical specimen, or autopsy material. A biological specimen also can be a biological fluid. Biological fluids include saliva, blood, derivatives and fractions of blood such as serum, and fluids of the respiratory tract, including the oropharyngeal tract, such as sputum, that has been expectorated or collected during bronchoscopy or bronchoalveolar lavage. Examples of appropriate specimens for use with the current disclosure for the detection of members of the Mycobacterium or Nocardia genera or individual species of Mycobacterium or Nocardia include conventional clinical samples, for instance blood or blood-fractions (for example, serum), bronchoalveolar lavage (BAL), sputum, and induced sputum samples. Techniques for acquisition of such samples are well known in the art. Blood and blood fractions (for example, serum) can be prepared in traditional ways. Oropharyngeal tract fluids can be acquired through conventional techniques, including sputum induction, BAL, and oral washing. Obtaining a sample from oral washing involves having the subject gargle with an amount of normal saline for approximately 10-30 seconds and then expectorate the wash into a sample cup. cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments
(introns) and transcriptional regulatory sequences. cDNA can also contain untranslated regions (UTRs) that are responsible for translational control in the corresponding RNA molecule. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Degenerate variant: A polynucleotide encoding a secA polypeptide, such as a secAl polypeptide, that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the sec A polypeptide, such as a secAl polypeptide, encoded by the nucleotide sequence is unchanged. Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, for instance, that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Specific, non-limiting examples of an epitope include a tetra- to penta-peptide sequence in a polypeptide, a tri- to penta- glycoside sequence in a polysaccharide. In the animal most antigens will present several or even many antigenic determinants simultaneously. Such a polypeptide may also be qualified as an immunogenic polypeptide. In one embodiment, the epitope is Mycobacterium species-specific. In another embodiment, the epitope is Nocardia species-specific. Expression Control Sequences: Nucleic acid sequences that control and regulate the expression of a nucleic acid sequence, such as a heterologous nucleic acid sequence, to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, polyA signals, a start codon (for instance, ATG) in front of a protein-encoding polynucleotide sequence, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter. A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included (see for example, Bitter et al, Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptφ, ptac (ptrp-lac-hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. In one embodiment, the promoter is a cytomegalovirus promoter. Fluorescence Resonance Energy Transfer (FRET): A spectroscopic process by which energy is passed between an initially excited donor molecule to an acceptor molecule that are separated by 10-100 A. The donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule. The efficiency of energy transfer is proportional to the inverse sixth power of the distance (R) between the donor and acceptor (1/R6) fluorophores and occurs without emission of a photon. In applications using FRET, the donor and acceptor dyes are different, in which case FRET either can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. For example, if the donor's fluorescence is quenched it indicates the donor and acceptor molecules are within the Fδrster radius (the distance where FRET has 50% efficiency, approximately 20-60 A), whereas if the donor fluoresces at its characteristic wavelength, it denotes that the distance between the donor and acceptor molecules has increased beyond the Fδrster radius. This occurs, for example, when a TaqMan® probe is degraded by Taq polymerase following hybridization of the probe to a target nucleic acid sequence or when a hairpin probe is hybridized to a target nucleic acid sequence. In another example, energy is transferred via FRET between two different fluorophores such that the acceptor molecule can emit light at its characteristic wavelength, which is always longer than the emission wavelength of the donor molecule. Examples of oligonucleotides using FRET that can be used to detect amplicons include linear oligoprobes, such as FRETprobes (also referred to as HybProbes), 5' nuclease oligoprobes, such as TaqMan® probes or TaqMan® probes with a minor groove binding moiety, hairpin oligoprobes such as molecular beacons, scorpion primers and UniPrimers, minor groove binding probes, and self-fluorescing amplicons, such as sunrise primers. Fluorescent marker or Fluorescent dye: A chemical compound, which when excited by exposure to a particular wavelength of light, emits light (for instance, fluoresces), for example at a different wavelength than that to which it was exposed. Fluorophores can be described in terms of their emission profile, or "color." Green fluorophores, for example Cy3, fluorescein isothiocyanate (FITC), and Oregon Green, are characterized by their emission at wavelengths generally in the range of 515-540 λ. Red fluorophores, for example LightCycler Red 640, LightCycler Red 705, Texas Red, Cy5 and tetramethylrhodamine, are characterized by their emission at wavelengths generally in the range of 590-7 lO λ. Encompassed by the term "fluorescent marker" as it is used herein are luminescent molecules, which are chemical compounds which do not require exposure to a particular wavelength of light to fluoresce; luminescent compounds naturally fluoresce. Therefore, the use of luminescent signals eliminates the need for an external source of electromagnetic radiation, such as a laser. An example of a luminescent molecule includes, but is not limited to, aequorin (Tsien, Ann. Rev. Biochem. 67:509, 1998). FRETProbes: A pair of adjacent, fluorogenic hybridization oligoprobes used for real-time
PCR, for example in a LightCycler™ (Roche Molecular Biochemicals; Mannheim, Germany) capillary-based microvolume fluorimeter and thermocycler with rapid temperature control. FRETProbes include an upstream probe labeled with a 3' donor fluorophore (for example, FITC) and a downstream probe labeled with an acceptor fluorophore at the 5' terminus. When the oligoprobes hybridize to an internal sequence in the amplified target sequence during the annealing phase of PCR, the two fluorophores are in close proximity to each other, for example within 1-10 nucleotides of each other. This adjacent hybridization results in a FRET signal due to the interaction between the donor and acceptor fluorophores that can be measured (see Mackay et al, Nucleic Acids Research, 30:1292-1305, 2002, incorporated herein by reference). Specific, non-limiting examples of FRETProbes include FastStart DNA Hybridization
Probes (Roche Molecular Biochemicals; Mannheim, Germany). With FastStart DNA Hybridization Probes, one probe is labeled at the 5' end with a LightCycler Red fluorophore, for example a LightCycler Red 640 or Red 705 fluorophore, and to avoid extension, the probe is modified at the 3' end by phosphorylation. The other probe is labeled at the 3' end with FITC. During FRET, FITC, the donor fluorophore, is excited by the light source of the LightCycler instrument, and part of the excitation energy is transferred to LightCycler Red, the acceptor fluorophore. The intensity of the light emitted by the acceptor fluorophore is measured by the apparatus, such as a LightCycler™. The increasing amount of measured fluorescence is proportional to the increasing amount of the amplified target nucleic acid generated during the ongoing PCR process. Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Also includes the cells of the subject. Hybridization: Oligonucleotides hybridize by hydrogen bonding, which includes Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleotide units. For example, adenine and thymine are complementary nucleotides which base pair through formation of hydrogen bonds. "Complementary" refers to sequence complementarity between two nucleotide units. For example, if a nucleotide unit at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide unit at the same position of a DNA or RNA molecule, then the oligonucleotides are complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units which can hydrogen bond with each other. A "hybridization complex" is formed when complementary nucleotides in a nucleic acid molecule(s) form base pairs. In one embodiment, a hybridization complex is formed when target nucleic acids, for example amplified Mycobacterium species-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium species-specific oligonucleotide probes, chemically linked to a polymeric solid support. In another embodiment, a hybridization complex is formed when target nucleic acids, for example amplified Nocardia species-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Nocardia species-specific oligonucleotide probes, chemically linked to a polymeric solid support. "Specifically hybridizable" and "complementary" are terms which indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. An oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, or under conditions in which an assay is performed. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (1989), chapters 9 and 11, herein incorporated by reference. For purposes of the present disclosure, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the primer or oligonucleotide probe and the target sequence. "Stringent conditions" can be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 5% mismatch will not hybridize. Stringent conditions are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5°C to 20°C lower than the Tm for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Tijssen ( Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993). Nucleic acid molecules that hybridize under stringent conditions to a Mycobacterium or a Nocardia secAl sequence will typically hybridize to a selected portion of the gene under wash conditions of 2x SSC at 50°C Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Labeled: A biomolecule is labeled if it is capable of being detected. For example, a nucleic acid or a polypeptide may be coupled covalently or noncovalently to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent markers or dyes, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989, and Ausubel et al, Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998. Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Minor groove binder (MGB) probe: A DNA probe with a conjugated minor groove binder group. Minor groove binders are a potent class of naturally occurring antibiotics. These long, flat molecules adopt a crescent shape that fits snugly into the minor groove formed by double stranded DNA. When attached to the terminal end of a DNA probe, the MGB group forms an extremely stable duplex with single-stranded DNA targets, allowing shorter probes (for example, as short as thirteen bases) to be used for hybridization based assays. In comparison with unmodified DNA probes, MGB probes have higher melting temperature and increased specificity, especially when a mismatch is in the MGB region of the duplex. Fluorogenic MGB probes are more specific for single base mismatches and fluorescence quenching is more efficient, giving increased sensitivity using these probes. In addition, A T rich duplexes are stabilized more than G/C rich duplexes. MGB probes are more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures. Thus, MGB probes are useful when designing assays for closely related sequences, such as species-specific assays. The MGB probes can also include a non- fluorescent quencher, which virtually eliminates the background fluorescence associated with traditional quenchers, providing better sensitivity and quantitation precision. Specific, non-limiting examples of MGB probes include the TaqMan® MGB probes (Applied Biosystems: Foster City, CA) and the MGB Eclipse™ Probes (Epoch Biosciences; Bothell, WA). Molecular beacon probe: Oligonucleotides with stem-loop structures that contain a fluorescent marker at the 5' end, such as FAM, TAMRA, TET, or ROX, and a quencher, such as DABCYL, at the 3' end. The degree of quenching via fluorescence energy resonance transfer
(FRET) is inversely proportional to the sixth power of the distance between the quencher and the fluorescent marker. Thus, FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye. After heating and cooling, molecular beacon probes reform a stem-loop structure that quenches the fluorescent signal from the marker. If a PCR product whose sequence is complementary to the loop sequence is present during the heating/cooling cycle, hybridization of the molecular beacon probe to one strand of the PCR product, or amplicon, will increase the distance between tlie marker and the quencher, resulting in increased fluorescence (FRET is interrupted). Molecular probes allow detection of multiple DNA species (multiplexing) using different reporter dyes on different probes or beacons. Multiplex PCR: Polymerase chain reaction that uses multiplex primers to produce more than one PCR product that can be detected in a single reaction tube. Multiplex primers: More than one pair of primers that are used simultaneously to amplify more than one target and/or control nucleic acid molecule in a single reaction tube. Multiplex probes: More than one probe, such as a molecular beacon probe, used simultaneously to detect more than one target and or control nucleic acid molecule in a single reaction tube. Mycobacterium: A genus of bacteria responsible for more disease world-wide than all other bacterial genera combined. Mycobacteria are small, 0.5 x 1 μm, weakly gram-positive bacilli that have two characteristic bacteriologic features. First, the cell walls of these organisms contain very long chain fatty acid esters known as mycolic acids (lipids typically having chain lengths of 70 to 90 carbons). This unusual cell envelope allows the mycobacteria to be impervious to many antiseptic solutions and antibiotics. Secondly, the pathogenic mycobacteria have an unusually slow growth rate. Unlike typical bacterial pathogens such as E. coli or Staphylococcus aureus, which double in 20-30 minutes and grow to high densities overnight in culture media, Mycobacterium species, such as Mycobacterium avium (M. avium) and M. tuberculosis, double approximately every 24 hours. Thus it can take three to four weeks to obtain a dense liquid culture or a visible colony of mycobacteria on a plate. The best known property of the genus is the ability of the bacteria to resist decolorization by weak acids after staining, hence the term "acid-fast bacilli" (Wayne "Cultivation of Mycobacterium tuberculosis for research purposes", in Tuberculosis: Pathogenesis. Protection, and Control. ASM Press, Bloom BR (ed), Washington, DC, 1994, pp. 73-84). Currently, over 70 species of mycobacteria have been well defined. Species other than M. tuberculosis andM. leprae have been termed the "environmental mycobacteria," many of which are known pathogens. For example, M. kansasii is an important pulmonary pathogen, along with the M. avium complex. Within the genus Mycobacterium, there are a number of closely related species that have been grouped into complexes. The Mycobacterium avium complex (MAC) includes two species, M. avium andM. intracellulare. Though M. avium was originally identified as a pathogen of birds, it is now known that both M. avium and M. intracellulare are environmental saprophytes that survive well in soil, water, and food, and can be carried by animals (Inderlied et al, Clin MicrobiolRev 6:266- 310, 1993; Shinnick and Good, Eur. J. Clin. Microbiol Infect. Dis. 13:884-901, 1994). MAC is an opportunistic, rather than innate, pathogen in humans; it causes disease primarily in immunocompromised patients. MAC causes three classical disease syndromes: disseminated MAC, pulmonary MAC, and MAC cervical lymphadenitis, the most common of which is disseminated MAC (Horsburgh, "Epidemiology of Mycobacterium avium complex," in Mycobacterium avium Complex Infection: Progress in Research and Treatment. Marcel Dekker, Inc., Korvick and Benson (eds), New York, 1996, pp. 1-22). The Mycobacterium tuberculosis complex (MTC) includes M. tuberculosis, M. bovis, M. africanum, andM microti. Each of these species, except M microti (a cause of rodent tuberculosis), causes tuberculosis in humans. Significantly, there are no environmental reservoirs of M. tuberculosis complex organisms, and only a few animals (cattle and occasionally deer) transmit tuberculosis to humans. Of all the culturable mycobacteria, only M tuberculosis is an obligate pathogen. The two laboratory techniques that have classically been used to detect a mycobacterial infection are the acid fast smear and cultivation of the organism. In addition, techniques have been developed to determine the species of the organism (for example, DNA blotting techniques). Generally, these techniques are performed on a body fluid, such as sputum or blood (Heifets and Good, "Current laboratory methods for the diagnosis of tuberculosis," in: Tuberculosis: Pathogenesis. Protection and Control. ASM Press. Bloom BR (ed), Washington, DC, 1994, pp. 85-110). Mycobacterium/Nocardia genera-specific primer: A primer that permits amplification of a Mycobacterium or Nocardia genus-specific nucleic acid, for example a conserved region of the secAl gene, present in virtually all members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus. In one embodiment, a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene that is conserved among members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus (a Mycobacterium/Nocardia genera-specific nucleic acid sequence). In one specific, non-limiting example, a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene that is approximately 700 base pairs in length, is in the 5' half of the secAl gene and includes the substrate specificity domain (SSD). In a further specific, non-limiting example, a pair of Mycobacterium/Nocardia genera-specific secAl primers amplifies a region of the secAl gene (including primers) that corresponds to a region between amino acid positions 138 and 391 of the secAl protein. In another specific, non-limiting example, a pair of Mycobacterium/Nocardia genera- specific secAl primers amplifies a region of the secAl gene (excluding primers) that corresponds to a region between amino acid positions 148 and 380 of the secAl protein. Mycobacterium/Nocardia genera-specific probe or probe oligonucleotide: A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secA gene, in virtually all members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus. In one embodiment, a Mycobacterium/Nocardia genera-specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Mycobacterium and Nocardia genera, but not in bacteria of any other genus (a Mycobacterium/Nocardia genera-specific nucleic acid sequence). Mycobacterium genus-specific probe or probe oligonucleotide: A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secAl gene, in virtually all members of the Mycobacterium genus, but not in bacteria of any other genus. In one embodiment, a Mycobacterium genus-specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Mycobacterium genus, but not in bacteria of any other genus (a Mycobacterium genus-specific nucleic acid sequence). Mycobacterium species-specific primer: A primer which permits amplification of a nucleic acid from a single species of Mycobacterium and not other species of Mycobacterium. In one embodiment, the nucleic acid primer can be used to amplify the secAl gene from clinically relevant mycobacterial pathogens. In one embodiment, a pair of Mycobacterium species-specific secAl primers amplifies a region of the secAl gene that is unique to a particular Mycobacterium species. In one specific, non-limiting example, a pair of Mycobacterium species-specific secAl primers amplifies a region of the secAl gene that is in the 5' half of the secAl gene. In another specific, non- limiting example, a pair of primers amplifies a region of the secAl gene that is unique to M gordonae. In other specific, non-limiting examples, a pair of primers amplifies a region of the secAl gene that is unique to Mycobacterium abscessus (M abscessus), M. africanum, M. asiaticum, M. aurum, M. avium, M. bovis, M. celatum, M. chelonae, M. flavescens, M. fortuitum, M. gastri, M. genavense, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. leprae, M. malmoense, M. marinum, M. mucogenicum, M. nonchrόmogenicum, M. peregrinum, M. scrofulaceum, M. shimoidei, M. simiae, M. smegmatis, M. szulgai, M. termoresistible, M. terrae, M. triviale, M. tuberculosis, M. ulcerans, M. vaccae, X-Cluster, or M xenopi. Mycobacterium species-specific probe or probe oligonucleotide: A probe oligonucleotide that hybridizes to a nucleic acid, for example the secAl gene, from one Mycobacterium species, but does not bind nucleic acid from other Mycobacterium species, under a specified set of hybridization conditions. In one specific, non-limiting example, the probe binds M avium nucleic acid under a specific set of hybridization conditions, but does not bind nucleic acid from any other Mycobacterium species under identical hybridization conditions. Nocardia: A genus of bacteria characterized as Gram-positive rods, which in old cultures or clinical specimens may appear as branching chains resembling fungal hyphae. Nocardia are weakly acid-fast following staining with the modified Ziehl-Neelsen or Kinyoun stain. Cultures may grow in a few days, but typically require two to three weeks of incubation. Most human infections (90%) are caused by inhalation of members of the N asteroides group, which includes three subgroups: (l)Nocardia asteroides complex (which contains multiple subspecies), (2) Nocardia farcinica, and (3) Nocardia nova. Nocardia brasiliensis, Nocardia caviae (otitidiscaviarum), and Nocardia transvalensis represent the remaining 10% of infections. Of these, Nocardia brasiliensis is the most important in tropical areas; it is most often seen as a cutaneous infection that can affect individuals with normal immune function (although 70% of cases of Nocardia brasiliensis are seen in immunocompromised individuals). Nocardia has been isolated from soil and organic material throughout the world. Human infection usually results from the inhalation of airborne bacilli or the traumatic inoculation of organisms into the skin. The infection is not transmissible between individuals. Natural resistance, mediated by intact mucous membranes and alveolar and tissue phagocytes, is quite strong. In immunocompromised hosts, pulmonary infection results in the formation of abscesses and, rarely, granulomas with hematogenous or lymphatic dissemination to the skin or central nervous system. Nocardia can subvert the antimicrobial mechanisms of phagocytes by inhibiting phagosome- lysosome fusion. Owing to the debilitated nature of the infected patients, mortality is high (up to 45 percent), even with appropriate therapy. Nocardiosis is usually associated with T-cell dysfunctions, immunoglobulin deficiencies, or leukocyte abnormalities. Although nocardiosis has been diagnosed in individuals with no detectable deficiency of humoral or cell-mediated immunity, it usually occurs in patients whose immune status has been compromised by post-transplant immunosuppressive therapy, leukemia, lymphoma, pancytopenia, dysgammaglobulinemia, humoral defects, chronic granulomatous disease, or steroid therapy. The male/female ratio in nocardiosis is approximately 2:1, and infections occur from infancy to old age. There is no apparent geographic clustering of cases in the United States, except for cutaneous infection with N. brasiliensis, which is more common in the south. Nocardia genus-specific probe or probe oligonucleotide: A nucleic acid probe that specifically hybridizes to a nucleic acid, for example the secAl gene, in virtually all members of the Nocardia genus, but not in bacteria of any other genus. In one embodiment, a Nocardia genus- specific secAl probe hybridizes to a region of the secAl gene that is conserved among members of the Nocardia genus, but not in bacteria of any other genus (a Nocardia genus-specific nucleic acid sequence). Nocardia species-specific primer: A primer which permits amplification of a nucleic acid from a single species of Nocardia and not other species of Nocardia. In one embodiment, the nucleic acid primer can be used to amplify the secAl gene from clinically relevant nocardial pathogens. In one embodiment, a pair of Nocardia species-specific secAl primers amplifies a region of the secAl gene that is unique to a particular Nocardia species. In one specific, non-limiting example, a pair of Nocardia species-specific secAl primers amplifies a region of the secAl gene that is in the 5' half of the secAl gene. In another specific, non-limiting example, a pair of primers amplifies a region of the secAl gene that is unique to Nocardia abscessus (N. abscessus). In other specific, non-limiting examples, a pair of primers amplifies a region of the secAl gene that is unique to N. Africana, N. asteroids drug pattern type IV, N asteroides drug pattern type VI, N asteroides type strain, N. beijingensis, N. brasiliensis, N. brevicatena, N carnea, N. corynebacteroides, N. cyriacigeorgica, N. farcinica, N. ignorata, N. kruczakiae, N. nova, N. otitidiscaviarum (N. caviae), N. paucivorans, N. pseudobrasiliensis, N seriolae, N. transvalensis, N. transvalensis new taxon I, N. vaccinii, N. veterana, oiN. vinacea. Nocardia species-specific probe or probe oligonucleotide: A probe oligonucleotide that hybridizes to a nucleic acid, for example the secAl gene, from one Nocardia species, but does not bind nucleic acid from other Nocardia species, under a specified set of hybridization conditions. In one specific, non-limiting example, the probe binds Nocardia brasiliensis nucleic acid under a specific set of hybridization conditions, but does not bind nucleic acid from any other Nocardia species under identical hybridization conditions. Nucleic acid sequence (or polynucleotide): A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, and includes polynucleotides encoding full length proteins and/or fragments of such full length proteins which can function as a therapeutic agent. A polynucleotide is generally a linear nucleotide sequence, including sequences of greater than 100 nucleotide bases in length. In one embodiment, a nucleic acid is labeled (for example, biotinylated, fluorescently labeled or labeled with radioisotopes). Nucleotide: Nucleotide includes, but is not limited to, a monomer that includes a base linked to a sugar, as in DNA and RNA, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide. In one embodiment, nucleotides are labeled (for example, biotinylated, fluorescently labeled, or labeled with radioisotopes). Oligonucleotide: A linear polynucleotide sequence of between 10 and 100 nucleotide bases in length. An oligonucleotide can be labeled (for example, using radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes) either at the 3' or the 5' end, or by the incorporation of labeled nucleotides into the oligonucleotide. Examples of oligonucleotides are those which are at least 15, 25, 50, 75 or 90 nucleotide bases in length. Oligonucleotides can be primers or probes. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide. Ortholog: Two nucleic acid or amino acid sequences are orihologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species. Orthologous sequences are also homologous sequences. Polymerase chain reaction (PCR): A method for amplifying specific DNA segments which exploits certain features of DNA replication. For instance replication requires a primer and specificity is determined by the sequence and size of the primer. One primer is complementary to the sense-strand at one end of the DNA sequence to be amplified and the other primer is complementary to the antisense-strand at the other end of the DNA sequence to be amplified. The method amplifies specific DNA segments by cycles of template denaturation; primer addition; primer annealing and replication using a thermostable DNA polymerase. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample. Polymeric solid support: The physical structural shape of a medium that forms a solid support. Thus, medium can generally include, but are not limited to: polymer films (for instance, polymers having a substantially non-porous surface); polymer filaments (for example, mesh and fabrics); polymer beads; polymer foams; polymer frits; and polymer threads. Polymers can include, but are not limited to, cellulosic substrates, such as nitrocellulose, nylon, silicon, metals, such as gold, Teflon, polypropylene, polyethylene, polybutylene, polyisobutylene, plybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene- propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroefhylene, polysulfornes, hydroxylated biaxially oriented polypropylene, aminated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof. Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred in nature. The term polypeptide or protein as used herein encompasses any amino acid sequence and includes, but may not be limited to, modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced. Substantially purified polypeptide as used herein refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine; or (d) a residue having an electropositive side chain, for example, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl. Variant amino acid sequences may, for example, be 80, 90 or even 95, 96, 97, 98 or 99% identical to the native amino acid sequence. Programs and algorithms for determining percentage identity can be found at the NCBI website. Primers: Primers are short nucleic acid molecules, such as DNA oligonucleotides, 15 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by PCR, real-time PCR, or other nucleic-acid amplification methods known in the art. An "upstream" or "forward" primer is a primer 5' to a reference point on a nucleic acid sequence. A "downstream" or "reverse" primer is a primer 3' to a reference point on a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction. Methods for preparing and using primers are described, for example, in Sambrook et al. (In
Molecular Cloning: A Laboratoiγ Manual, Cold Spring Harbor, New York, 1989), Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992), and
Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). The nucleic acid can be attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example, in Sambrook et al. (In Molecular Cloning: A Laboratoiy Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992). The secAl gene is approximately 2849 nucleotides in length. The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the secAl gene sequences. Such molecules can comprise at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of these sequences, and can be obtained from any region of the disclosed sequences. One of ordinary skill in the art will appreciate that the specificity of a particular primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides of the nucleic acid encoding a Mycobacterium or a Nocardia secAl protein will anneal to a target sequence, such as a Mycobacterium or a Nocardia nucleic acid, respectively, in a sputum sample, with a higher specificity than a corresponding primer of only 15 nucleotides. Probes: Nucleic acid probes can also be readily prepared based on the nucleic acid molecules provided herein. A probe comprises an isolated nucleic acid. Methods for preparing and using probes are described, for example, in Sambrook et al. (la Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1992), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990). One of ordinary skill in the art will appreciate that the specificity of a particular probe also increases with its length. Thus, for example, an oligonucleotide probe comprising 20 consecutive nucleotides of the nucleic acid encoding a Mycobacterium or Nocardia secAl protein will anneal to a target sequence, such as an amplified Mycobacterium or Nocardia nucleic acid, respectively, with a higher specificity than a corresponding oligonucleotide probe of only 15 nucleotides. Thus, in order to obtain greater specificity, oligonucleotide probes and primers can be selected that comprise at least 15, 20, 25, 30, 35, 40 or more consecutive nucleotides of Mycobacterium ox Nocardia secAl gene sequences. The disclosure thus includes isolated nucleic acid molecules that comprise specified lengths of the Mycobacterium and Nocardia secAl gene sequences. Such molecules can comprise at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of these sequences, and can be obtained from any region of the disclosed sequences. Nucleic acid molecules can be selected as probe sequences that comprise at least 15, 20, 25, 30, 35, 40 or more consecutive nucleotides of any of portions of the Mycobacterium or Nocardia secAl gene. Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein is more pure than the protein in its natural environment within a cell. For example, a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation. Similarly, a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment comprising a complex mixture of oligonucleotides. Quencher: A molecule, such as DABCYL or TAMRA, which is capable of absorbing fluorescence emitted from a fluorescent marker or dye thereby reducing the level of fluorescence detected in a sample. A quencher molecule can be coupled to an oligonucleotide to generate, for example a molecular beacon probe, a minor groove binding probe, or a TaqMan® probe. In molecular beacon probes, a quencher is coupled to the 3' end of the probe. Real-time PCR: Real-time PCR detects amplification products as they accumulate in a sample during the PCR amplification process. In real-time PCR, the formation of a PCR product is monitored either continuously during amplification, or during each annealing or extension step of each cycle, by means of fluorescent probes, fluorescent primers or fluorescent dyes that bind to double-stranded DNA. In one embodiment, real-time PCR is a two-step process based on 1) hybridization of the PCR product with an oligomer probe, the potential of which to emit fluorescence is inactivated by a covalently linked quencher, and 2) separation of the fluorophore from the quencher, or digestion of the hybridized probe, resulting in a fluorescent signal enhancement. By recording the amount of fluorescence emission at each cycle, it is possible to monitor a PCR reaction during the exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target nucleic acid. In another embodiment, real-time PCR with FRETProbes involves two probes with different (acceptor and donor) fluorophores. Interaction between the donor and acceptor fluorophores produces FRET and the intensity of emission due to the acceptor fluorophore in the sample is subsequently measured. Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. secA: An essential, highly conserved, ATP-driven protein component of the sec-dependent protein export pathway in bacteria. The sec-dependent protein export pathway is responsible for binding presecretory proteins and promoting their translocation, via a translocase, across the bacterial plasma membrane. The translocase core is composed of integral membrane proteins and the peripheral membrane protein secA. secA is a soluble, cytoplasmic, multifunctional, 100 kDa homodimer that interacts with acidic phospholipids in the bacterial cell membrane, the SecYEG pore (a membrane channel formed by SecE (9 kDa), SecG (11 kDa), and SecY (47 kDa)) and molecular chaperones, such as SecB (17 kDa), that deliver precursor proteins to the translocase. secA is an ATPase that provides energy for protein translocation and is composed of a helicase motor domain (belonging to the DEAD family) and a translocation domain. Binding of secA to presecretory proteins and their translocation requires the presence of a substructure called the specificity domain (SSD). The SSD is essential for protein translocation, is unique to sec A, and is absent from other members of the DEAD family of proteins (Baud et al, J. Biol Chem, 277:13724-13731, 2002). Some Gram-positive bacteria have two homologous secA proteins with non-overlapping functions, secAl and secA2. Genes encoding secAl and secA2 have been identified in M. leprae, M. avium, M. tuberculosis, andM smegmatis. Both mycobacterial secAl and secA2 proteins exhibit significant sequence homology to other bacterial and plant secA homologs, and both contain the conserved ATP-binding motifs. The two secA proteins are not very similar to each other, although each is highly conserved in different mycobacterial species. secAl is the essential housekeeping secA protein, whereas secA2 is an accessory secretion factor. In M. tuberculosis, secAl is believed to be functionally equivalent to the very well characterized Escherichia coli secA. As Nocardia secA is similar to Mycobacterium secAl (see the disclosure herein) it is also referred to herein as Nocardia secAl. The M. tuberculosis H37Rv secAl protein is a 949 amino acid molecule (GenBank Accession No. NP_217757) that has been crystallized and its structure elucidated (Sharma et al, Proc. Natl. Acad. Sci. USA, 100:2243-2248, 2003).' The motor domain of secAl is composed of residues 2-221 and 360-387, whereas the translocation domain is composed of residues 222-359 and 587-835 of the secAl protein. The SSD is located between residues 222-359 of the secAl protein and is inserted in nucleotide binding domain 1 (NBD1; Sharma et al, Proc. Nat. Acad. Sci. USA, 100:2243-2248, 2003). NBD1 is located between amino acids 1 and 426 and the nucleotide binding domain 2 (NBD2) is located between amino acids 426 and 641 in the 5' half of the secAl protein. The nucleic acid sequence encoding secAl has also been identified for M tuberculosis H37Rv (GenBank Accession No. BX842582; nucleotides 150019 to 152868). Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44; 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et α/., Nuc. Acids Res. 16:10881-90, 1988; Huang et al, Computer Appls. Biosc. 8:155-65, 1992; and Pearson et al, Meth. Mol Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10; 1990, presents a detailed consideration of sequence alignment methods and homology calculations. One specific, non-limiting example of alignment software includes DNASTAR Lasergene. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site. For comparisons of nucleic acid sequences, sequence identity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLAST sequence analysis software, for instance, the NCBI BLAST 2.0 program gapped blastn set to default parameters. (One example of such default settings would be: expect = 10, filter = default, descriptions = 500 pairwise, alignments = 500, alignment view = standard, gap existence cost = 11, per residue existence = 1, per residue gap cost = 0.85). These methods can be used to determine sequence identity over short windows, such as over the first 500 or 700 base pairs of the Mycobacterium or Nocardia secAl gene. An indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater identity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity, when using gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, Comput. Appl. Biosci. 10:67-70, 1994). Other programs use SEG. When less than the entire sequence is being compared for sequence identity, homologs typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that significant homologs can be obtained that fall outside of the ranges provided. TaqMan® probes: A linear oligonucleotide probe with a 5' reporter fluorophore and a 3' quencher fluorophore, such as TAMRA. In the intact TaqMan probe, energy is transferred (via FRET) from the short- wavelength fluorophore to the long- wavelength fluorophore on the other end, quenching the short-wavelength fluorescence. After hybridization, the probe is susceptible to degradation by the endonuclease activity of a processing Taq polymerase. Upon degradation, FRET is interrupted, increasing the fluorescence from the short- wavelength fluorophore and decreasing fluorescence from the long-wavelength fluorophore. Target nucleic acid sequence: A nucleic acid sequence to which an antisense or sense oligonucleotide specifically hybridizes. In one embodiment, a Mycobacterium species-specific secA primer specifically hybridizes to a target nucleic acid sequence in a Mycobacterium species secA gene. In another embodiment, a Nocardia species-specific secA primer specifically hybridizes to a target nucleic acid sequence in a Nocardia species secA gene. The target nucleic acid sequence of a secAl gene can include a substrate specificity domain (SSD) responsible for binding of secAl to the signal sequence of proteins to be exported. A specific, non-limiting example of a target nucleic acid sequence is a region of the secAl gene that is approximately 700 base pairs in length in the 5' half of the secAl gene that includes a substrate specificity domain located between amino acid positions 222 and 359 of the secAl protein. A further specific, non-limiting example of a target nucleic acid sequence is a region of the secAl gene that corresponds to a region between amino acid positions 148 and 380 of the secAl protein. Variant oligonucleotides and variant analogs: A variation of an oligonucleotide or an oligonucleotide analog is an oligomer having one or more base substitutions, one or more base deletions, and/or one or more base insertions, so long as the oligomer substantially retains the activity of the original oligonucleotide or analog, or has sufficient complementarity to a target sequence. A variant oligonucleotide or analog may also hybridize with the target DNA or RNA, under stringency conditions as described above. A variant oligonulceotide or analog also exhibits sufficient complementarity with the target DNA or RNA of the original oligonucleotide or analog as described above.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Mycobacterium and Nocardia secA Nucleic Acids Of the more than seventy known species of Mycobacterium, approximately thirty species of Mycobacterium have been associated with human disease. Examples of disease-associated Mycobacterium include Mycobacterium abscessus (M abscessus), M. africanum, M. asiaticum, M. avium, M. bovis, M. chelonae, M.flavescens, M. fortuitum, M. gastri, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. leprae, M. malmoense, M. marinum, M. mucogenicum, M. peregrinum, M. scrofulaceum, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans, X-Cluster, and xenopi. Of the approximately twenty known species of Nocardia, three species are responsible for most human infections. Examples of disease-associated Nocardia include Nocardia asteroids, Nocardia brasiliensis, and Nocardia caviae. This disclosure provides the nucleic acid sequences encoding secA polypeptide fragments from different Mycobacterium and Nocardia species, including disease-associated Mycobacterium and Nocardia. In specific embodiments, these sequences are used for generating oligonucleotide primers and probes for the detection of bacteria of the Mycobacterium and Nocardia genera in samples. In other specific embodiments, these sequences are used for generating oligonucleotide primers and probes for the detection of Mycobacterium and Nocardia species in samples. Nucleic acid sequences encoding secA polypeptides, such as secAl polypeptide fragments, from different species of Mycobacterium and Nocardia are disclosed herein. Specific, non-limiting examples of Mycobacterium secAl nucleic acid sequences encoding a Mycobacterium secAl polypeptide fragment include, but are not limited to, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID
NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 221. Specific, non-limiting examples of Nocardia secAl nucleic acid sequences encoding a Nocardia secAl polypeptide fragment include, but are not limited to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245,
SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. These polynucleotides include DNA, cDNA, and RNA sequences that encode a Mycobacterium or a Nocardia secAl polypeptide fragment. It is understood that all polynucleotides encoding a Mycobacterium or a Nocardia secA polypeptide, such as a secAl polypeptide fragment, are also included herein. In one embodiment, a Mycobacterium secAl nucleic acid sequence is at least 80% homologous to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO:221. In other embodiments, a Mycobacterium secAl nucleic acid sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO:221. In one embodiment, a Nocardia secAl nucleic acid sequence is at least 80% homologous to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. In other embodiments, a Nocardia secAl nucleic acid sequence is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244,
SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. The Mycobacterium and Nocardia secA polynucleotides disclosed herein include a region encoding the substrate specificity domain. In some embodiments, the sequence encoding the substrate specificity domain is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the substrate specificity domain of M tuberculosis. The Mycobacterium and Nocardia secA polynucleotides, such as secAl polynucleotides, may be a recombinant DNA molecule which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA) independent of other sequences. DNA sequences encoding Mycobacterium or Nocardia secA polypeptide fragments, such as secAl polypeptide fragments, can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Mycobacterium and Nocardia secA polynucleotide sequences, such as secAl polynucleotide sequences, can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (for instance, ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with secA polynucleotide sequences, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Gluzman ed., 1982). The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the sec A polypeptide encoded by the nucleotide sequence is functionally unchanged. Also disclosed herein are Mycobacterium and Nocardia secA oligonucleotides, for example secAl probes and secAl primers. The Mycobacterium and Nocardia secA oligonucleotides, for example secAl oligonucleotides, can be genera- or species-specific. Mycobacterium/Nocardia genera-specific secA oligonucleotides include those oligonucleotides encoding a secA protein present in any Mycobacterium or Nocardia species found in clinical samples. Mycobacterium or Nocardia species- specific secA oligonucleotides include those oligonucleotides encoding a secA protein present in specific Mycobacterium or Nocardia species, respectively, found in clinical samples. Mycobacterium species found in clinical samples can be Mycobacterium known to be associated with disease, such as M tuberculosis, M. bovis, M. africanum, M. asiaticum, M. avium, M. celatum, M. chelonae, M. fortuitum, M. genavense, M. haemophilum, M. intracellulare, M. kansasii, M. malmoense, M. marinum, M. scrofulaceum, M. shimoidei, M. simiae, M. szulgai, M. ulcerans, andM. xenopi. Nocardia species found in clinical samples can be Nocardia known to be associated with disease, such as Nocardia asteroides complex, Nocardia farcinica, Nocardia nova, Nocardia brasiliensis, Nocardia otitidiscaviarum, and Nocardia transvalensis. The methods disclosed herein take advantage of the fact that under appropriate conditions oligonucleotides, such as probes and primers, form base-paired duplexes with oligonucleotides which have a complementary base sequence. The stability of the duplex is dependent on a number of factors, including the length of the oligonucleotides, the base composition, and the composition of the solution in which hybridization is effected. The effects of base composition on duplex stability may be reduced by carrying out the hybridization in particular solutions, for example in the presence of high concentrations of tertiary or quaternary amines. The thermal stability of the duplex is also dependent on the degree of sequence similarity between the sequences. By carrying out the hybridization at temperatures close to the anticipated Tm's of the type of duplexes expected to be formed between the target sequence(s) and the oligonucleotides, for example amplification primers, real-time PCR primers and probes, or oligonucleotides bound to an array, the rate of formation of mis-matched duplexes may be substantially reduced. The length of each oligonucleotide sequence can be selected to optimize binding of target Mycobacterium or Nocardia nucleic acid sequences, for example secA nucleic acid sequences, such as secAl nucleic acid sequences. An optimum length for use with a particular Mycobacterium or Nocardia nucleic acid sequence under specific screening conditions can be determined empirically. Oligonucleotides, for example probes or primers, of the disclosed secAl nucleic acid sequences may be comprised of at least 15 consecutive nucleic acids, which is sufficient to permit the oligonucleotide to selectively hybridize to DNA that encodes the disclosed Mycobacterium secAl polypeptide or polypeptide fragment (for example a polynucleotide set forth as SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO:221) or a Nocardia secAl polypeptide or polypeptide fragment (for example a polynucleotide set forth as SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252) under physiological conditions. In other embodiments, oligonucleotides comprising at least 15, 20, 25, 30, 35, 40, or more consecutive nucleotides of the Mycobacterium or Nocardia secA sequences, such as secAl sequences, can be used. Jn specific non-limiting examples, the oligonucleotide includes nucleotides 1-15, 16-30, or 31-45, etc., 2-16, 17-31, or 32-46, etc., or 3-17, 18-32, or 33-47, etc. of a Mycobacterium or Nocardia secAl sequence. In other specific non-limiting examples, the oligonucleotide includes nucleotides 1-20, 21-40, or 41-60, etc., 2-21, 22-41, or 42-61, etc., or 3-22, 23-42, or 43-62, etc. of 'a Mycobacterium or Nocardia secAl sequence. In further specific non- limiting examples, the oligonucleotide includes nucleotides 1-25, 26-50, 51-75, etc., 2-26, 27-51, or 52-76, or 2-27, 28-52, or 53-77, etc. of a Mycobacterium or Nocardia secAl sequence. In one embodiment, the Mycobacterium secAl sequence is a target sequence for amplification. In another embodiment, the Nocardia secAl sequence is a target sequence for amplification. In further embodiments, the target sequence for amplification is in the 5' half of the Mycobacterium or Nocardia secAl gene and includes the substrate specificity domain. In one specific, non-limiting example, the target sequence for amplification is a region of the secAl gene that is at least about 700 base pairs in length in the 5' half of the secAl gene and includes the substrate specificity domain. In other specific, non-limiting examples, the target sequence for amplification is a region of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 base pairs in length in the 5' half of the secAl gene and includes the substrate specificity domain. Mycobacterium/Nocardia genera-specific secA oligonucleotides, such as primers and probes, selectively hybridize to a region that is present in all Mycobacterium and Nocardia secA genes, but that is not present in a sec A nucleic acid in any other genus of bacteria. In one embodiment, a Mycobacterium/Nocardia genera-specific probe hybridizes to a region of the secAl gene that is common to all Mycobacterium and Nocardia secAl nucleic acids. Mycobacterium/Nocardia genera-specific probes thus can be used to distinguish Mycobacterium and Nocardia from other genera of bacteria. Specific, non-limiting examples of
Mycobacterium/Nocardia genera-specific secAl probes include probes that selectively hybridize to a nucleic acid sequence within a region in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain. A Mycobacterium/Nocardia genera-specific primer can be used to sequence a Mycobacterium or Nocardia secAl nucleic acid in order to identify a Mycobacterium or Nocardia secAl nucleic acid. Alternatively, two, or more, genera-specific primers can be used to amplify a region within the secAl gene, for example by polymerase chain reaction (PCR) or more specifically by real-time PCR, that is common to all Mycobacterium and Nocardia, but that is not present in secAl in bacteria of another genus. Specific, non-limiting examples of Mycobacterium/Nocardia genera-specific secAl primers include primers that selectively hybridize to a nucleic acid sequence within a region in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain. Mycobacterium and Nocardia species-specific secAl oligonucleotides, such as primers and probes, can be used to identify a Mycobacterium or Nocardia species of interest. Thus, these species- specific probes and primers specifically hybridize to a region in a secAl gene which is unique to a particular Mycobacterium or Nocardia species and not to other species. Specific, non-limiting examples of Mycobacterium or Nocardia species-specific secAl probes include probes that selectively hybridize to a nucleic acid sequence within the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain. In one embodiment, a Mycobacterium species-specific primer is used to sequence a Mycobacterium secAl nucleic acid in order to identify a Mycobacterium species-specific secAl nucleic acid. In another embodiment, a Nocardia species-specific primer is used to sequence a Nocardia secAl nucleic acid in order to identify a Nocardia species-specific secAl nucleic acid. In other embodiments two, or more, species-specific primers can be used to amplify a region, for example by PCR or more specifically by real-time PCR, within the secAl gene that is unique to a particular Mycobacterium or Nocardia species. Specific, non-limiting examples of Mycobacterium or Nocardia species-specific secAl primers include primers that selectively hybridize to a nucleic acid sequence in the 5' half of the secAl gene that is at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 base pairs in length and includes the substrate specificity domain. The primers and probes disclosed herein can be end-labeled (for example, radiolabeled, enzymatically-Iabeled, fluorescently-labeled, or biotinylated). One specific, non-limiting example of a primer label is a fluoresceinated Mycobacterium species specific primer. Another specific, non- limiting example of a primer label is a biotinylated Nocardia species specific primer. The probes disclosed herein can be fluorescently-labeled, such as for use in real-time PCR. In one embodiment, the oligonucleotide probes in the sample are labeled to render them readily detectable. Detectable labels may be any species or moiety that maybe detected either visually or with the aid of an instrument. Detectable labels can be radioisotopes, chemiluminescent tags, haptens, or fluorescent markers. Specific, non-limiting examples of fluorescent markers include FITC, LightCycler Red 640, Light Cycler Red 705, 6-carboxy-X-rhodamine (ROX), 5-carboxyfluorescein (FAM), 2'7'-dimethoxy- 4'5'-dichloro-6-carboxyfluorescein (JOE), and 6-carboxy-2',4,7,7'-tetrachlorofluorescein (TET). In one embodiment, the fluorescent markers coupled to the oligonucleotide probes have spectrally distinct emission spectra such that the amplified DNA sequences to which they specifically hybridize can be distinguished within the same reaction tube. In some embodiments, four, five, six, seven, or more probes that are sufficiently complementary to a nucleic acid sequence, such as a Mycobacterium and/or a Nocardia secAl nucleic acid sequence, may be used in a single reaction tube. FRETProbes include an upstream probe labeled with a 3' donor fluorophore, such as FITC, and a downstream probe labeled with an acceptor fluorophore, such as LightCycler Red 640 or Red 705, at the 5' terminus. The nucleic acid sequence of a FRETProbe includes a nucleic acid sequence that detects the amplified product from the target nucleic acid sequence of interest, such as a Mycobacterium secA sequence, for example, a Mycobacterium species-specific secAl sequence.
When the FRETProbes are not hybridized to the target sequence, the donor fluorophore is excited by a filtered light source, such as by a LightCycler's light emitting diode (LED), and a green fluorescent light is emitted at a slightly longer wavelength. However, when the pair of FRETProbes hybridize to the target sequence, the two fluorophores are in close proximity to each other, for example within 1- 10 nucleotides of each other, and the energy emitted by the excitation of the donor fluorophore excited the acceptor fluorophore, for example a LightCycler Red 640 attached to the probe. The resultant energy transfer via FRET results in the emission of a red fluorescent light at an even longer wavelength. The intensity of the light emitted by the acceptor fluorophore is measured by the apparatus, such as a LightCycler. The increasing amount of measured fluorescence is proportional to tlie increasing amount of the amplified target nucleic acid generated during the ongoing PCR process. Since the acceptor fluorophore only emits a signal when both labeled probes are hybridized to the target nucleic acid sequence, the fluorescence measurement is performed after the annealing step in the PCR process. Molecular beacon probes include probes coupled to a fluorescent marker in combination with a quencher molecule. The nucleic acid sequence of a molecular beacon probe includes a nucleic acid that detects the amplified product from the DNA sequence of interest, and sequences that permit the molecular beacon probe to form a hairpin structure. Attached to opposite ends (the 5' and the 3' end of the molecular beacon) are a fluorescent reporter molecule and a quencher molecule. When the molecular beacon is in the hairpin conformation (not hybridized to product) any fluorescence emitted by the fluorescent label is absorbed (quenched) by the quencher molecule via FRET and no fluorescence is detected. When a molecular beacon hybridizes to an amplified target nucleic acid with a complementary nucleic acid sequence the fluorescent label and the quencher molecule are separated, and fluorescence is detected and can be measured during each PCR cycle. These probes are known to one of skill in the art (see the Molecular-Probes, Eugene, OR website). TaqMan® probes include linear oligonucleotide probes with a 5' reporter fluorophore and a
3' quencher fluorophore, such as TAMRA. In the intact Taqman® probe, energy is transferred (via FRET) from the short-wavelength fluorophore to the long- wavelength fluorophore on the other end, quenching the short-wavelength fluorescence. After hybridization, the probe is susceptible to degradation by the endonuclease activity of a processing Taq polymerase. Upon degradation, FRET is interrupted, increasing the fluorescence from the short- wavelength fluorophore and decreasing fluorescence from the long-wavelength fluorophore. Specific, non-limiting examples of quencher molecules include N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA) and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL). Many suitable forms of these fluorescent markers and quenchers are widely available commercially with substituents on their phenyl moieties which can be used as the site for coupling or as the coupling functionality for attachment to an oligonucleotide.
Mycobacterium and Nocardia secA Polypeptides SecAl polypeptide fragments from different Mycobacterium and Nocardia species are disclosed herein. Mycobacterium secAl polypeptide fragments include, but are not limited to, polypeptides having a sequence as set forth as one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 220. Nocardia secAl polypeptide fragment sequences can be translated from the Nocardia nucleic acid sequences as set forth as one of SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. SecAl polypeptide fragments also include, but are not limited to, an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence as set forth as one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 220, or to the translated Nocardia nucleic acid sequences as set forth as SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. The Mycobacterium and Nocardia secA polypolypeptides disclosed herein include a region encoding the substrate specificity domain which is between amino acids 222 and 359 of the secAl protein. In some embodiments, the sequence encoding the substrate specificity domain is at least 80%, 85%, 90%>, 95%, 96%, 97%, 98%, or 99% homologous to the substrate specificity domain of M. tuberculosis. Fragments, variants, and fusions of the Mycobacterium and Nocardia secAl polypeptide fragments identified above are disclosed herein and can readily be prepared by one of skill in the art using molecular techniques. In one embodiment, a fragment of a Mycobacterium secAl polypeptide fragment includes at least 8, 10, 15, or 20 consecutive amino acids of a Mycobacterium secAl polypeptide fragment. In another embodiment, a fragment of 'a Nocardia secAl polypeptide fragment includes at least 8, 10, 15, or 20 consecutive amino acids of a Nocardia secAl polypeptide fragment. In other embodiments, a fragment of a Mycobacterium or a Nocardia secAl polypeptide fragment includes a species-specific antigenic epitope. In one embodiment, a fragment is at least 17 amino acids, at least 23 amino acids, at least 25 amino acids, or at least 30 amino acids in length from a sec A polypeptide fragment. The polypeptides include polypeptides as set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 220, conservative variants thereof, and homologues thereof. In one embodiment, a fragment retains at least one species-specific antigenic epitope. One skilled in the art, given the disclosure herein, can purify a Mycobacterium or a Nocardia secAl polypeptide fragment using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the Mycobacterium or Nocardia secAl polypeptide can also be determined by amino- terminal amino acid sequence analysis. Minor modifications of the Mycobacterium or Nocardia secAl polypeptide primary amino acid sequences may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein. Isolation and purification of recombinantly expressed polypeptides may be carried out by conventional means including preparative chromatography and immunological separations.
Mycobacterium and Nocardia secA Antibodies A secA polypeptide or a fragment or conservative variant thereof can be used to produce antibodies which are immunoreactive or bind to a Mycobacterium or Nocardia species-specific epitope of secA, such as secAl. Polyclonal antibodies, antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are included. The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al, "Production of Polyclonal Antisera, in: Immunochemical Protocols pages 1-5, Manson, ed., Humana Press 1992; Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1, 1992. The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al, sections 2.5.1-2.6.7; and Harlow et al, in: Antibodies: a Laboratory Manual, page 726, Cold Spring Harbor Pub., 1988. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion- exchange chromatography. See, for example, Coligan et al, sections 2.7.1-2.7.12 and sections 2.9.1 - 2.9.3; Barnes et al, Purification of Immunoglobulin G (IgG), in: Methods in Molecular Biology, Vol. 10, pages 79-104, Humana Press, 1992. Methods of in vitro and in vivo multiplication of monoclonal antibodies are well known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large-scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, for example, syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal. Antibodies can also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in WO 91/11465, 1991, and Losman et al, Int. J. Cancer 46:310, 1990. Alternatively, an antibody that specifically binds a secA polypeptide, such as a secAl polypeptide, can be derived from a humanized monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 86:3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321 :522, 1986; Riechmann et al, Nature 332:323, 1988; Verhoeyen et al, Science 239:1534, 1988; Carter et al, Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandliu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al, J. Immunol 150:2844, 1993. Antibodies can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 119, 1991; Winter et al, Ami. Rev. Immunol. 12:433, 1994. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from Stragene Cloning Systems (La Jolla, CA). In addition, antibodies can be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of Hie endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody- secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al, Nature Genet. 7:13, 1994; Lonberg et al, Nature 368:856, 1994; and Taylor et al, Int. Immunol 6:579, 1994. Antibodies include intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor and are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with tlie enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). An epitope is any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patents No. 4,036,945 and No. 4,331,647, and references contained therein; Nisonhoff et al, Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al, Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL chains. This association maybe noncovalent (Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659, 1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, for example, Sandhu, supra. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No.4,946,778; Pack et al. , Bio/Technology 11 : 1271 , 1993 ; and Sandhu, supra) . Another form of an antibody fragment is a peptide coding for a single complementarity- determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (Larrick et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 1991). Antibodies can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or a peptide used to immunize an animal can be derived from substantially purified polypeptide produced in host cells, in vitro translated cDNA, or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used earners which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (for example., a mouse, a rat, or a rabbit). Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991). It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first mono-clonal antibody. Effector molecules, for example, therapeutic, diagnostic, or detection moieties, can be linked to an antibody that specifically binds a secA polypeptide, such as a secAl polypeptide, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (for example, through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids. In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site. When the target site is a tumor, a linker which is cleavable under conditions present at the rumor site (for example, when exposed to tumor-associated enzymes or acidic pH) may be used. In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (for example, enzymes or fluorescent molecules) drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.
Detection and Identification of Mycobacterium and Nocardia Genera Using secA Nucleic Acids Probes and primers directed to a secA nucleic acid sequence can be used for the detection and/or identification of the Mycobacterium and Nocardia genera in clinical samples using a variety of detection and identification methods. The disclosed methods for detecting and identifying a Mycobacterium genus or a Nocardia genus are an advantage over previous methods because they provide rapid and accurate detection and identification of both Mycobacterium and Nocardia in a clinical sample. For example, the disclosed methods may be used to rapidly and accurately identify whether the causative agent of a pulmonary disease is either Mycobacterium or Nocardia, when it is otherwise not possible to distinguish the cause of the infection.
Amplification-Based Detection and Identification Methods The presence of a Mycobacterium and Nocardia (for example, any member of the Mycobacterium and Nocardia genera) may be detected with the use of any one of a number of techniques. In one embodiment, the presence of a Mycobacterium can be detected by amplification of a Mycobacterium nucleic acid sequence. In another embodiment, the presence of a Nocardia can be detected by amplification of a Nocardia nucleic acid sequence. Any nucleic acid amplification method can be used to detect the presence of a Mycobacterium or a Nocardia in a sample. In one specific, non-limiting example, PCR is used to amplify the mycobacterial nucleic acid sequences. In another specific, non-limiting example, PCR is used to amplify the norcardial nucleic acid sequences. In other specific, non-limiting examples, real-time PCR, RT-PCR, transcription-mediated amplification (TMA), or ligase chain reaction can be used to amplify mycobacterial or nocardial nucleic acid sequences. Techniques for nucleic acid amplification are well-known to those of skill in the art. In some embodiments, Mycobacterium, Nocardia, or Mycobacterium and Nocardia DNA sequences are amplified. In other embodiments, Mycobacterium, Nocardia, or Mycobacterium and Nocardia RNA sequences are reverse transcribed prior to amplification using reverse transcription- polymerase chain reaction (RT-PCR). In one specific, non-limiting example, amplification of a Mycobacterium secA nucleic acid sequence, such as a secAl nucleic acid sequence, can be used to detect the presence of a member of the genus Mycobacterium in a sample. In another specific, non- limiting example, amplification of 'a Nocardia secA nucleic acid sequence, such as a secAl nucleic acid sequence, can be used to detect the presence of a member of the genus Nocardia in a sample. Any type of thermal cycler apparatus, for example a PTC- 100® Peltier Thermal Cycler (MJ Research, Inc.; San Francisco, CA), a Robocycler® 40 Temperature Cycler (Stratagene; La Jolla, CA), or a GeneAmp® PCR System 9700 (Applied Biosystems; Foster City, CA) can be used to amplify nucleic acid sequences. For real-time PCR, any type of real-time thermocycler apparatus, for example a LightCycler (Roche; Mannheim, Germany), a 7700 Sequence Detector (Perkin Elmer/Applied Biosystems; Foster City, CA), ABI7700 (Applied Biosystems; Foster City, CA), or an MX4000 (Stratagene; La Jolla, CA) can by used to amplify nucleic acid sequences in real-time. Any region of a secA gene that is common to all Mycobacterium and Nocardia, but not in bacteria in another genus, can be amplified. For example, secA Mycobacterium/Nocardia genera- specific primers can be used to amplify a region that is at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 1000, or more base pairs in length to produce amplified Mycobacterium and/or Nocardia secA nucleic acids. The target region of the secAl gene can include a substrate specificity domain (responsible for binding of secAl to the signal sequence of proteins to be exported) located between amino acid positions 222 and 359 of the secAl protein. In one embodiment, Mycobacterium/Nocardia genera-specific primers, which specifically bind to a conserved region in the 5' half of the secAl gene, are used to produce an amplified Mycobacterium/Nocardia genera-specific nucleic acid sequence that is approximately 700 base pairs in length and includes the substrate specificity domain. In other embodiments,
Mycobacterium/Nocardia genera-specific primers, which specifically bind to a conserved region in the 5' half of the secAl gene, are used to produce an amplified Mycobacterium/Nocardia genera- specific nucleic acid sequence that is approximately 200, approximately 300, approximately 400, approximately 500, or approximately 600 base pairs in length and includes the substrate specificity domain. In some embodiments, the Mycobacterium/Nocardia genera-specific nucleic acid sequence is amplified from Mycobacterium, Nocardia, or both Mycobacterium and Nocardia. At least two primers are utilized in the amplification reaction. One or both of the primers can be end-labeled (for example, radiolabeled, fluorescently-labeled, enzymatically-labeled, or biotinylated). In one embodiment, the resulting amplicon is labeled. The pair of primers includes an upstream primer (which binds 5' to the downstream primer) and a downstream primer (which binds 3' to the upstream primer). In one embodiment, either the upstream primer or the downstream primer is labeled. One specific, non-limiting example of a primer label is a fluoresceinated downstream primer. The amplified Mycobacterium/Nocardia genera-specific product, for example a
Mycobacterium or a Nocardia secA nucleic acid sequence, can be detected in real-time, for example by real-time PCR, in order to determine the presence, the identity, and/or the amount of a Mycobacterium and or Nocardia genus in a sample. In this manner, an amplified DNA sequence, such as an amplified Mycobacterium or Nocardia secAl nucleic acid sequence, can be detected using a probe specific for the product amplified from the DNA sequence of interest. Detecting the amplified product includes the use of labeled probes that are sufficiently complementary and hybridize to the amplified nucleic acid sequence. Thus, the presence, amount, and/or identity of the amplified product can be detected by hybridizing a labeled probe, such as a fluorescently labeled probe, complementary to the amplified product. In one embodiment, the detection of a target nucleic acid sequence of interest includes the combined use of PCR amplification and a labeled probe such that the product is measured using real-time PCR. In another embodiment, the detection of an amplified target nucleic acid sequence of interest includes the transfer of the amplified target nucleic acid to a solid support, such as a blot, for example a Northern blot, and probing the blot with a probe, for example a labeled probe, that is complementary to the amplified target nucleic acid sequence. In yet another embodiment, the detection of an amplified target nucleic acid sequence of interest includes the hybridization of a labeled amplified target nucleic acid to probes that are attached, for example chemically-linked, to an array and that are complementary to the amplified target nucleic acid. In one embodiment, an assay system including a primer pair complementary to a Mycobacterium/Nocardia secA nucleic acid sequence (for example, for the amplification of a
Mycobacterium/Nocardia genera-specific nucleic acid) also includes a labeled probe that hybridizes to the amplified Mycobacterium/Nocardia genera-specific product in order to detect the amplified product. In one specific, non-limiting example, the primer pair includes Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium/Nocardia genera-specific probe. In other specific, non-limiting examples, the primer pair includes
Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium or Nocardia genus-specific probe. The amplified target nucleic acid can be detected using fluorescently-labeled genus-specific probes in real time. In one embodiment, the fluorescently-labeled probes rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time. For example, FRET that occurs between fluorogenic labels on different probes (for example, using FRETProbes) or between a fluorophore and a non-fluorescent quencher on the same probe (for example, using a molecular beacon or a TaqMan® probe) can identify a probe that specifically hybridizes to the DNA sequence of interest and in this way, using Mycobacterium/Nocardia genera- specific probes, Mycobacterium genus-specific probe, or Nocardia genus-specific probes can detect the presence, identity, and/or amount of Mycobacterium and/or Nocardia genera in a sample. In one embodiment, the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube. In one embodiment, real-time PCR can be performed in a microfluidics device, such as in an I-CORE® module (Cepheid; Sunnyvale, CA). Microfluidics involves manipulating minute volumes of liquids and is used to miniaturize biological assays. Real-time PCR using microfluidic technology offers several benefits over standard real-time PCR techniques, for example reduced sample volume and increased performance. In another embodiment, a melting curve analysis of the amplified target nucleic acid can be performed subsequent to the amplification process. The Tm of a nucleic acid sequence depends on the length of the sequence and its G/C content. Thus, the identification of the Tm for a nucleic acid sequence can be used to identify the amplified nucleic acid and/or single nucleotide polymorphisms (SNPs) between amplified nucleic acids. One specific, non-limiting example of an apparatus that can perform real-time PCR and melting curve analysis is the LightCycler™ (Roche; Mannheim, Germany).
Airay-Based Detection and Identification In other embodiments, devices, such as arrays, can be used to detect the presence of Mycobacterium and/or Nocardia, using specific oligonucleotide probes. The arrays herein termed "Mycobacterium/Nocardia detection arrays," are used to detect the presence of Mycobacterium and Nocardia genera. Other arrays are specific for the detection of either the Mycobacterium genus or the Nocardia genus. A pre-determined set of probes, for example Mycobacterium/Nocardia genera- specific secA oligonucleotide probes, are attached to the surface of a solid support for use in detection of the amplified Mycobacterium and Nocardia nucleic acid sequences. Alternatively, Mycobacterium genus-specific or Nocardia genus-specific probes are attached to the surface of a solid support for use in the detection of the amplified Mycobacterium or Nocardia genus-specific nucleic acid sequences, respectively. Additionally, if an internal control nucleic acid sequence was amplified in the amplification reaction (see above), an oligonucleotide probe can be included to detect the presence of this amplified control nucleic acid. The oligonucleotide probes attached to the array specifically hybridize to target nucleic acids. In one specific, non-limiting example, the anay includes a plurality of Mycobacterium/Nocardia genera-specific oligonucleotide probes. In another specific, non-limiting example, the anay includes a plurality of Mycobacterium genus-specific and/or Nocardia genus- specific probes. A hybridization complex is formed when target nucleic acids, for example amplified secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium/Nocardia genera-specific oligonucleotide probes, Mycobacterium genus-specific probes, or Nocardia genus-specific probes, chemically linked to a polymeric solid support. In one embodiment the hybridization complex forms a hybridization pattern of the sample. In one embodiment, a sample containing a target nucleic acid is hybridized to an array. In another embodiment, the target nucleic acid sequences (for example an approximately 700 base pair region in the 5' half of the secAl gene that includes a substrate specificity domain) are first amplified in an amplification reaction before hybridizing to the array. Thus, probe sequences of use with the method when detecting, for example, secAl nucleic acid, are Mycobacterium/Nocardia genera- specific oligonucleotide probes, Mycobacterium genus-specific probes, or Nocardia genus-specific probes. The sequences forming the array can be directly attached or linked to the support.
Alternatively, the oligonucleotide probes can be attached to the support by non-Mycobacterium or Nocardia sequences such as oligonucleotides or other molecules that serve as spacers or linkers to the solid support. The solid support can be formed from an organic polymer (a polymeric support). Suitable materials for the solid support include, but are not limited to: polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene, polyisoprene, polyvinylpyrrolidine, polytetrafluroethylene, polyvinylidene difluroide, polyfluoroethylene-propylene, polyethylenevinyl alcohol, polymethylpentene, polycholorotrifluoroethylene, polysulfornes, hydroxylated biaxially oriented polypropylene, animated biaxially oriented polypropylene, thiolated biaxially oriented polypropylene, etyleneacrylic acid, thylene methacrylic acid, and blends of copolymers thereof (see U.S. Patent No. 5,985,567, herein incorporated by reference). In general, suitable characteristics of the material that can be used to form the solid support surface include: being amenable to surface activation such that upon activation, the surface of the support is capable of covalently attaching a biomolecule such as an oligonucleotide thereto; amenability to "in situ" synthesis of biomolecules; being chemically inert, such that at the termination of biomolecular synthesis, areas on the support not occupied by the biomolecules are not amenable to non-specific binding, or when non-specific binding occurs, such materials can be readily removed from the surface without removing the biomolecules. In one embodiment, the solid support surface is polypropylene. Polypropylene is chemically inert and hydrophobic. Non-specific binding is generally avoidable, and detection sensitivity is improved. Polypropylene has good chemical resistance to a variety of organic acids (for instance, formic acid), organic agents (for instance, acetone or ethanol), bases (for instance, sodium hydroxide), salts (for instance, sodium chloride), oxidizing agents (for instance, peracetic acid), and mineral acids (for instance, hydrochloric acid). Polypropylene also provides a low fluorescence background, which minimizes background interference and increases the sensitivity of the signal of interest. A surface activated organic polymer can be the solid support surface. In one specific non- limiting example, the surface activated organic polymer is a polypropylene material aminated via radio frequency plasma discharge. Such materials are easily utilized for the attachment of nucleotides, and thus are well suited for oligonucleotide synthesis. The amine groups on the activated organic polymers are reactive with nucleotides such that the nucleotides can be bound to the polymers. Alternatively, other reactive groups can be used (for example, carboxylated, hydroxylated, thiolated, or active ester groups). A wide variety of array formats can be employed in accordance with the present disclosure.
One example includes a linear anay of oligonucleotide bands, generally referred to in the art as a dipstick. As would be readily appreciated by those skilled in the art, other anay formats including, but not limited to slot (rectangular) and circular anays are equally suitable for use (see U.S. Patent No. 5,981,185, herein incorporated by reference). In one embodiment, the array is formed on a polymer medium, which is a thread, membrane or film. Particularly disclosed for preparation of anays at this time are biaxially oriented polypropylene (BOPP) films; in addition to their durability, BOPP films exhibit a low background fluorescence. The anay formats of the present disclosure can be included in a variety of different types of formats. A "format" includes any format to which the solid support can be affixed, such as microtiter plates, test tubes, inorganic sheets, dipsticks, etc. For example, when the solid support is a polypropylene thread, one or more polypropylene threads can be affixed to a plastic dipstick-type device; polypropylene membranes can be affixed to glass slides. The particular format is, in and of itself, unimportant. All that is necessary is that the solid support can be affixed thereto without affecting the functional behavior of the solid support or any biopolymer absorbed thereon, and that the format (for example, the dipstick or slide) is stable to any materials into which the device is introduced (for example, clinical samples, hybridization solutions, etc.). The arrays of the present disclosure can be prepared by a variety of approaches which are known to those working in the field. Pursuant to one type of approach, the complete oligonucleotide probe sequences are synthesized separately and then attached to a solid support (see U.S. Patent No. 6,013,789, herein incorporated by reference). In another embodiment, the sequences can be synthesized directly onto the support to provide the desired anay (see U.S. Patent No. 5,554,501, herein incorporated by reference). Suitable methods for covalently coupling oligonucleotides to a solid support and for directly synthesizing the oligonucleotides onto the support would be readily apparent to those working in the field; a summary of suitable methods can be found in Matson et al, Anal. Biochem. 217:306-10, 1994. In one embodiment, the oligonucleotides are synthesized onto the support using conventional chemical techniques for preparing oligonucleotides on solid supports (for example, see PCT applications WO 85/01051 and WO 89/10977, or U.S. Patent No. 5,554,501, herein incorporated by reference). In one specific, non-limiting example, a polypropylene support (for example, a BOPP) is first surface animated by exposure to an ammonia plasma generated by radiofrequency plasma discharge. The reaction of a phosphoramidite-activated nucleotide with the animated membrane followed by oxidation with, for example, iodine provides a base stable amidate bond to the support. A suitable array can be produced using automated means to synthesize oligonucleotides in the cells of the array by laying down the precursors for the four bases in a predetermined pattern. Briefly, a multiple-channel automated chemical delivery system is employed to create oligonucleotide probe populations in parallel rows (conesponding in number to the number of channels in the delivery system) across the substrate. Following completion of oligonucleotide synthesis in a first direction, the substrate can then be rotated by 90° to permit synthesis to proceed within a second (2°) set of rows that are now perpendicular to the first set. This process creates a multiple-channel anay whose intersection generates a plurality of discrete cells. The oligonucleotides can be bound to the polypropylene support by either the 3' end of the oligonucleotide or by the 5' end of the oligonucleotide. In one embodiment, the oligonucleotides are bound to tlie solid support by the 3' end. However, one of skill in the art will be able to determine whether the use of the 3' end or the 5' end of the oligonucleotide is suitable for bonding to the solid support. In general, the internal complementarity of an oligonucleotide probe in the region of the 3' end and the 5' end determines binding to the support. In general, the end of the probe with the most internal complementarity is bound to the support, thereby leaving the end with the least internal complementarity to bind to amplified Mycobacterium and/or Nocardia sequences.
Other Detection and Identification Methods The presence, identity and/or amount of a Mycobacterium or a Nocardia can be determined by other hybridization methods, such as Northern or Southern blot analysis, using labeled oligonucleotides specific to a nucleic acid sequence, such as a conserved portion of the secAl gene that is common only to Mycobacterium and Nocardia (a Mycobacterium/Nocardia genera-specific nucleic acid sequence). These oligonucleotides, such as those described above, can be labeled radioactively with isotopes (such as 32P) or non-radioactively, with tags such as fluorophores or biotin (Ward and Langer, Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981), and hybridized to individual Mycobacterium and Nocardia nucleic acid samples transfened and immobilized on membranes or other solid supports by dot-blot or transfer from gels after electrophoresis. Visualization methods such as autoradiography or fluorometric (Landegren et al, Science 242:229-237, 1989) or colorimetric reactions (Gebeyehu et al, Nucleic Acids Res. 15:4513-4534, 1987) can be used to detect a signal and the signals then quantitated using, for instance, a spectrophotometer, a scintillation counter, a densitometer or a Phosphorimager (Amersham Biosciences). The Phosphorimager is able to analyze both DNA samples from blots and gels using autoradiographic, direct fluorescence or chemifluorescence detection. Since the Phosphorimager is more sensitive than ordinary x-ray film, exposure times can be reduced up to ten-fold and signal quantitation of both weak and strong signals on the same blot is possible. Images can be visualized and evaluated with the aid of computer programs such as ImageQuant™. The identification of an amplified Mycobacterium/Nocardia genera-specific product, for example a Mycobacterium/Nocardia genera-specific secA nucleic acid sequence, also can be determined by sequencing the amplified product in order to determine if the amplified product is from a Mycobacterium, a Nocardia, or both a Mycobacterium and a Nocardia. Techniques for sequencing nucleic acids are well known to those of skill in the art. Antibodies that specifically bind secAl polypeptides also can be used to detect a Mycobacterium or a Nocardia in a sample. In one embodiment, antibodies against secAl polypeptides can be attached or linked to a support medium, such as a blot or a dish, and a protein sample applied. In another embodiment, antibodies that specifically bind secAl polypeptides can be used to detect secAl polypeptides by using them to probe blots containing a protein sample.
Methods of using antibodies to detect proteins, such as Western blot analysis and ELISA, are well known in the art.
Detection and Identification of Mycobacterium and Nocardia Species Using sec A Nucleic Acids Methods for the detection and/or identification of the Mycobacterium and Nocardia g lenera. as described above, can be used for the detection and/or identification of a Mycobacterium or a Nocardia species using Mycobacterium and Nocardia species-specific probes and primers. The disclosed methods for detecting and identifying a Mycobacterium species or a Nocardia species are an advantage over previous methods because they provide rapid and accurate detection and identification of Mycobacterium and Nocardia species in a clinical sample. For example, the disclosed methods may be used to rapidly and accurately identify whether the causative agent of pulmonary disease is a particular Mycobacterium or Nocardia species, when it is otherwise not possible to distinguish the cause of the infection.
Amplification-Based Detection and Identification Methods In one embodiment, the amplification-based detection and identification methods described above are used to detect and/or identify a Mycobacterium or a Nocardia species in a sample. For example, a pair of primers used in an amplification reaction can be Mycobacterium species-specific primers which permit amplification of a nucleic acid from a clinically relevant mycobacterial pathogen. In one specific non-limiting example, secAl species-specific primers which specifically bind to a unique region of the Mycobacterium secAl gene are used to produce an amplified Mycobacterium species-specific nucleic acid in order to detect the presence of, or identify, a particular Mycobacterium species in a sample, such as M tuberculosis, M. avium, or M kansasii. Alternatively, a pair of primers used in an amplification reaction can be Nocardia species-specific primers which permit amplification of a nucleic acid from a clinically relevant nocardial pathogen. In one specific non-limiting example, secAl species-specific primers which specifically bind to a unique region of the Nocardia secAl gene are used to produce an amplified Nocardia species- specific nucleic acid in order to detect the presence of, or identify, a particular Nocardia species in a sample, such as TV. asteroids, TV brasiliensis, or TV caviae. Any region of a secA gene, such as a secAl gene, that is unique to a Mycobacterium or a Nocardia species may be amplified. For example, secAl species-specific primers can be used to amplify a region that is at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 700, at least 1000, or more base pairs in length. Specific, non-limiting examples of a Mycobacterium species-specific secAl nucleic acid sequence that can be amplified to detect and or identify a Mycobacterium species in a sample include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 221. Specific, non-limiting examples of a Nocaria species-specific secAl nucleic acid sequence that can be amplified to detect and/or identify a Nocardia species in a sample include, but are not limited to, the nucleic acid sequence set forth in SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252. More than one pair of primers can be included in an amplification reaction (multiplex primers) in order to simultaneously amplify more than one target and/or control nucleic acid molecule in a single reaction tube. In one embodiment, more than one pair of primers is included in the amplification reaction in order to amplify more than one Mycobacterium species-specific secA nucleic acid sequence present in a sample. In other embodiments, more than one pair of primers is included in the amplification reaction in order to amplify more than one Nocardia species-specific secA nucleic acid sequence in a sample, or to amplify at least one Nocardia and at least one Mycobacterium species-specific secA nucleic acid sequence in a sample. In additional embodiments, at least one pair of Mycobacterium/Nocardia genera-specific primers is included in an amplification reaction with at least one pair of Mycobacterium or Nocardia species-specific primers. In a further embodiment, an additional pair of primers is included in the amplification reaction as an internal control. Internal control primers are designed to amplify nucleic acid from all samples, and serve to provide confirmation of appropriate amplification. One of skill in the art will readily be able to identify primer pairs to serve as internal control primers. An amplified Mycobacterium species-, Nocardia species-, or a Mycobacterium/Nocardia genera-specific product, for example a species- or genera-specific secA nucleic acid sequence, can be detected in real-time, for example by real-time PCR (see above) using Mycobacterium and/or Nocardia species-specific probes, in order to determine the presence, the identity, and/or the amount of a Mycobacterium species or a Nocardia species in a sample. In one embodiment, an assay system including a primer pair complementary to a Mycobacterium secA nucleic acid sequence (for example, for the amplification of a Mycobacterium/Nocardia genera- or Mycobacterium species-specific nucleic acid) also includes a labeled probe that hybridizes to the amplified Mycobacterium species-specific nucleic acid in order to detect a Mycobacterium species. In another embodiment, an assay system includes a labeled probe that hybridizes to an amplified Mycobacterium/Nocardia genera-specific nucleic acid or to an amplified Nocardia species-specific nucleic acid in order to detect a Nocardia species. In specific, non-limiting examples, the primer pair includes Mycobacterium/Nocardia genera-specific primers and the probe includes a fluorescently-labeled Mycobacterium species-specific probe or a fluorescently-labeled Nocardia species-specific probe. In another specific, non-limiting example, the primer pair includes Mycobacterium species-specific primers and the probe includes a fluorescently- labeled Mycobacterium species-specific probe. In a further specific, non-limiting example, the primer pair includes Nocardia species-specific primers and the probe includes a fluorescently-labeled Nocardia species-specific probe. The amplified target nucleic acid can be detected using fluorescently-labeled species-specific probes in real time. In another embodiment, the fluorescently-labeled probes rely upon fluorescence resonance energy transfer (FRET), or in a change in the fluorescence emission wavelength of a sample, as a method to detect hybridization of a DNA probe to the amplified target nucleic acid in real-time. For example, FRET that occurs between fluorogenic labels on different probes (for example, using FRETProbes) or between a fluorophore and a non-fluorescent quencher on the same probe (for example, using a molecular beacon or a TaqMan® probe) can identify a probe that specifically hybridizes to the DNA sequence of interest and in this way, using Mycobacterium or Nocardia species-specific probes, can detect the presence, identity, and/or amount of a Mycobacterium species or a Nocardia species in a sample. In one embodiment, the fluorescently-labeled DNA probes used to identify amplification products have spectrally distinct emission wavelengths, thus allowing them to be distinguished within the same reaction tube.
Array-Based Detection and Identification Methods The anay-based detection and identification methods described above can be used to detect and/or identify a Mycobacterium and/or a Nocardia species in a sample. The Mycobacterium or Nocardia species-specific oligonucleotide probes attached to the array specifically hybridize to target nucleic acids. In one specific, non-limiting example, the anay includes a plurality of Mycobacterium and Nocardia species-specific oligonucleotide probes. In other specific, non-limiting examples, the array includes a plurality of Nocardia ox Mycobacterium species-specific oligonucleotide probes. A hybridization complex is formed when target nucleic acids, for example amplified Mycobacterium/Nocardia genera-specific secAl nucleic acids, hybridize to a plurality of oligonucleotide probes, for example Mycobacterium species-specific oligonucleotide probes or Nocardia species-specific probes, attached, for example chemically linked, to a polymeric solid support. In one embodiment the hybridization complex forms a hybridization pattern of the sample. In another embodiment, a hybridization pattern of the sample is compared to a control hybridization pattern in order to identify a Mycobacterium species or a Nocardia species in a sample. Probe sequences of use with the array method are species-specific oligonucleotide probes that recognize a secAl nucleic acid only from a particular Mycobacterium species or a particular Nocardia species. Such sequences can be determined by examining the sequences of the different species, and choosing probes that specifically hybridize to a particular species, but not others (see species-specific probes listed above). One of skill in the art will be able to identify other Mycobacterium and Nocardia oligonucleotide probes that can be attached to the surface of a solid support for the detection of other amplified Mycobacterium and Nocardia species-specific nucleic acid sequences.
Other Detection and Identification Methods The presence, identity and/or amount of a Mycobacterium species or a Nocardia species can be determined by other hybridization methods, as described above. Such methods would include Northern or Southern blot analysis, using labeled oligonucleotides specific to a Mycobacterium or a Nocardia species-specific nucleic acid sequence. The identification of an amplified Mycobacterium/Nocardia genera-specific product, an amplified Mycobacterium species-specific product, or a Nocardia species-specific product, in order to identify the Mycobacterium or Nocardia species in a sample, also can be determined by sequencing the amplified product. Techniques for sequencing nucleic acids are well known to those of skill in the art. As described above for the detection of a Mycobacterium genus or a Nocardia genus in a sample, antibodies that specifically bind secAl polypeptides also can be used to detect a Mycobacterium species or a Nocardia species in a sample. In one embodiment, antibodies against secAl polypeptides can be linked to a support medium, such as a blot or a dish, and a protein sample applied. In another embodiment, antibodies that specifically bind a species specific secAl epitope can be used to probe blots containing a Mycobacterium protein sample or a Nocardia protein sample. Methods of using antibodies to detect proteins, such as Western blot analysis and ELISA, are well known in the art.
Methods of Treating a Subject Suspected of Being Infected with Mycobacterium or Nocardia Disclosed herein are methods of treating subjects suspected of being infected with Mycobacterium or Nocardia. Using the detection and identification methods described above a subject can be given the appropriate treatment once it is confirmed that the subject is infected with a Mycobacterium or a Nocardia and the nature of the treatment customized to the exact species of Mycobacterium or Nocardia infecting the subject. Methods of treatment for subjects infected with various species of Mycobacterium or Nocardia are well known to those of skill in the art. Appropriate specimens for use with the current disclosure in the detection and identification of Mycobacterium genus and species, or Nocardia genus and species, include any conventional clinical samples, for instance abscess samples, blood or blood- fractions (for example, serum), bone manow, bronchoalveolar lavage (BAL) samples, bronchial wash or bronchial brush samples, cerebrospinal fluid, esophageal samples, tissue drainage samples, eye conjunctiva samples, gastric biopsy or washing samples, nasopharyngeal swabs, saliva, sputum, induced sputum samples, stool samples, synovial fluid, skin lesions, sinus aspirates, tissue biopsies, transtracheal aspirates, surgical specimens, or autopsy material. Techniques for acquisition of such samples are well known in the art. See, for instance, Schluger et al (J. Exp. Med. 176:1327-33, 1992) (collection of serum samples); Bigby et al (Am. Rev. Respir. Dis. 133:515-8, 1986) and Kovacs et al. (NEJM 318:589-93, 1988) (collection of sputum samples); and Ognibene et al (Am. Rev. Respir. Dis. 129:929-32, 1984) (collection of bronchoalveolar lavage, BAL). In addition to conventional methods, oral washing sequences (Helweg-Larsen et al, J. Clin. Microbiol. 36:2068-72, 1998) provide a non-invasive technique for acquiring appropriate samples to be used in nucleic acid hybridization or amplification of a nucleic acid sequence from a Mycobacterium or a Nocardia. Oral washing involves having the subject gargle with 50 cc of normal saline for 10-30 seconds and then expectorate the wash into a sample cup. Serum or other blood fractions can be prepared in the conventional manner. About 200 μl of serum is an appropriate amount for the extraction of DNA for use in hybridization or amplification reactions. See also, Schluger et al, J. Exp. Med. 176:1327-33, 1992; Ortona et al, Mol. Cell Probes 10:187-90, 1996. Once a sample has been obtained, the sample can be used directly, or concentrated (for example, by centrifugation or filtration) and a hybridization reaction or an amplification reaction performed. Alternatively, the nucleic acid can be extracted using any conventional method prior to hybridization or amplification. For example, rapid DNA preparation can be performed using a commercially available kit (for example, the InstaGene Matrix, BioRad, Hercules, CA; the NucliSens isolation kit, Organon Teknika, Netherlands). In one embodiment, the DNA preparation technique chosen yields a nucleotide preparation that is accessible to, and amenable to, nucleic acid hybridization or amplification.
Kits Kits are provided which contain the necessary reagents for detecting bacteria of the Mycobacterium and Nocardia genera and/or particular Mycobacterium and Nocardia species. Instructions provided in the diagnostic kits can include calibration curves, diagrams, illustrations, or charts or the like to compare with the determined (for example, experimentally measured) values or other results. The nucleotide sequences disclosed herein, and fragments thereof, can be supplied in the form of a kit for use in detection of bacteria of the Mycobacterium and Nocardia genera or specific Mycobacterium and Nocardia species. In such a kit, an appropriate amount of one or more of the Mycobacterium/Nocardia genera specific, Mycobacterium species-specific, or Nocardia species- specific oligonucleotide primers or probes is provided in one or more containers. The oligonucleotide primers and probes may be provided suspended in an aqueous solution or as a freeze- dried or lyophilized powder, for instance. The container(s) in which the oligonucleotide(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles. In some applications, pairs of primers may be provided in pre- measured single use amounts in individual, typically disposable, tubes or equivalent containers. The sample to be tested for the detection of bacteria of the Mycobacterium or Nocardia genera, or a specific Mycobacterium or Nocardia species can be added to the individual tubes described above and amplification, such as by real-time PCR, carried out directly. The amount of each oligonucleotide primer or probe supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each oligonucleotide primer provided would likely be an amount sufficient to prime several in vitro amplification reactions. Those of ordinary skill in the art know the amount of oligonucleotide primer that is appropriate for use in a single amplification reaction. General guidelines may for instance be found in Innis et al (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, CA, 1990), Sambrook et al. (In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In some embodiments, kits may also include the reagents necessary to carry out PCR in vitro amplification reactions, including, for instance, DNA sample preparation reagents, appropriate buffers (for example, polymerase buffer), salts (for example, magnesium chloride), and deoxyribonucleotides (dNTPs). Kits may in addition include either labeled or unlabeled oligonucleotide probes for use in detection of a target nucleic acid, such as an amplified nucleic acid. The appropriate sequences for such a probe will be any sequence that falls between the annealing sites of the two provided oligonucleotide primers, such that the sequence the probe is complementary to is amplified during the amplification reaction. In some applications, a pair of primers and a probe may be provided in a single tube, or equivalent container, in a pre-measured single use. The amount of each probe provided would likely be in an amount sufficient to detect a target nucleic acid, such as an amplified nucleic acid. In another embodiment, the kit includes a Mycobacterium, a Nocardia, or a Mycobacterium/Nocardia detection anay, a buffer solution, a conjugating solution for developing the signal of interest, and a detection reagent for detecting the signal of interest, each in separate packaging, such as a container. The kit can also optionally include an effective amount of a pre- treatment solution for application to the probe biomolecules to produce an amplified signal. It may also be advantageous to provide in the kit one or more control sequences. The design of appropriate positive control sequences is well known to one of ordinary skill in the appropriate art. The invention is illustrated by the following non-limiting Examples.
EXAMPLES
Example 1 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid in a Sample This example demonstrates how a Mycobacterium/Nocardia genera-specific nucleic acid sequence encoding a fragment of the secAl protein can be amplified from a sample, thereby identifying the presence of Mycobacterium or Nocardia in the sample.
DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfuls of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at
10,500 x g for 5 minutes. The supernatant was removed and the pellet was washed in Tris-HCL, pH 8.0. The suspension was centrifuged at 10,500 x g for 5 minutes, resuspended in ultra-pure water, and vortexed for 10 minutes. The tube was then heated at 95 °C for 25 minutes. The extracted DNA was stored at -15 to -25 °C until ready to use.
PCR Amplification The Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a region of a secAl gene that is conserved in both Mycobacterium and Nocardia. For example, in one experiment genera-specific primers were designed based on the fact that the target sequence for amplification is a region within the Mycobacterium tuberculosis secAl gene (located between base pair 150019 and base pair 152868 in the Mycobacterium tuberculosis genome; Genbank Accession No. BX842582). More specifically, primers were designed against a target sequence located between base pair 151730 and base pair 152428 of the Mycobacterium tuberculosis genome in order to generate an amplified sequence approximately 700 base pairs in length, excluding the primers. In other experiments, the target sequence of the secAl gene conesponded to a sequence encoding a region between amino acid 148 and 380 (or amino acid positions 138 and 391, if including the primers) of the secAl protein, which included the substrate specificity domain (located between amino acid positions 222 and 359 of the secAl protein). In further experiments, the amplified sequence was approximately 500 base pairs in length. Any combination of Mycobacterium/Nocardia genera-specific forward and reverse primers, for example pairs derived from the primers listed in Table 1, can be used to generate Mycobacterium/Nocardia genera-specific secAl nucleic acid sequences (amplicons). Genus-specific secAl primers, used to generate amplicons that will be sequenced, are tailed with M13 sequencing primers (M13 forward tail: 5'GTA AAA CGA CGG CCA G-3' (SEQ ID NO: 229); M13 reverse tail: 5'CAG GAA ACA GC TAT GAC-3' (SEQ ID NO: 230)). These include the Mtu.Forward primer (5'GTAAAACGACGGCCAGGACAGYGAGTGGATGGGYCGSGTGCACCG-3'; SEQ ID NO: 13; Y can be T or C, and S can be G or C) and the Mtu.Reverse primer (5'CAGGAAACAGCTATGACACCACGCCCAGCTTGTAGATCTCGTGCAGCTC-3'; SEQ ID NO: 14), which were synthesized by Midland Certified Reagent Company. The PCR amplification was performed using 1 pmol of forward primer (see Table 1), 1 pmol of reverse primer (see Table 1), 1 Unit UNG, ultra pure water, and 5 μl of extracted DNA. The PCR thermocycling program consisted of either (i) 12 two-step cycles of 1 minute at 95 °C and 2 minutes at 72 °C, then 29 cycles of 1 minute at 96 °C, 1 minute at 65 °C and 1 minute at 72 °C, or (ii) an initial step of 10 minutes incubation at 30°C (UNG), followed by 10 minutes at 95°C, 49 cycles of 1 minute at 95°C, 1 minute at 65°C and 1 minute at 72°C, and a final step of 10 minutes incubation at 72°C.
Table 1. Mycobacterium/Nocardia genera-specific forward and reverse primers.
Figure imgf000055_0001
Figure imgf000056_0001
2 S can be G or C 3 R can be G or A
Gel electrophoresis confirmed that the Mycobacterium/Nocardia genera-specific secAl target sequence was consistently amplified from multiple species of Mycobacterium and Nocardia (Tables 2 and 3). These results indicate that the Mycobacterium/Nocardia genera-specific secAl primers were capable of Mycobacterium and Nocardia genera-wide amplification of the target sequence.
Table 2. Detection of Mycobacterium/Nocardia genera-specific secAl amplicons by gel electrophoresis I
Figure imgf000056_0002
Table 3. Detection of Mycobacterium/Nocardia genera-specific secAl amplicons by gel electrophoresis II
Figure imgf000057_0001
The amplicons were then purified using the Microcon-100 Microconcentrator columns and eluted in 100 μl TE. The products showing strong gel bands were diluted 1 : 10 in ultra pure water. Clinical samples were then screened to detect the presence of Mycobacterium. Mycobacterium genus-specific primer sets used in these experiments included: (primer set 1) Mtu.For.l (SEQ ID NO: 8) and Mtu.Rev.2 (SEQ ID NO: 10), (primer set 2) Mtu.For.l (SEQ ID NO: 8) and Mtu.Rev.3 (SEQ ID NO: 11), and (primer set 3) Mtu.For.l. edit (SEQ ID NO: 9) and Myco.Rev.490 (SEQ ID NO: 24). The results are presented in Table 4. Positive PCR results by gel electrophoresis conelated with the presence of acid-fast bacilli in the sample, indicating that the identification of Mycobacterium in a sample, using secA genus-specific primers, was consistent and specific for Mycobacterium. This method was specific since no amplification of the secAl region was observed when negative respiratory samples or when a control (human genomic DNA) were used. In some instances, the amplicon was sequenced to confirm that it was from the conesponding species of Mycobacterium present in the sample.
Table 4. Detection of Mycobacterium in patient samples
Figure imgf000058_0001
AFB acid-fast bacilli QNS quantity not specified
Example 2 Sequencing of Mycobacterium/Nocardia secAl Amplicons Once the secAl amplicons were generated, they were sequenced in order to identify Mycobacterium and Nocardia species-specific differences in the secAl sequence. One forward and one reverse sequencing reaction were prepared for each product in a 0.2 μl PCR tube. These reactions contained ABI Big Dye mix (Applied Biosystems Inc., Foster City, CA), IX sequencing buffer, 3.2 pmoles of forward or reverse M13 sequencing primers, ultra pure water, and 1 to 2 μl purified PCR product. The thermocychng parameters for cycle sequencing consisted of 25 cycles of 10 seconds at 96 °C, 5 seconds at 50 °C, and 4 minutes at 60 °C.
Sequence analysis Upon completion of cycle sequencing, excess dye terminators were removed using the Clean Seq Reaction Clean-Up Method, which contains a magnetic particle solution to bind sequencing extension products. The sequences were analyzed using the 3100 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA). The DNASTAR Lasergene software was used to assemble each individual secAl DNA sequence, and then align all the sequences produced to compare differences and similarities between the various species of Mycobacterium (FIG. 1) and Nocardia (not shown). The Mycobacterium nucleic acid sequences were then translated in the corresponding amino acid sequence (FIG. 2).
Example 3 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid Sequence and Detection of a Mycobacterium in a Sample This example shows how Mycobacterium genus-specific probes can be used to detect a Mycobacterium secAl amplicon.
DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfuls of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at
10,500 x g for 5 minutes. The supernatant was removed and the pellet was washed in Tris-HCL, pH 8.0. The suspension was centrifuged at 10,500 x g for 5 minutes, resuspended in ultra-pure water, and vortexed for 10 minutes. The tube was then heated at 95 °C for 25 minutes. The extracted DNA was stored at -15 to -25 °C until ready to use.
PCR Amplification The Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene (see Table 1). For example, in one experiment genera-specific primers were designed based on the fact that the target sequence for amplification is a region within the Mycobacterium tuberculosis secAl gene (located between base pair 150019 and base pair 152868 in the Mycobacterium tuberculosis genome; Genbank Accession No. BX842582). More specifically, primers were designed against a target sequence located between base pair 151730 and base pair 152428 of the Mycobacterium tuberculosis genome in order to generate an amplified sequence approximately 700 base pairs in length, excluding the primers. In other experiments, the target sequence of the secAl gene conesponded to a sequence encoding a region between amino acid positions 138 and 391 of the secAl protein, which included the substrate specificity domain (located between amino acid positions 222 and 359 of the secAl protein). In yet other experiments, the primers amplified a target sequence approximately 500 base pairs in length. In one experiment, real-time PCR amplification was performed in a 25 μl volume containing 2.5 mM MgCl2, IX MGB Eclipse Probes (Epoch Biosciences, Bothell, WA; SEQ ID NO: 7; Table 5) that will only hybridize to a Mycobacterium genus-specific secAl nucleic acid sequence, 1 pmol of Mtu.For.l forward primer (SEQ ID NO: 8; Table 1), 1 pmol of Mtu.rev.3 reverse primer (SEQ ID NO: 11; Table 1), 1 Unit UNG, ultra pure water, and 5 μl of extracted DNA (Table 6). In a second experiment (Table 7), the primer set included Mtii.For.l.edit (SEQ ID NO: 9) and Mtu.Rev.3 (SEQ ID NO: 11).
Table 5. Mycobacterium genus-specific MGB probes
Figure imgf000060_0001
The PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 8 minutes at 95 °C, 50 cycles of 10 seconds at 95 °C, 20 seconds at 56 °C and 30 seconds at 76 °C. The results in Tables 6 and 7 demonstrate that the Mycobacterium secA genus-specific MGB probe consistently detected the amplification product, regardless of the Mycobacterium species from which it was amplified. Thus, the genus-specific MGB probes were capable of detecting the presence of various species of Mycobacterium.
Table 6. Mycobacterium genus-wide detection with genus-specific MGB probes.
Figure imgf000060_0002
Figure imgf000061_0002
Table 7. Mycobacterium detection with different genus-specific probes
Figure imgf000061_0003
Figure imgf000061_0001
♦Detection at 60 °C
♦♦Detection at 65 °C
Mycobacteuum/Nocardia genera-specific amplicons, from samples containing different species of Mycobacterium, were generated usmg the Mycobacterium/Nocardia genera-specific pnmer pair Mtu.Forl.Edit (SEQ ID NO: 9; Table 1) and Mtu.Rev490 (SEQ ID NO: 24; Table 1) and then detected with a mix of FRETProbes (see Table 7, footnotes). Table 8. Mycobacterium and Nocardia FRETProbes.
Figure imgf000062_0002
Figure imgf000062_0001
The PCR thermocycling program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C. The results in Table 7 demonstrate that the Mycobacterium secA FRETProbe mix consistently detected the amplification product, regardless of the Mycobacterium species from which it was amplified. Thus, the Mycobacterium FRETProbes were also capable of detecting tlie presence of various species of Mycobacterium.
Example 4 Amplification of a Mycobacterium/Nocardia Genera-Specific secAl Nucleic Acid Sequence in a Sample and Identification of a Mycobacterium species This example demonstrates how a Mycobacterium/Nocardia genera-specific nucleic acid sequence encoding a fragment of the secAl protein can be amplified and then used to identify the Mycobacterium species present in the sample.
DNA extraction Bacterial samples were grown on Middlebrook 7H11 agar and Dubos Tween Albumin broth for 10 to 21 days. The DNA of the bacteria grown on the agar and broth was extracted as follows using the alkaline wash and heat lysis method: Two small loopfiils of organism were suspended in 1.0 ml of alkaline wash solution (0.5 M NaOH, 0.05 M sodium citrate), and vortexed for 10 minutes. The cell lysate was centrifuged at 10,500 x g for 5 minutes. The supernatant was removed and the pellet was washed in Tris-HCL, pH 8.0. The suspension was centrifuged at 10,500 x g for 5 minutes, resuspended in ultra-pure water, and vortexed for 10 minutes. The tube was then heated at 95 °C for 25 minutes. The extracted DNA was stored at -15 to -25 °C until ready to use. PCR Amplification The Mycobacterium/Nocardia genera-specific secAl PCR primers (such as those listed in Table 1) were selected from a conserved region of the secAl gene to produce an amplified sequence approximately 700 base pairs in length, excluding the primers. The real-time PCR amplification was performed in a 25 μl volume containing 2.5 mM MgCl2, IX LightCycler-Fast Start DNA Hybridization Probes (a pair of FRETProbes, such as those listed in Table 9, including one FITC-labeled "up" probe and one Red640-labeled "down" probe; Roche; Mannheim, Germany) specific for the Mycobacterium species of interest, 1 pmol of Mtu.For.l forward primer (SEQ ID NO: 8), 1 pmol of Mtu.rev.3 reverse primer (SEQ ID NO: 11), 1 Unit UNG, ultra pure water, and 5 μl of extracted DNA. Other pairs of Mycobacterium/Nocardia genera-specific forward and reverse primers, for example pairs derived from the primers listed in Table 1, above, can be used to generate Mycobacterium secAl amplicons. The PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C.
Table 9. Mycobacterium species-specific FRETprobes.
Figure imgf000063_0001
Figure imgf000064_0001
The numbers in each probe name (for example 85-63 in M.abscessusScAFRET85-63) represent the nucleotide position within the amplified target nucleic acid sequence that is complementary to the probe sequence.
A positive signal using the species-specific real-time PCR probes indicated the presence of a secAl nucleic acid sequence from the conesponding Mycobacterium species.
Example 5 Amplification of a Shorter Mycobacterium Nocardia Genera-Specific secAl Nucleic Acid and Identification of a Mycobacterium species in a Sample This example demonstrates how a shorter nucleic acid sequence encoding the genus-specific fragment of the secAl protein from Mycobacterium can be amplified from a sample, in order to identify the presence of a Mycobacterium in the sample. Once a Mycobacterium is detected, the amplified nucleic acids can be sequenced to identify the specific Mycobacterium species present. DNA extraction, PCR amplification and sequencing were performed essentially as described in Examples 1 and 2, above. The Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene to produce a sequence of approximately 513 base pairs (470 base pairs, excluding the primers). The genus-specific secAl primers were tailed with M13 sequencing primers and include the Mtu.For.l.editM13 primer (5'- GTAAAACGACGGCCAGGACAGYGAGTGGATGGGYCGSGT 3'; SEQ ID NO: 15) and the Myco.Rev490-467M13 primer (5'CAGGAAACAGCTATGACGCGGACGATGTARTCCTTGTCSCG-3'; SEQ ID NO: 25; Y can be T or C, S can be G or C, and R can be G or A). Once it was determined that this set of generic primers could specifically amplify the target sequence from various mycobacteria, real-time PCR amplification was performed in a 25 μl volume containing 2.5 mM MgCl2, IX LightCycler-Fast Start DNA Hybridization Probes (Roche; Mannheim, Germany) specific for the Mycobacterium species of interest (a pair of species-specific primers including one FITC-labeled "up" primer and one Red640-labeled "down" primer; see Table 9), 1 pmol of forward primer (SEQ ID NO: 9), 1 pmol of reverse primer (SEQ ID NO: 24), 1 Unit UNG, ultra pure water, and 5 μl of extracted DNA. The PCR thermocycling program consisted of 10 minutes at 30 °C (UNG digestion), 10 minutes at 95 °C, 12 two-step cycles of 10 seconds at 95 °C and 30 seconds at 72 °C, then 29 cycles of 10 seconds at 95 °C, 20 seconds at 60 °C and 30 seconds at 72 °C, with a final hold of 10 minutes at 72 °C. In some experiments, 65 °C was used instead of 60 °C. The results are demonstrated in Table 10. If the baseline (real-time PCR) data were not conclusive as to which species of Mycobacterium was present in the sample, melting curves were generated, according to LightCycler instructions, in order to identify the species in the sample.
Figure imgf000065_0001
Figure imgf000066_0001
Example 6 Identification of Mycobacterium Species in a Sample Using Mycobacterium Species-Specific secA Minor Groove Binder (MGB) Probes This example demonstrates how species-specific secAl MGB probes can be used to identify a Mycobacterium species in a sample. DNA was extracted from cultures of various species of Mycobacterium and a Mycobacterium genus-specific region of the secAl gene was amplified essentially as described in Example 3, above. The Mycobacterium/Nocardia genera-specific secAl PCR primers were selected from a conserved region of the secAl gene to produce an amplified sequence either 761 base pairs or 638 base pairs in length. The real-time PCR amplification was perfoπned in a 25 μl volume containing 2.5 mM MgCl2, IX MGB Eclipse Probes (Epoch Biosciences, Bothell, WA) specific for the Mycobacterium species of interest (Table 9), 1 pmol of forward primer (SEQ ID NO: 8 for the 761 base pair product or SEQ ID NO: 9 for the 638 base pair product; see Table 1), 1 pmol of reverse primer (SEQ ID NO: 11 for the 761 base pair product or SEQ ID NO: 12 for the 638 base pair product; see Table 1), 1 Unit UNG, ultra pure water, and 5 μl of extracted DNA.
Table 11. Mycobacterium species-specific MGB probes
Figure imgf000066_0002
Figure imgf000067_0001
The PCR thermocychng program consisted of 10 minutes at 30 °C (UNG digestion), 8 minutes at 95 °C, 50 cycles of 10 seconds at 95 °C, 20 seconds at 56 °C and 30 seconds at 76 °C. A positive signal during the real-time amplification procedure indicated the presence of the conesponding Mycobacterium species in the sample (Table 12).
Table 12. Identification oi Mycobacterium Species in a Sample Using Mycobacterium Species- Specific secA Minor Groove Binder (MGB) Probes
Figure imgf000067_0002
Example 7 Detection and Identification of Mycobacterium and Nocardia by secAl Sequences This example demonstrates how nucleic acids encoding the secAl protein from bacteria of the genus Mycobacterium and Nocardia can be specifically amplified from a clinical sample and then sequenced in order to identify the particular Mycobacterium and Nocardia species present in the sample.
Mycobacterial reference strains and clinical isolates The 47 reference strains and 59 clinical isolates used in this Example are listed in Table 13. Conventional identification of clinical isolates included an initial assessment of pigment production, growth rate and colony characteristics. Slowly growing mycobacteria were identified using the AccuProbe assays (Gen-Probe, San Diego, CA), which are specific for M avium complex (MAC), M avium, M. intracellulare, M. tuberculosis (MTB) complex, M gordonae, and M kansasii. Additional biochemical testing of MTB complex isolates included niacin and nitrate tests. AccuProbe-negative slowly growing isolates were identified by 16S rDNA sequencing. Rapidly growing isolates were identified by hsp65 PRA (Telenti et al., J. Clin. Microbiol., 31:175-178, 1993), and 16S rDNA sequencing when necessary.
Table 13. List of mycobacterial reference strains and clinical isolates Species Reference strains No. of clinical isolate: and source M abscessus ATCC 19977 (T) 6 M. africanum ATCC 25420 (T) ATCC 35711 M. asiaticum ATCC 25276 (T) 1 M. avium ATCC 25291 (T) 5 M. bovis ATCC 19210 (T) ATCC 35734 M. celatum ATCC 51131 (T) ATCC 51130 M. chelonae ATCC 35752 (T) 2 M.flavescens ATCC 14474 (T) ATCC 23008 ATCC 23033 M. fortuitum ATCC 6841 (T) 5 M. gastri ATCC 15754 (T) ATCC 25157 M. gordonae ATCC 14470 (T) 6 M. haemophilum ATCC 29548 (T) ATCC 33206 M. intracellulare ATCC 13950 (T) 6 M. kansasii ATCC 12478 (T) 6 M. leprae AL583919.1 M. malmoense ATCC 29571 (T) 1 M. marinum ATCC 927 (T) 5 M. mucogenicum ATCC 49650 (T) 5 M. nonchromogenicum ATCC 19530 (T) ATCC 25142 M. peregrinum ATCC 14467 (T) 1 M. scrofulaceum ATCC 19981 (T) 1 M. shimoidei ATCC 27962 (T) ATCC 49773 M. simiae ATCC 25275 (T) 1 M. smegmatis ATCC 19420 (T) ATCC 14468 M. szulgai ATCC 35799 (T) 1 M. terrae ATCC 15755 (T) ATCC 25267 ATCC 25268 M. triviale ATCC 23292 (T) ATCC 23290 ATCC 23291 M. tuberculosis ATCC 27294 (T) 6 ATCC 25177 M. ulcerans ATCC 19423 (T) ATCC 25900 ATCC 25897 ATCC 35839 M. xenopi ATCC 19250 (T) 1 ATCC = American Type Culture Collection, T = type strain
Non-mycobacterial reference strains Seven American Type Culture Collection (ATCC) non-mycobacterial strains were also included in this study: Gordonia bronchialis [ATCC 25592 (T)], Gordonia terrae [ATCC 25594 (T)], Nocardia brasiliensis [ATCC 19296 (T)], Nocardia farcinica [ATCC 3318 (T)], Rhodococcus equi [ATCC 6939 (T)], Rhodococcus rhodochrous [ATCC 13808 (T)}, and Tsukamurella paurometabola [ATCC 8368 (T)].
DNA extraction Mycobacterial cells were disrupted by an alkaline wash and heat lysis method (Baele et al, J. Clin. Microbiol. 39:1436-1442, 2001), with a few modifications: one small loopful of organisms grown on Middlebrook 7H11 agar (Remel, Lenexa, KS) for 7 to 21 days was resuspended in 20 μl of lysis buffer [(0.25% sodium dodecyl sulfate (QB Inc, Gaithersburg, MD), 0.05 N NaOH (Sigma, St. Louis, MO)], mixed by vortexing and heated at 95°C for 5 minutes. After the addition of 180 μl of water and mixing by vortexing, samples were incubated at 95°C for 25 minutes. Samples were then spun at 16,000 x g for 5 minutes to remove cell debris. Nucleic acid concentration in the supernatant was determined spectrophotometrically (A260). DNA extraction from the non-mycobacterial reference strains was performed by a phenol-chloroform-isoamyl alcohol method as described elsewhere (Conville et al., J. Clin. Microbiol, 38:158-164, 2000).
Primer design and PCR Two conserved regions were identified after aligning secAl sequences from five Mycobacterium species, which were available in the NCBI GenBank database at the time this study was initiated. These regions were used to design two primers (Mtu.For.l; SEQ ID NO: 8 and Mtu.rev.3; SEQ ID NO: 11), which correspond to tuberculosis secAl gene positions 412-440, and 1141-1172, respectively [GenBank accession no. BX842582.1, nucleotides 150019-152868]. A schematic representation of the primer design is shown in FIG. 3. Mtu.For.l (5'GAC AGY GAG TGG ATG GGY CGS GTG CAC CG-3 ', and Mtu.Reverse3 (5 'ACC ACG CCC AGC TTG TAG
ATC TCG TGC AGC TC-3'), were commercially synthesized (Midland Certified Reagent Company, Midland, TX). Primers used for sequencing of the secAl gene regions were tailed with M13 sequencing primer sites: M13 forward tail (5'GTA AAA CGA CGG CCA G-3'), M13 reverse tail (5'CAG GAA ACA GC TAT GAC-3'). The PCR amplification using Mtu.For.l (M13 tailed) and Mtu.Rev.3 (M13 tailed) generated a product of 700 base pahs (excluding the primers, FIG. 3). PCR reactions were performed in a Perkin-Elmer 9600 Thermocycler with a reaction mix containing 2.5 mM MgC12, IX LightCycler-Fast Start DNA Hybridization Probes (Roche, Mannheim, Germany), 1 pmol of forward primer, 1 pmol of reverse primer, 1 Unit of Uracil TV-glycosylase (UNG) enzyme (Roche), 5 μl of extracted DNA and ultra pure water to a final volume of 25 μl. The PCR thermocycling program consisted of an initial step of 10 minutes incubation at 30°C (UNG), followed by 10 minutes at 95°C, 49 cycles of 1 minute at 95°C, 1 minute at 65°C and 1 minute at 72°C, and a final step of 10 minutes incubation at 72°C. A negative control of ultra pure water was included with every amplification reaction. DNA samples of selected strains were also amplified and sequenced for the 16S rDNA gene using the MicroSeq Full Gene 16S rRNA Bacterial Isolation Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.
PCR product detection and purification PCR products were visualized by UV illumination of an ethidium bromide-stained 2% agarose gel following electrophoresis. The purification of the remaining PCR product was achieved with Microcon-100 microconcentrators (Millipore, Bedford, MA), following the manufacturer's instructions.
DNA sequencing The ABI Prism BigDye Terminator v 1.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used for the sequencing of the PCR product. The sequencing reaction contained 4 μl of Big Dye premix, 0.5X buffer, 3.2 pmol of 10 sequencing primer, and approximately 150 ng of PCR product template in a total volume of 20 μl. The following M13 primers were used for sequencing: M13 Forward, 5'GTA AAA CGA CGG CCA G-3 ' (SEQ ID NO: 229) and M13 Reverse, 5'CAG GAA ACA GCT ATG AC-3 (SEQ ID NO: 230). The sequencing reaction and template preparation were performed in accordance with the instructions of the manufacturer. Sequencing products were purified with CleanSEQ Sequencing Reaction Clean- Up system (Agencourt, Beverly, MA) and analyzed using the 3100 Genetic Analyzer (Applied Biosystems), following the manufacturer's instructions.
Sequence and phylo genetic analysis The Lasergene program (version 5.51; DNASTAR, Inc. Madison, WI) was used for sequence assembly and alignment. Multiple sequence alignment of sec A 1 700 base pair sequences was done by the CLUSTAL W method (Thompson et al., Nucleic Acids Res. 22:4673-4680, 1994). Phylogenetic analyses were performed with the PHYLIP version 3.5c package (Felsenstein, 1993). Distance matrices based on Kimura's two-parameter model (Kimura et al, J. Mol. Evol, 16:111-120, 1980) were produced with the DNADIST program, and a neighbor-joining tree constructed with the NEIGHBOR program. The resulting trees were depicted using the TreeView version 1.4 package (Page, Comput. App. Biosci. 12:357-358, 1996). The stability of the grouping was assessed by the bootstrap method using SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs. A total of 1,000 bootstrapped trees were generated. Assembled 16S rDNA sequences from selected isolates were compared with 16S rDNA sequences available in GenBank databases using the standard nucleotide-nucleotide Basic Local Alignment Search Tool (Blast) program (National Center for biotechnology Information, Bethesda, MD). The isolate was identified as most closely related to the reference species if its 16S rDNA sequence demonstrated the highest relatedness and had over 98.5% identical bases compared to the respective reference sequence, based on the data from a previous study (Stackebrandt and Goebe, Int. J. Syst. Bacteriol, 44:846-849, 1994).
Nucleotide sequence accession numbers Partial sequences of mycobacterial and nocardial secAl were deposited in GenBank under accession numbers AY724701 to AY724734, and AY781799 to AY781800, respectively. 16S rDNA sequences were deposited in GenBank under accession numbers AY734991 to AY734996.
SecAl partial sequences of the mycobacterial type strains A 700 base pair section of secAl was amplified from 29 Mycobacterium type strains, and the nucleotide sequences were determined and compared pairwise. No insertions or deletions were detected. A range of 83.3 to 100% interspecies similarity was observed (FIG. 4). Sequence variability occurred throughout the 700 base pair region targeted. However, a particularly hypervariable region was observed at the beginning of the 700 base pair region, between nucleotides 7-70. The members of the MTB complex (M tuberculosis, M. africanum, and M bovis) had identical sequences. Members of the following closely related species could be differentiated: M gastri and M kansasii (96.9% similarity), M abscessus andM chelonae (91.7% similarity), M marinum and M ulcerans (98.1%). No amplification of the secAl gene was ever observed with the negative control, which was included with every amplification reaction.
Amino acid sequences of the mycobacterial type strains The deduced amino acid sequences of the amplified 700 base pair fragment of secAl comprised 233 amino acid residues (F148 to A375, M tuberculosis numbering, protein Accession no. CAE55574.1). Each type strain examined displayed unique amino acid sequences in this region, except for the members of the MTB complex, which shared the same protein sequence. Interspecies similarity at the amino acid level ranged from 87.6% to 100% (100% for the members of the MTB complex). High variability was observed in the first half of the 233 deduced amino acid sequence, with three particularly hypervariable regions between residues 5-23, 46-57, and 84-118. The following pairs of closely related species could also be differentiated from one another at the protein level: M gastri and kansasii (99.1% similarity), M abscessus andM chelonae (96.6% similarity), M marinum andM ulcerans (98.3% similarity).
Evaluation of the SecAl identification procedure on non-mycobacterial reference strains In addition to amplifying and sequencing the secAl gene from mycobacteria, seven non- mycobacterial isolates in genera considered related to the genus Mycobacterium were tested. No amplification of secAl was observed with the type strains of Gordonia bronchialis, Gordonia terrae, Rhodococcus equi, Rhodococcus rhodochrous, and Tsukamurella paurometabola. In contrast, amplification of a 700 base pair region of secAl was observed with the type strains of Nocardia brasiliensis and Nocardia farcinica. Sequencing of the amplified product allowed a clear differentiation of the Nocardia isolates from each other and from all Mycobacterium species strains tested. The ranges of similarity between TV. brasiliensis and the type strains of Mycobacterium species were 82.6-87.6% and 82.4-88.8%, for nucleotide and amino acid sequences respectively. For TV. farcinica these values were 82.0-89.4 and 83.7-89.7, respectively.
Phylogenetic tree A phylogenetic tree of 34 sequenced mycobacterial reference strains, as well as M leprae, and two sequenced nocardial reference strains, was constructed by the neighbor-joining method, using Corynebacterium efficiens as the outgroup (FIG. 5). All mycobacterial species studied showed good separation. Within the consensus tree, four major clusters of mycobacteria could be defined. Most of the slow growing species grouped together in the first cluster. The rapid growers M flavescens, M. fortuitum, M mucogenicum, M. peregrinum, andM smegmatis grouped together in the second cluster. Interestingly, M. triviale also appeared in that group. A third cluster included the closely related species M terrae and M nonchromogenicum. Finally, a long separate branch included M abscessus and M chelonae. The members of the M tuberculosis complex were distantly separated from all the other species. Slowly growing pathogenic M kansasii and nonpathogenic M gastri could be easily distinguished, although they cannot be separated by 16S rDNA. Two other pairs of species closely related by 16S rDNA sequences, M abscessus - M. chelonae, andM marinum - M. ulcerans, were separated as well. TV. brasiliensis and TV. farcinica clustered together as an independent branch, which was clearly separated from all the mycobacteria. In addition, the two Nocardia species could be easily differentiated from one another.
Correlation of additional mycobacterial isolates with the type strains In addition to the type strains, the 700 base pair fragment of secAl was sequenced from one or more reference strains or well characterized clinical isolates (Table 13). Five or six clinical isolates were obtained and sequenced from each of 9 commonly isolated species of mycobacteria (Table 13 and separately in Table 14). Table 14. Range of similarity to the type strain of 50 clinical isolates from nine commonly isolated Mycobacterium species
Species Number of Range of % Range of % clinical similarity similarity isolates ΓDNAI (amino acid)
M abscessus 6 100 100
M. avium 5 98.4-99.1 99.6-100
M. fortuitum 5 99.0-99.9 100 M. gordonae 6 96.0-99.0 99.6-100 M. intracellulare 6 99.4-100 100
M. kansasii 6 97.3-100 99.6-100
M. marinum 5 99.1-100 99.6-100
M. mucogenicum 5 99.4-100 100 M. tuberculosis 6 100 100 ,
For species of mycobacteria listed in Table 13 only (not in Table 14), the level of similarity with respective type strains was typically higher than 99%. All isolates were correctly identified by the secAl gene, and also clustered together with their respective type strains in the phylogenetic tree (not shown), with the exception of two reference strains of M. flavescens (ATCC 23008 and 23033) that appeared more closely related to M smegmatis (FIG. 5). These two M flavescens reference strains are more closely related to each other (96.3% similarity) than to the type strain (91.0, 90.4%) according to their secAl gene sequences (FIGS. 4 and 5). In order to compare the results obtained by secAl with 16S rDNA sequences, sequencing of the full 16S rDNA gene on the three M flavescens reference strains used in this study was performed. 16S rDNA sequencing agreed with the sequences appearing in GenBank for those isolates. The percentage of similarity among the three species was lower than one would expect for 16S rDNA sequences of isolates of the same species (range 98.1- 98.4%). Similarly, although all three reference strains of M triviale grouped together in a separate branch in the phylogenetic tree (FIG. 5), secAl sequences of the M triviale ATCC 23290 and 23291 isolates were almost identical to each other (99.9% similarity), but quite divergent from that of the type strain (91.9 and 91.7% similarity, respectively). Interestingly, 16S rDNA sequences confirmed these findings, with 99.9% similarity between M triviale ATCC 23290 and 23291, but only 98.3% similarity of each of them with the type strain. To evaluate the performance of this method for the identification of mycobacteria, 50 clinical isolates were obtained and sequenced from 9 species of Mycobacterium (Table 14). These organisms were selected as representative of the most commonly isolated clinical species. In general, few nucleotide differences were observed between the clinical isolates of a given species and the corresponding type strain. The intraspecies percentage of similarity at the DNA level was very high (usually >99%, range 96.0%-100%). Comparison of the deduced amino acid sequences among isolates of the same species showed between 99.6 to 100% similarity for all species tested. M gordonae showed the highest degree of intraspecies variability, with percentages of similarity at the DNA level ranging from 96.0 to 99.0, however, all six clinical isolates of M gordonae tested matched most closely with the type strain. Four clinical isolates of M kansasii showed 100% similarity to the type strain, while two other isolates had identical sequences showing 97.3% similarity to the type strain. However, all six clinical isolates of M kansasii were conectly identified.
Example 8 Identification of Nocardia species Using Sequence Analysis of a Portion of the secA Gene This example demonstrates how nucleic acids encoding the secAl protein from bacteria of the genus Nocardia can be specifically amplified from clinical samples and then sequenced in order to identify Nocardia isolates to the species level. SecAl is a key component of the major pathway of protein secretion across the cytoplasmic membrane. A 520-base pair region of the secAl gene of various type or reference strains of pathogenic Nocardia species was amplified using Mycobacterium/Nocardia genera-specific primers (Mtu.For.l.editM13 primer and MycoRev490-467M13 primer; see Table 1). Gel electrophoresis confirmed that the Mycobacterium/Nocardia genera-specific secAl target sequence was consistently amplified from multiple species of Nocardia (Table 15). These results indicate that the Mycobacterium/Nocardia genera-specific primers were capable of Nocardia genus-wide amplification of the target sequence. Sequence analysis of 434 base pairs allowed clear differentiation of all Nocardia species, with a range of interspecies similarity of 85.3% to 99.1%. Corresponding 16S rDNA sequences for the same isolates showed a range of similarity of 95.2% to 99.8%. The higher interspecies sequence variability seen with the secAl gene allows finer discrimination of closely related species, such as members of the "TV. nova complex."
Figure imgf000074_0001
Figure imgf000075_0001
In addition to the type and reference strains, a 434-base pair fragment of the secAl gene was sequenced from 15 clinical isolates of Nocardia species and showed a range of 99.1 to 100% similarity to the secAl sequences of the type or reference strain to which their identification corresponded. Comparison of the deduced 145 amino acid sequences of the SecAl proteins showed between 99.3 and 100% similarity to the conesponding type or reference strain. Sequencing the secAl gene, and using deduced amino acid sequences of the secAl protein, provides a more discriminative method for identification of Nocardia isolates than 16S rDNA sequencing. Example 9 Identification oi Mycobacterium Species in a Sample Using Mycobacterium Species-Specific secAl Primers This example demonstrates how nucleic acids encoding the secAl protein of a Mycobacterium species can be specifically amplified and identified. DNA is extracted from cultures of various species of Mycobacterium and is amplified essentially as described in Example 1, above, with the exception that Mycobacterium species-specific secAl PCR primers are used to amplify the target nucleic acid sequence. The Mycobacterium species-specific secAl PCR primers are selected from unique regions (species-specific) of the Mycobacterium secAl gene. The species-specific secAl primers are all reverse primers (Table 16) and were designed to be used with the Mtu.For.ledit forward primer (SEQ ID NO: 9; Table 1).
Table 16. Mycobacterium species-specific primers
Figure imgf000075_0002
Figure imgf000076_0001
The numbers in each probe name (for example 358-337 in M.abscessScAprim358-337) represent the nucleotide position within the amplified target nucleic acid sequence that is complementary to the probe sequence.
Real-time PCR amplification is performed as described in Example 3. A positive signal due to the hybridization of a species-specific probe (for example, the species specific probes listed in Table 9 or Table 11) indicates the presence of the conesponding Mycobacterium species in the sample. Once the secAl amplicons are detected with the Mycobacterium species-specific probes, the secAl amplicons are sequenced in order to confirm the origin of the Mycobacterium secAl sequence. Sequencing reactions are performed essentially as described in Example 2. Example 10 Isolation oi Mycobacterium and Nocardia Genera from a Clinical Sample and the Subsequent Identification of Particular Mycobacterium and Nocardia Species in the Sample This example demonstrates how nucleic acids encoding the secAl protein from bacteria of Mycobacterium and Nocardia genera can be specifically amplified from a clinical sample and then sequenced in order to identify the particular Mycobacterium or Nocardia species present in the sample. DNA is extracted from clinical samples, a Mycobacterium/Nocardia genera-specific secAl nucleic acid sequence is amplified, and Mycobacterium and Nocardia species-specific probes are used to identify the origin of the secAl nucleic acid (see the protocol described in Examples 3 or 4). A positive signal due to the hybridization of a species-specific probe indicates the presence of the conesponding Mycobacterium or Nocardia species in the sample. Once Mycobacterium or Nocardia species-specific secA amplicons are detected in the sample, the amplicons are sequenced to confirm the origin of the amplified secA nucleic acid generated from the sample. Sequencing reactions are performed as described in Example 2.
This disclosure provides methods of detecting the presence of Mycobacterium and Nocardia in a sample using secA nucleic acid sequences that are conserved in Mycobacterium and Nocardia. The disclosure also provides methods of identifying a Mycobacterium or a Nocardia species involved in infection of a subject. The disclosure further provides Mycobacterium and Nocardia species-specific secAl nucleic acid sequences. It will be apparent that the precise details of the methods and sequences described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim: 1. A method for detecting a Mycobacterium or Nocardia in a sample, comprising: amplifying a Mycobacterium/Nocardia genera-specific secAl nucleic acid from the sample utilizing at least two Mycobacterium/Nocardia genera-specific primers that bind within a region of a nucleic acid sequence encoding a secAl protein that is conserved among members of the Mycobacterium and Nocardia genera, and wherein the conserved region is in the 5' half of a secAl gene and includes a substrate specificity domain, thereby detecting a Mycobacterium or Nocardia in the sample.
2. The method of claim 1, wherein detecting comprises hybridizing the amplified genera-specific secAl nucleic acid to a secAl probe oligonucleotide.
3. A method of distinguishing between a Mycobacterium or Nocardia species in the sample using the method of claim 2, wherein the secAl probe oligonucleotide is Mycobacterium species-specific or Nocardia species-specific.
4. The method of claim 1, wherein the amplified genera-specific secAl nucleic acid is at least 400 base pairs in length.
5. The method of claim 1, wherein the genera-specific secAl nucleic acid is amplified by real-time PCR.
6. The method of claim 1, wherein one of tlie genera-specific primers comprises a sequence as set forth as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.
7. The method of claim 1, wherein the sample is from a subject.
8. The method of claim 5, wherein the subject is a human.
9. The method of claim 3, wherein the species-specific probe oligonucleotide is fluorescently-labeled and detecting hybridization comprises measuring an intensity of emission of fluorescence in the sample.
10. The method of claim 3, wherein the Mycobacterium species-specific probe oligonucleotide has a sequence as set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, or SEQ ID NO: 206.
11. The method of claim 3 , wherein the Nocardia species-specific probe oligonucleotide has a sequence as set forth as SEQ ID NO: 228.
12. The method of claim 2, wherein the probe oligonucleotide further comprises a • minor groove binder.
13. The method of claim 1 , wherein the amplified genera-specific secAl nucleic acid is a radiolabeled, a fluorescently-labeled, a biotin-labeled, an enzymatically-labeled, or a chemically- labeled nucleic acid.
14. The method of claim 3, wherein the Mycobacterium or the Nocardia species- specific secAl probe oligonucleotide is attached to a polymeric solid support in a predetermined array pattern and wherein hybridization of the amplified genera-specific secAl nucleic acid to the species-specific probe oligonucleotide attached to the solid support forms a hybridization pattern of the sample.
15. The method of claim 14, further comprising comparing the hybridization pattern of the sample to a control hybridization pattern of Mycobacterium or Nocardia species.
16. The method of claim 3, wherein hybridizing the amplified genera-specific secAl nucleic acids to a Mycobacterium or Nocardia species-specific secAl probe oligonucleotide comprises: transferring the amplified genera-specific secA nucleic acid to a polymeric solid support; and contacting the genera-specific secA nucleic acid transfened to the polymeric solid support with the Mycobacterium or Nocardia species-specific secAl probe oligonucleotide.
17. The method of claim 16, wherein the Mycobacterium species-specific probe oligonucleotide has a sequence as set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, or SEQ ID NO: 206.
18. The method of claim 16, wherein the Nocardia species-specific probe oligonucleotide has a sequence as set forth as SEQ ID NO: 228.
19. The method of claim 16, wherein the species-specific probe oligonucleotide is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled.
20. A method of distinguishing between the Mycobacterium or Nocardia species in the sample using the method of claim 1, wherein detecting comprises sequencing the amplified genera- specific secAl nucleic acid.
21. A method for identifying a species of Mycobacterium in a sample, comprising: detecting an amplified Mycobacterium species-specific secAl nucleic acid, wherein the amplified Mycobacterium species-specific secAl nucleic acid is amplified from the sample utilizing at least two Mycobacterium species-specific primers that bind within a region of the Mycobacterium species specific secAl nucleic acid sequence containing at least one nucleic acid difference, as compared to a secAl nucleic acid sequence present in another species of
Mycobacterium, and wherein the region is in the 5' half of a Mycobacterium secAl gene and includes a substrate specificity domain, thereby identifying a species of Mycobacterium in the sample.
22. The method of claim 21 , wherein the amplified Mycobacterium species-specific secAl nucleic acid is at least about 400 base pairs in length.
23. The method of claim 21 , wherein the Mycobacterium species-specific sec A 1 nucleic acid is amplified by polymerase chain reaction (PCR), real-time PCR, reverse transcriptase- polymerase chain reaction (RT-PCR), ligase chain reaction, or transcription-mediated amplification (TMA).
24. The method of claim 23, wherein the Mycobacterium species-specific secAl nucleic acid is amplified by real-time PCR.
25. The method of claim 21, wherein detecting comprises hybridizing a fluorescently- labeled Mycobacterium species-specific probe to the amplified Mycobacterium genus-specific secAl nucleic acid and measuring an intensity of emission of fluorescence in the sample.
26. The method of claim 25, wherein the Mycobacterium species-specific probe oligonucleotide has a sequence as set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ED NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, or SEQ ID NO: 206.
27. The method of claim 25, wherein the probe further comprises a quencher.
28. The method of claim 21, wherein the primer is a Mycobacterium species-specific secA primer having a sequence as set forth as SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, or SEQ ID NO: 219.
29. The method of claim 21 , wherein the sample is from a subject.
30. The method of claim 29, wherein the subject is a human.
31. The method of claim 29, wherein the sample comprises an abscess sample, blood, a blood fraction, bone manow, a bronchoalveolar lavage sample, a bronchial wash sample, a bronchial brush sample, cerebrospinal fluid, an esophageal sample, a tissue drainage sample, an eye conjunctiva sample, gastric biopsy, a gastric washing sample, a nasopharyngeal swab, saliva, sputum, an induced sputum sample, a stool sample, synovial fluid, a skin lesion sample, a sinus aspirate, a tissue biopsy, a transtracheal aspirate, a surgical specimen, or autopsy material.
32. The method of claim 21, wherein the amplified Mycobacterium species-specific secA nucleic acid is a labeled nucleic acid.
33. The method of claim 32, wherein the labeled nucleic acid comprises a radiolabeled, a fluorescently-labeled, a chemically-labeled, an enzymatically-labeled, or a biotin-labeled nucleic acid.
34. The method of claim 21 , further comprising sequencing the amplified Mycobacterium species-specific secAl nucleic acid.
35. The method of claim 21, wherein detecting comprises hybridizing the amplified Mycobacterium species-specific secAl nucleic acid to at least one Mycobacterium species-specific probe oligonucleotide attached to a polymeric support in a predetermined pattern, and wherein hybridization of the amplified Mycobacterium species-specific secAl nucleic acid to the probe oligonucleotide chemically-linked to the solid support forms a hybridization pattern of the sample.
36. The method of claim 25, wherein the probe oligonucleotide further comprises a minor groove binder.
37. The method of claim 35, further comprising comparing the hybridization pattern of the sample to a control hybridization pattern of Mycobacterium species.
38. A method for identifying a species of Mycobacterium or Nocardia in a sample, comprising: contacting the sample, under hybridizing conditions, to a plurality of probe oligonucleotides attached to a polymeric solid support in an array, wherein each of the probe oligonucleotides is specific to a Mycobacterium or Nocardia species, wherein the Mycobacterium species-specific probe oligonucleotides hybridizes to a Mycobacterium species-specific secAl nucleic acid sequence in the sample and the Nocardia species-specific probe oligonucleotides hybridizes to a Nocardia species-specific secAl nucleic acid sequence in the sample; and detecting the Mycobacterium or Nocardia species-specific secAl nucleic acid hybridization if it occurs, thereby identifying a species of Mycobacterium or Nocardia in the sample.
39. The method of claim 38, further comprising amplifying Mycobacterium or
Nocardia species-specific secA nucleic acids in the sample prior to the hybridization step.
40. A method of treating a subject suspected of being infected with a Mycobacterium or a Nocardia, comprising: distinguishing between a Mycobacterium or a Nocardia species in a sample from the subject using the method of claim 3; and treating the subject for the Mycobacterium or Nocardia species identified in the sample, thereby treating the subject suspected of being infected with a Mycobacterium or a Nocardia.
41. A method of treating a subject suspected of being infected with a Mycobacterium, comprising: identifying a Mycobacterium species in a sample from the subject using the method of claim 21; and treating the subject for the Mycobacterium species identified in the sample, thereby treating the subject suspected of being infected with a Mycobacterium.
42. A device for detecting a Mycobacterium species in a sample, comprising: a solid phase nucleic acid array comprising at least one Mycobacterium species- specific probe oligonucleotide attached to a solid polymeric support surface in a predetermined pattern, wherein the at least one Mycobacterium species-specific probe oligonucleotide comprises one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO 34, SEQ ID NO: 35, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, or SEQ ID NO: 206.
43. The device of claim 42, wherein the solid polymeric support surface is a polypropylene surface.
44. An isolated Mycobacterium polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 220.
45. The isolated Mycobacterium polypeptide of claim 44, consisting essentially of an amino acid sequence as set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 220.
46. An isolated nucleic acid encoding the polypeptide of claim 44.
47. The nucleic acid of claim 46, wherein the nucleic acid comprises a sequence as set forth in SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ED NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 221.
48. The nucleic acid of claim 46, wherein the nucleic acid consists essentially of a sequence as set forth in SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ED NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ED NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ED NO: 188, SEQ ID NO: 189, or SEQ ID NO: 221.
49. A vector comprising the nucleic acid of claim 47.
50. A kit for detecting a Mycobacterium or a Nocardia species in a sample, comprising: a solid phase nucleic acid array comprising a plurality of Mycobacterium or Nocardia species-specific oligonucleotides attached to a solid polymeric support surface in a predetermined pattern; a Mycobacterium genus-specific primer or a Mycobacterium species-specific primer, in separate packaging; a Nocardia genus-specific primer or a Nocardia species-specific primer, in separate packaging; and instructions for using the kit.
51. The kit of claim 50, further comprising an amplification enzyme, in separate packaging.
52. The kit of claim 50, further comprising a buffer solution, in separate packaging.
PCT/US2005/006609 2004-02-27 2005-02-28 Detection and identification of mycobacterium and nocardia using primers and probes directed to the seca1 gene Ceased WO2005085478A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54837104P 2004-02-27 2004-02-27
US60/548,371 2004-02-27

Publications (2)

Publication Number Publication Date
WO2005085478A2 true WO2005085478A2 (en) 2005-09-15
WO2005085478A3 WO2005085478A3 (en) 2006-01-05

Family

ID=34919358

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/006609 Ceased WO2005085478A2 (en) 2004-02-27 2005-02-28 Detection and identification of mycobacterium and nocardia using primers and probes directed to the seca1 gene

Country Status (1)

Country Link
WO (1) WO2005085478A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009087688A3 (en) * 2008-01-04 2009-10-15 Premas Biotech Pvt.Ltd Method for detection of nucleic acid of mycobacterium and uses thereof
CN110079620A (en) * 2019-04-27 2019-08-02 中国疾病预防控制中心传染病预防控制所 Identify three kinds of nocardial methods simultaneously using high-resolution melting curve method

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996026276A1 (en) * 1995-02-22 1996-08-29 THE GOVERNMENT OF THE UNITED STATES OF AMERICA,_represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES SecA GENE OF MYCOBACTERIUM TUBERCULOSIS AND RELATED METHODS AND COMPOSITIONS
KR100451108B1 (en) * 2000-05-30 2004-10-06 주식회사 바이오메드랩 diagnosis kit for Mycobacterial gene to determine strain identity and drug-resistance and the manufacturing method thereof
EP1227152A1 (en) * 2001-01-30 2002-07-31 Société des Produits Nestlé S.A. Bacterial strain and genome of bifidobacterium
WO2002077183A2 (en) * 2001-03-21 2002-10-03 Elitra Pharmaceuticals, Inc. Identification of essential genes in microorganisms

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009087688A3 (en) * 2008-01-04 2009-10-15 Premas Biotech Pvt.Ltd Method for detection of nucleic acid of mycobacterium and uses thereof
CN110079620A (en) * 2019-04-27 2019-08-02 中国疾病预防控制中心传染病预防控制所 Identify three kinds of nocardial methods simultaneously using high-resolution melting curve method

Also Published As

Publication number Publication date
WO2005085478A3 (en) 2006-01-05

Similar Documents

Publication Publication Date Title
Williams et al. The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae
Kraus et al. Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR
CA2322617A1 (en) Compounds and methods for diagnosis of tuberculosis
Mijs et al. Evaluation of a commercial line probe assay for identification of Mycobacterium species from liquid and solid culture
WO2005103249A1 (en) Probe and primer for tubercle bacillus detection, and method of detecting human tubercle bacillus therewith
WO2014003583A2 (en) Detection of pathogens
Phetsuksiri et al. Comparison of loop-mediated isothermal amplification, microscopy, culture, and PCR for diagnosis of pulmonary tuberculosis
US10962540B2 (en) Beta lactamase as biomarker for the specific detection of tuberculosis-complex bacteria
US7271781B2 (en) Multiplex hybridization system for identification of pathogenic mycobacterium and method of use
CA2477195A1 (en) Deleted sequence in m. tuberculosis, method for detecting mycobacteria using these sequences and vaccines
Fauville-Dufaux et al. Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85
CN110168109B (en) Diagnostic method for detecting and identifying tubercle bacillus and non-tubercle bacillus infection, confirmation of drug resistance of tubercle bacillus to rivastigmine and reagent group thereof
Dilworth et al. Comparison of polymerase chain reaction for IS6110 and Amplicor in the diagnosis of tuberculosis.
WO2005085478A2 (en) Detection and identification of mycobacterium and nocardia using primers and probes directed to the seca1 gene
CN1914334B (en) Primer pairs and kits for detecting Mycobacterium tuberculosis
EP2284263A2 (en) Primer and probe for use in detection of mycobacterium kansasii and method for detection of mycobacterium kansasii using the same
CA2481517A1 (en) Detection of mycobacteria in clinical material
Souza et al. Development and evaluation of a single tube nested PCR based approach (STNPCR) for the diagnosis of plague
US7960106B2 (en) Diagnostic method and products useful therein
US20130338018A1 (en) Primers, sequences and recombinant probes for identification of mycobacterium species
Palacios et al. Comparison of solid and liquid culture media with the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples
Chaiprasert et al. One-tube multiplex PCR method for rapid identification of Mycobacterium tuberculosis
JP4806749B2 (en) Bordetella pertussis gene detection method using LAMP method and primer set used in this method
JP3360736B2 (en) Probe specifically recognizing mycobacterial microorganism and method for detecting mycobacterial microorganism
RU2795987C1 (en) Set of oligonucleotide primers and fluorescently labeled probes and a method for detecting brucellosis pathogen dna

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase