RU2832754C1 - Polymerase chain reaction primers and probes for detecting mycobacterium tuberculosis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology. Described is a set of oligonucleotides for amplifying a fragment of the M. tuberculosis region. Also described is a kit for detecting drug resistance in M. tuberculosis, containing said kit. Also described is a method for detecting drug resistance in M. tuberculosis using a set of oligonucleotides for amplifying a fragment of the M. tuberculosis region.

EFFECT: reliable and effective detection of drug resistance of Mycobacterium tuberculosis to the most common first- and second-line drugs.

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Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/062,351, filed October 10, 2014, the disclosure of which is incorporated herein by reference.

STATE SUPPORT

The invention described herein was made, at least in part, with government support under National Institutes of Health Grants Nos. U01AI082174 and R01AI080653. Accordingly, the United States Government has certain rights in this invention.

AREA OF TECHNOLOGY

The present invention provides novel primers and probes based on sloppy molecular beacons (SMB) and molecular beacons (MB) for amplification and detection of segments of various genes of Mycobacterium tuberculosis ( M. tb ) for the purpose of detecting the presence of M. tb DNA and detecting resistance to anti-tuberculosis drugs.

LEVEL OF TECHNOLOGY

Tuberculosis (TB) was declared a global threat approximately two decades ago (WHO Global TB Report 2013). Although the incidence of new TB cases is declining worldwide, the Millennium Development Goal of a 50% incidence reduction by 2015 is unlikely to be achieved (WHO Global TB Report 2013). The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB poses a major challenge to achieving these incidence reduction targets (WHO Global TB Report 2013). MDR TB is defined as TB that is resistant to treatment with at least rifampicin and isoniazid, and XDR TB is defined as MDR TB that is additionally resistant to treatment with fluoroquinolone antibiotics and the injectable drugs amikacin, kanamycin and capreomycin. Patients with drug-resistant tuberculosis are best identified as early as possible so that appropriate infection control and treatment can be initiated quickly. Boehme, CC, et. al, 2011, Lancet 377: 1495-1505.

Complete determination of the drug resistance pattern of Mycobacterium tuberculosis ( M. tb ) by conventional phenotypic methods can take weeks to months because of the very slow growth of this bacterium (Heifets, L., et al., J Clin Microbiol 38:1227–1230; Kim, S. J., 2005, Eur Respir J 25:564–569; and PT, K., and K. GP., 1985, Public health Mycobacteriology: A guide for level III laboratory. Center for Disease Control, US Department of Health and Human Services, Atlanta, Georgia.). Molecular analysis offers the prospect of more rapid detection of drug resistance. M. tb does not naturally harbor drug resistance plasmids; thus, molecular analysis is directed toward chromosomal DNA. Genotypic assays are relatively simple to develop because the M. tb genome is highly sequence conserved. Virtually all drug-susceptible clinical isolates of M. tb contain identical drug resistance target DNA sequences, with the exception of a few easily identifiable "natural polymorphisms". It follows that any deviation from the wild-type sequence of a drug resistance target gene indicates the presence of drug resistance to the corresponding drug. Genotypic assays are more rapid and sensitive than phenotypic assays because target DNA can be amplified by PCR. Biological threats can be minimized by killing infectious organisms at early stages.

The genetic targets responsible for most cases of drug resistance in TB are now well known. Real-time PCR remains the most sensitive, rapid and reliable method for detecting mutations in bacteria. Almost all other mutation detection methods, including PCR-MS, microarrays, miniarrays and next-generation sequencing, require nucleic acid amplification as the first step in the detection process. In contrast, real-time PCR allows for amplification, detection and analysis of a sample in a single well. No tube opening or complex fluidic systems are required. However, a broad methodology for drug resistance analysis that is simple and reliable enough to be performed outside reference laboratories has not yet been developed. Thus, there is a need for new primers and probes for the detection of M.tb and for the detection of drug resistance in M.tb to the most commonly used first- and second-line drugs.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to primers, probes and related applications for detecting M.tb and M.tb resistance to drugs (drug resistance).

In one aspect, the present invention relates to an isolated set of oligonucleotides or a set of primers for amplifying a portion of a region of M tuberculosis selected from the group consisting of the rpoB gene, the gyrA gene, the gyrB gene, the inhA promoter, the rrs gene, the eis promoter, the embB gene, the katG gene, the dosR gene, the IS6110 gene, the IS1081 gene. The set comprises a pair of forward and reverse primers specific for said fragment, wherein each primer has a sequence substantially identical to the sequence of an oligonucleotide selected from the oligonucleotides listed below in Tables 1A and 1B. Accordingly, each primer has a sequence substantially complementary to the complementary strand of the sequence of an oligonucleotide selected from the oligonucleotides listed in said Tables. In some embodiments, the primer sequence is identical to the sequence of an oligonucleotide selected from the oligonucleotides listed in Tables 1A and 1B.

In a second aspect, the present invention relates to an isolated nucleic acid having a sequence substantially identical to a sequence selected from the sequences listed in Table 2. In some embodiments, the nucleic acid comprises a sequence selected from the sequences listed in Table 2. The nucleic acid can be labeled, for example, with a fluorophore and a quencher attached to its two ends, respectively, or a fluorophore linked to an internal nucleotide in the probe. Examples of fluorophores include fluorescein, cyanine 5 or TexasRed, and TAMRA. Examples of quenchers include BHQ1, BHQ2, and DABCYL.

The present invention provides a kit comprising one or more of the above-described sets of oligonucleotides and a nucleic acid. Said kit may further comprise a DNA polymerase, nucleotides for extending the sequence, and a buffer.

In a third aspect of the present invention, a method for detecting drug resistance in M. tuberculosis is provided. The method comprises the steps of amplifying a first target nucleotide sequence using a first pair of primers and obtaining a first amplicon, wherein (i) the first pair of primers is specific for a fragment of a region selected from the group consisting of the rpoB gene, the gyrA gene, the gyrB gene, the inhA promoter, the rrs gene, the eis promoter, the embB gene and the katG gene, and (ii) each primer has a sequence substantially identical to the sequence of an oligonucleotide selected from the oligonucleotides listed in Tables 1A and 1B, and detecting a mutation in the first amplicon. The presence of a mutation is indicative of drug resistance. In the said method, the detection step can be performed using various methods for detecting nucleic acids known in the art, including, for example, sequencing-based methods and techniques based on hybridization of nucleotide or peptide-nucleic acid probes.

In one embodiment, the detection step is performed by a process comprising (i) contacting a first amplicon with a first probe specific for the mutation under conditions that favor hybridization to form a probe-target hybrid; (ii) performing a melting temperature (Tm) assay to determine a test Tm value for the probe-target hybrid; and (iii) comparing the test Tm value to a predetermined Tm value. A test Tm value that differs from the predetermined Tm value indicates the presence of a mutation. For example, a shift in the test Tm value by at least 3 (e.g., 3, 4, or 5) standard deviations from a reference Tm value indicates the presence of a mutation. Conversely, a shift in the test Tm value by less than 3 standard deviations from the reference Tm value indicates the absence of a mutation. As used herein, the predetermined reference Tm value may be the mean Tm value of the wild type. In one example, a situation in which the test value Tm is less than a predetermined value of Tm, for example, by at least 3 standard deviations, indicates the presence of a mutation. In other cases, a situation in which the test value Tm is not less than a predetermined value of Tm, for example, by 3 standard deviations, indicates the absence of a mutation.

The method may further comprise amplifying a second target nucleotide sequence using a second pair of primers and obtaining a second amplicon, wherein the second pair of primers is specific for a fragment of a second region selected from the group consisting of the rpoB gene, the gyrA gene, the gyrB gene, the inhA promoter, the rrs gene, the eis promoter, the embB gene and the katG gene. In some embodiments, the first region is the rrs gene or the eis promoter. For example, the first region may be the rrs gene and the second region may be the eis promoter. The two regions may be amplified independently or in the same reaction system using techniques such as nested PCR. In this case, the mutation may be the A1401G or C1402T mutation in the rrs gene. The mutation may be within the eis promoter region interrogated by the eis primer sequences.

The above-described method detects resistance to a drug selected from the group consisting of isoniazid, rifampicin, amikacin, kanamycin, capreomycin, ethambutol and drugs of the fluoroquinolone class. The pair of primers may be a pair of primers selected from the primers listed in Tables 1A and 1B. The probe may have a sequence that is substantially or completely identical to a sequence selected from the sequences listed in Table 2.

In a fourth aspect of the present invention, there is provided a method for detecting the presence of M. tuberculosis in a test sample, for example, obtained from a subject. The method comprises contacting the test sample with a first pair of primers under conditions favouring an amplification reaction to produce a first amplicon and detecting the presence of the amplicon, which allows detecting the presence of Mycobacterium tuberculosis in the test sample. The first pair of primers may be a set of oligonucleotides for amplifying a part of a region of M. tuberculosis selected from the group consisting of the gyrB gene, the inhA promoter, the eis promoter, the embB gene, the katG gene, the dosR gene, the IS6110 gene, the IS1081 gene. Each primer of the first pair of primers has a sequence which may be substantially identical to the sequence of an oligonucleotide selected from the oligonucleotides listed in Table 1B. The method may further comprise contacting the test sample or the amplicon obtained using the first pair of primers with a second pair of primers under conditions favourable for the amplification reaction to obtain a second amplicon, and detecting the presence of the second amplicon. In this case, the presence of both the first and second amplicon indicates the presence of Mycobacterium tuberculosis in the test sample.

In a fifth aspect, the present invention provides another method for detecting the presence of M. tuberculosis in a test sample. The method comprises contacting a test sample with a first molecular beacon probe under conditions that favor a hybridization reaction to form a probe-target hybrid and detecting the presence of the probe-target hybrid, which allows detecting the presence of Mycobacterium tuberculosis in the test sample. In this method, the first molecular beacon probe has a sequence substantially identical to a sequence selected from the sequences listed in Table 2. In an example, the first molecular beacon probe is selected from the group consisting of probe IS1081, dosR2 and IS6110 (SEQ ID Nos. 67-69).

The details of at least one embodiment of the present invention are set forth in the description below. Other features, objects and advantages of the present invention will be apparent from the description and drawings, as well as from the claims.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a graph showing the detection of resistance to AMK and/or KAN in 603 clinical DNA samples using a three-spot Tm profile generated by an SMB probe. Each of the three SMBs used in the analysis was tested against all M.tb DNA samples in a multilocus PCR assay. The results for each sample are shown as a three-spot Tm plot on the x-axis, with the Tm value of each SMB indicated on the y-axis. Isolates were sorted left to right from phenotypically susceptible to resistant. For each resistant isolate, clear Tm shifts are seen for at least one of the three probes.

Figures 2A, 2B, and 2C are plots showing the first derivatives of the melting peak profiles of the three SMB probes. The melting peak profiles of wild-type, mutant, and mixed DNA samples are shown for the rrs -1400 SMB probe (2A), the eis 1 SMB probe (2B), and the eis 2 SMB probe (2C). Each melting curve represents a different strain.

Figure 3 is a graph showing the MIC values of rrs and eis mutant strains and the wild-type strains. The mean MIC values of AMK and KAN for rrs and eis mutants and the wild-type strains are shown. Error bars represent ± one standard deviation of the MIC values. eis -P represents the eis gene promoter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on the unexpected discovery of novel primers, SMB probes and MB probes for amplifying segments of eleven different M. tb genes to detect the presence of M. tb DNA and resistance to anti-tuberculosis drugs, such as isoniazid, rifampin, amikacin, kanamycin, capreomycin, ethambutol and fluoroquinolone drugs.

Primers and probes

The primers described herein for amplification of the rpoB , gyrA , gyrB genes, the inhA promoter region, the rrs gene, the eis promoter region, the embB and katG genes allow for sensitive amplification of mutagenesis hotspots causing drug resistance in M. tb . The corresponding SMB probes specifically bind to and detect these mutations causing drug resistance to the most common first- and second-line drugs. These primers can be used with very high efficiency in both symmetric and asymmetric PCR assays. The inventors have used the primer and probe sequences described herein to develop rapid and accurate molecular assays for drug susceptibility of M. tb . In addition to the obvious utility in molecular diagnostic assays for M. tb , these primers should also find application in sequencing target genes for detection of resistance mutations, in surveillance assays, and in any other probe-based assays that aim to specifically and sensitively detect mutations that cause resistance in M. tb to commonly used drugs. The sequences of the primers for amplification of the dosR, IS6110, and IS1081 genes allow highly sensitive and specific detection of M. tb and can be used in any PCR assay format that aims to provide highly specific and sensitive molecular diagnostics of tuberculosis. The following Tables list representative primers and probes according to the present invention.

One or more of the probe sequences listed in Table 2 can be prepared in a variety of detection formats, such as dual-labeled probes, including linear probes, Taqman-type probes, molecular beacon probes, and low-selectivity molecular beacon probes. The term "low-selectivity" probe refers to a mismatch-tolerant probe. Mismatch-tolerant probes hybridize to more than one target sequence and generate a detectable signal at the detection temperature of the assay, and the different hybrids formed in this manner are characterized by different melting temperatures. Linear or randomly coiled single-stranded probes are typically mismatch-tolerant. Examples of such probes are hairpin or linear probes with an internal fluorescent group whose fluorescence level increases upon hybridization with a particular target strand. See, for example, US Patents 7,662,550 and 5,925,517. US 20130095479.

The low selectivity probes are preferably dual-labeled hairpin probes or molecular beacon probes as described in U.S. Patents 7,662,550 and 5,925,517. These hairpin probes comprise a target binding sequence flanked by a pair of arms complementary to each other. They may be DNA, RNA, or PNA, or a combination of all three types of nucleic acids. They may also comprise modified nucleotides and modified internucleotide linkages. They may comprise a first fluorophore in one arm and a second fluorophore in the second arm, wherein the absorption spectrum of the second fluorophore substantially overlaps the emission spectrum of the first fluorophore. In the most preferred case, such hairpin probes are "molecular beacon probes" comprising a fluorophore in one arm and a quencher in the other arm, so that the probes do not fluoresce when free in solution. They can also be spectrum-shifting molecular beacon probes comprising, for example, several fluorophores in one arm that interact via fluorescence resonance energy transfer (FRET) and a quencher in the other arm. The length of the target-binding sequence can be, for example, 12-50 or 25-50 nucleotides, and the length of the hybridizing arms can be 4-10 or 4-6 (e.g., 5 or 6) nucleotides. Molecular beacon probes can be linked to primers as described in U.S. Patents 7,662,550 and 5,925,517 and WO 01/31062.

Thus, the term "low-selectivity molecular beacon probes" refers to this class of hairpin-shaped oligonucleotide hybridization probes labeled with a fluorescent label. Such probes generate a detectable signal in a homogeneous assay, i.e., an assay without the need to separate probes hybridized to the target from unbound probes. Due to their ability to bind more than one variant of a given target sequence, these probes can be used in assays to detect the presence of a single variant of a nucleotide sequence segment of interest among a number of possible variants, or even to detect the presence of two or more variants. Thus, the probes can be used in combination of two or more in the same assay. Since their target-binding sequences differ, the relative avidity differs for different variants. For example, the first probe may bind strongly to the wild-type sequence, moderately to the first allele, weakly to the second allele, and not at all to the third allele; while the second probe may bind weakly to the wild-type and the first variant, and moderately to the second variant and the third variant. Additional low-selectivity probes should exhibit even more distinct binding patterns due to differences in the target-binding sequences. Thus, the fluorescence emission spectra of a combination of low-selectivity probes detect different strains or species of microorganisms, as well as allelic variants/mutations of genes.

Since low-selectivity probes reproducibly fluoresce with variable intensity after binding to different DNA sequences, their combinations can be used, for example, in simple, rapid and sensitive assays based on nucleic acid amplification reactions (e.g., PCR-based assays) that allow the detection of multiple pathogens or variants in a single reaction container. At the same time, it should be understood that said assays can also be performed on samples suspected of containing directly detectable amounts of unamplified target nucleic acids. This detection assay is based on the analysis of the spectra of a set of partially hybridizing low-selectivity signal probes, e.g., low-selectivity molecular beacon probes, each of which is labeled with a fluorophore that emits light at its own wavelength optimum in order to obtain a "signature spectrum" of species-specific or variant-specific DNA sequences.

Multilocusity can be achieved using probes, for example, by designing different allelic discrimination molecular beacon probes for each target and differentially labeling each probe. (See, for example, U.S. Patents 7,662,550 and 5,925,517, WO 01/31062, and Tyagi et al. (2000) Nature Biotechnology 18: 1191-1196.) Mixtures of allelic discrimination probes, each containing aliquots of different colors, expand the number of probe signatures. In this regard, each molecular beacon-target hybrid with a unique melting temperature should be characterized by a corresponding unique signal intensity at a particular temperature and probe-amplicon concentration. In this way, a limited number of low-selectivity probes can be used as probes to detect many different possible target sequences by real-time PCR. Probes can be added to the amplification reaction mixture before, during, or after amplification. See U.S. Patent No. 7,662,550.

The present invention further provides kits containing reagents for performing the above-described methods, including PCR and/or probe-target hybridization reactions. For this purpose, one or more of the reaction components, such as PCR primers, polymerase and probes for the methods described herein, can be supplied as a kit for an application. Such a kit contains an appropriate amount of one or more of the reaction components in one or more containers or on a support.

The kit also contains additional materials for implementing the above-described methods. In some embodiments, the kit contains some or all of the reagents, materials for performing a method that uses the primers and/or probes according to the present invention. Some or all of the components of the kits may be contained in containers separate from the container(s) containing the primers and/or probes according to the present invention. Examples of additional components of the kits include, but are not limited to, one or more of the various polymerases, one or more of the control reagents (e.g., probes or PCR primers or control templates) and reaction buffers (in IX or concentrated form). The kit may also contain one or more of the following components: supports, reagents for stopping the reaction, modification or hydrolysis, osmolytes and a detection apparatus.

The reaction components used may be presented in various forms. For example, the components (e.g., enzymes, probes, and/or primers) may be suspended in an aqueous solution or presented as a lyophilized powder, pellet, or granule. In the latter case, the components upon reconstitution form a complete mixture of components for use in the assay. The kit components may be presented at any suitable temperature. For example, for storing kits containing protein components (e.g., enzyme) in liquid, delivery and storage at a temperature below 0°C, preferably at -20°C or lower, or otherwise in a frozen state is preferred.

The kit or system of the present invention may comprise any combination of the components described herein in an amount sufficient to perform at least one assay. In some applications, one or more of the reaction components may be provided in a pre-measured amount for a single use in an individual, typically in disposable tubes or equivalent containers. In this arrangement, the PCR may be performed by adding the target nucleic acid or the sample/cell containing the target nucleic acid directly to individual tubes. The number of components supplied in the kit may be any suitable amount and may depend on the target market for which the product is intended. The container(s) in which the components are supplied may be any conventional container capable of holding the form being supplied, such as microcentrifuge tubes, ampoules, bottles, or integral assay devices, such as fluidic devices, cartridges, lateral flow devices, or other similar devices.

The kits may also contain packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid materials (e.g., glass, plastic, paper, foil, microparticles, etc.) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, a well of a microtiter plate, a microchip, etc.). The kits may also further contain instructions recorded in tangible form for using the components.

Definitions

Nucleic acid refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA), an RNA molecule (such as, but not limited to, mRNA), or an analog of DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. A nucleic acid molecule can be double-stranded or single-stranded. An "isolated" nucleic acid is a nucleic acid that is not structurally identical to any naturally occurring nucleic acid or to any fragment of a naturally occurring genomic nucleic acid. Thus, the term includes, for example, (a) DNA that contains the sequence of a fragment of a naturally occurring genomic DNA molecule but is not flanked by both coding sequences that flank said fragment of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule, such as a cDNA, a fragment of a genome, a fragment obtained by PCR, or a restriction fragment; and (d) a recombinant nucleotide sequence that is a fragment of a hybrid gene, i.e. a gene encoding a hybrid protein.

As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid comprising a target nucleotide sequence of interest. The target nucleic acid may be single-stranded or double-stranded, and is often double-stranded DNA. "Target nucleotide sequence", "target sequence" or "target region" means a specific sequence comprising, in whole or in part, a single-stranded nucleic acid sequence. The target sequence may be in a nucleotide template or in the genome of a cell, which may be any form of single-stranded or double-stranded nucleic acid. The template may be purified or isolated nucleic acid, or may be unpurified or unisolated.

"Complementary" sequences as used herein may comprise or be formed by fully Watson-Crick base pairs (e.g., A-T/U and C-G), non-Watson-Crick base pairs, and/or base pairs formed by non-natural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. A fully complementary strand or fully complementary may mean 100% (fully) complementary or substantially complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

"Substantially complementary" means that the nucleic acid or oligonucleotide has a sequence comprising at least 10 contiguous bases that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%) complementary to at least 10 contiguous bases in a target nucleotide sequence, such that the nucleic acid or oligonucleotide can hybridize or anneal to the target nucleotide sequence, such as under PCR annealing conditions or probe-target hybridization conditions. Complementarity between sequences can be expressed as the number of mismatched bases in each set of at least 10 contiguous bases compared. The term "substantially identical" means that the first nucleic acid is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%) complementary to the second nucleic acid, such that the first nucleic acid is substantially complementary to the complementary strand of the second nucleic acid and is capable of hybridizing thereto under PCR annealing or probe-target hybridization conditions.

The terms "hybridization" or "hybridizable" or "hybridize" or "anneal" refer to the ability of fully or partially complementary nucleic acid strands to join together under specified hybridization conditions in a parallel or preferably antiparallel orientation to form a stable double-stranded structure or region (sometimes called a "hybrid" or "duplex" or "stem") in which the two component strands are joined by hydrogen bonds. Although hydrogen bonds are typically formed between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs can also be formed (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

The terms "nucleotide duplex", "duplex", "stem", "nucleotide hybrid" or "hybrid" refer to a stable nucleotide structure comprising a double-stranded nucleotide structure with hydrogen bonds, such as RNA:RNA, RNA:DNA and DNA:DNA double-stranded molecules and their analogs. Such a structure can be detected by any known means, such as using a labeled probe, an optically active support coated with a probe sensitive to changes in mass on its surface (U.S. Patent No. 6,060,237) or binding agents (U.S. Patent No. 5,994,056).

As used herein, the term "amplification" and its variants include any process for producing multiple copies or complementary strands of at least a fragment of a polynucleotide, said polynucleotide being commonly referred to as a "template". The template polynucleotide may be single-stranded or double-stranded. The template may be a purified or isolated nucleic acid, or may be unpurified or unisolated. Amplification of a given template may result in a population of polynucleotide amplification products, collectively referred to as an "amplicon". The polynucleotides of the amplicon may be single-stranded or double-stranded, or a mixture of single-stranded and double-stranded polynucleotides. Typically, the template contains a target sequence, and the resulting amplicon should contain polynucleotides having a sequence substantially identical to or substantially complementary to the target sequence. In some embodiments, the polynucleotides of a given amplicon are substantially identical or substantially complementary to one another; alternatively, in some embodiments, the nucleotide sequences of the polynucleotides within a given amplicon may differ from one another. The amplification may occur in a linear or exponential fashion and may involve repeated or sequential replication of a given template to form two or more amplification products. Some exemplary amplification reactions involve sequential and repeated rounds of nucleic acid synthesis from a template to form a plurality of progeny polynucleotides comprising at least a portion of the nucleotide sequence of the template and having at least some overall degree of nucleotide sequence identity (or complementarity) to the template. In some embodiments, each instance of nucleic acid synthesis, which may be referred to as an amplification "round," includes the creation of a free 3' end (e.g., by nicking one strand of dsDNA), resulting in primer formation and primer extension steps; optionally, additional denaturation steps may be included, in which the template is completely or partially denatured. In some embodiments, one amplification run includes a specified number of repetitions of an amplification cycle. For example, an amplification run may include 5, 10, 15, 20, 25, 30, 35, 40, 50 or more repetitions of a particular cycle. In one exemplary embodiment, amplification includes any reaction in which a particular polynucleotide template is subjected to two successive rounds of nucleic acid synthesis. The synthesis may include template nucleic acid synthesis.

The amplification according to the present invention may also include isothermal amplification. The term "isothermal" means performing a reaction at a substantially constant temperature, i.e., without changing the reaction temperature at which the nucleic acid polymerization reaction occurs. Isothermal temperatures for isothermal amplification reactions depend on the nucleic acid displacement polymerase used in the reactions. Typically, isothermal temperatures below the melting temperature (Tm; the temperature at which half of the potentially double-stranded molecules in a mixture are in a single-stranded, denatured state) of the predominant reaction product, i.e., typically 90°C or lower, are typically between about 20°C and 75°C, preferably between about 30°C and 60°C, and more preferably about 37°C.

The term "primer" or "primer oligonucleotide" refers to a nucleotide chain or oligonucleotide capable of hybridizing with a nucleotide template and acting as an initiation site for nucleotide incorporation during chain extension according to the composition of the nucleotide template for nucleic acid synthesis. "Chain extension nucleotides" refers to any nucleotides (e.g., dNTPs) and their analogs capable of incorporating into the elongation product during amplification, i.e., DNA, RNA, or a derivative of DNA or RNA that may contain a label.

As used herein, the term "oligonucleotide" refers to a short polynucleotide, typically less than or equal to 300 nucleotides in length (e.g., in the range of 5 to 150, preferably in the range of 10 to 100, more preferably in the range of 15 to 50 nucleotides). However, as used herein, the term is also intended to encompass longer or shorter polynucleotide chains. An "oligonucleotide" can hybridize with other polynucleotides, thereby acting as a probe for detecting a polynucleotide or a primer for extending a polynucleotide chain.

The term "probe" as used herein refers to an oligonucleotide capable of binding to a target nucleic acid with a complementary sequence through one or more types of chemical bonds, typically through the formation of complementary base pairs, typically through hydrogen bonding. Probes may bind target sequences that are not completely complementary to the probe sequence, depending on the stringency of the hybridization conditions. Any number of mismatched base pairs may be present, which should interfere with hybridization between the target sequence and the single-stranded nucleic acids described herein. However, if the number of mutations is so great that hybridization is not possible even under the least stringent hybridization conditions, the sequence is not complementary to the target sequence. The probe may be single-stranded or partially single-stranded and partially double-stranded. The number of strands in the probe is determined by the structure, composition and properties of the target sequence. Probes can be directly or indirectly labeled with a tag, such as biotin, to which a streptavidin complex can later be linked.

The term "detection probe" refers to an oligonucleotide having a sequence substantially complementary to a target sequence and capable of forming a stable probe:target hybrid for detection under stringent hybridization conditions. The probe is typically a synthetic oligomer that may contain bases complementary to a sequence outside the target region that do not interfere with hybridization under stringent hybridization conditions to the nucleotide target. The sequence non-complementary to the target may be a homopolymer region (e.g., polyA or polyT), a promoter sequence, a sequence recognized by a restriction endonuclease, or a sequence that imparts a desired secondary or tertiary structure (e.g., a catalytic site or hairpin structure), or a marker region that can facilitate detection and/or amplification. The term “stable” or “stable for detection” means that the temperature of the reaction mixture is at least 2°C below the melting temperature (Tm) of the nucleotide duplex contained in the mixture, more preferably at least 5°C below the Tm, and even more preferably at least 10°C below the Tm.

"Label" or "reporter molecule" is a chemical or biochemical entity that can be used to label a nucleic acid (including a single nucleotide), a polynucleotide, an oligonucleotide, or a protein ligand, such as an amino acid or an antibody. Examples include fluorescent agents, chemiluminescent agents, chromogenic agents, quenchers, radionuclides, enzymes, substrates, cofactors, inhibitors, magnetic particles, and other chemical entities known in the art. Labels or reporter molecules can generate a measurable signal and can be covalently or non-covalently attached to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide) or ligand.

As used herein, the term "contacting" and variations thereof, when used with respect to any set of components, includes any process wherein the components to be contacted are mixed into the same mixture (e.g., added to the same compartment or solution) and does not necessarily require actual physical contact between said components. Said components may be contacted in any order or any combination (or subcombination), including situations where one or more of said components are subsequently removed from the mixture, optionally before the other said components are introduced. For example, "contacting A with B and C" includes any of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, then C is added to the mixture; and (iii) A is added to the mixture of B and C. "Contact" of a nucleotide target or cell with one or more reaction components, such as a polymerase, a primer set, or a probe, includes any or all of the following situations: (i) the target or cell is contacted with a first component of the reaction mixture to form a mixture; other components of the reaction mixture are then added to the mixture in any order or combination; and (ii) the reaction mixture is completely formed before mixing with the target or cell.

The term "mixture" as used herein refers to a combination of elements that are intermingled with each other but not in a particular order. A mixture is heterogeneous and is not spatially separated into components. Examples of mixtures of elements include a number of different elements dissolved in the same solution, or a number of different elements attached to a solid support in a random order or without any order, wherein the different elements are not spatially separated. In other words, the mixture is not addressable.

In this document, the term "subject" refers to any organism possessing a genome, preferably a living animal, such as a mammal, which is the object of diagnosis, treatment, observation or experiment. An example of a subject may be a human, a food animal (beef and dairy cattle, sheep, poultry, pigs, etc.) or a companion animal (dogs, cats, horses, etc.).

"Sample" as used herein means any biological fluids or tissues obtained from an organism (e.g., a patient) or components of an organism (e.g., blood). A sample may be any biological tissue, cell(s), or fluid. A sample may be a "clinical sample," which is a sample obtained from a subject, such as a human patient or an animal subject. Biological samples that may be used include, but are not limited to, whole blood, saliva, urine, synovial fluid, bone marrow, cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions, sputum, semen, amniotic fluid, bronchoalveolar lavage, and other cellular exudates from a patient or subject. Such samples may be further diluted with a NaCl solution, a buffer, or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means. Biological specimens may also include sections, such as frozen tissue sections for histological purposes. A biological specimen may also be referred to as a "patient specimen." A biological specimen may also include a substantially purified or isolated protein, a membrane preparation, or a cell culture.

The terms determination, measurement, evaluation, and analysis are used interchangeably and include both quantitative and qualitative measurements and the determination of the presence or absence of characteristics, attributes, or properties. Evaluation may be relative or absolute. Evaluation of the presence of a target involves determining the amount of target present as well as determining its presence or absence.

As used herein, the term "reference" value refers to a value that is statistically correlated with a specific result when compared to an assay result. In preferred embodiments, the reference value may be determined based on a statistical analysis examining the mean value of the wild type. The reference value may be a threshold estimate or a limit estimate. Typically, the reference value is an upper (or lower) threshold value, depending on which result is more likely and how likely the alternative result is less than the specified value.

As used herein, a difference in these values indicates the presence or absence of a pathogen (e.g., Mycobacterium tuberculosis) or mutation. The phrase "difference" in a level or value refers to differences in the value of a variable (e.g., Tm) of an analyte (e.g., a probe-target hybrid) in a sample compared to a control or reference level or value. In one embodiment, a difference in a value or level may be a statistically significant difference between the amount of analyte in a sample compared to a control. For example, a difference may be statistically significant if the measured level of the analyte is greater than 1.0, 2.0, 3.0, 4.0, or 5.0 standard deviations from the mean of any control or reference group.

A number of ranges of values are presented herein. It should be understood that any intermediate value, down to tenths of the lower limit value (unless the context clearly indicates otherwise), located between the upper and lower limits of a stated range, is also included in the description of the invention. Each smaller range located between any stated values, or an intermediate value in a stated range, and any other stated or intermediate value in a stated range are included in the scope of the present invention. The upper and lower limits of these smaller ranges may independently be included in these ranges or not included in them, and each range where any or both of the end values are included or not included in the smaller ranges are also included in the scope of the present invention, with the exception of any explicitly excluded end value in the stated range. If a stated range includes one or both end values, ranges that do not include one or both of these end values are also included in the scope of the invention. The term "about" generally means plus or minus 10% of the stated numerical value. For example, “approximately 10%” may mean a range of 9 to 11%, and “approximately 20” may mean 18 to 22%. Other meanings of the term “approximately” (such as rounding) may be apparent from the context, for example, “approximately 1” may also mean 0.5 to 1.4.

Application

Recently, two separate SMB-based assays have been described that can rapidly and reliably detect M. tb mutations that predominantly confer resistance to rifampin and fluoroquinolones (FQs) (Chakravorty, S. B. et al., 2011, J Clin Microbiol 49:932-940; Chakravorty, S. H., et al., 2012, J Clin Microbiol 50:2194-2202). The assays described herein have the advantage of being in a real-world PCR format, so they are easy to use and are not susceptible to amplicon cross-contamination. Furthermore, mutation detection has been shown to be robust and suitable for high-throughput assays. The examples below present an SMB-based assay system for detecting TB drug resistance with the addition of assays for detecting AMK and KAN resistance. In addition, the reasons for the discrepancy between the assay described in this document and phenotypic susceptibility testing methods are investigated. The results show that some of the most commonly used phenotypic assays may miss M. tb isolates with resistance-conferring mutations if these mutations only moderately increase KAN minimum inhibitory concentration (MIC) values. In addition, new mutations in whiB7 associated with low-level resistance to KAN are identified.

As described herein, multilocus SMB-PCR and melting analysis accurately detected mutations in the rrs gene and eis promoter associated with resistance to AMK and/or KAN. The assay did not generate false positive resistance results when testing NTM, Gram-positive, and Gram-negative bacteria. Most heterogeneous resistance cases were also detected by the assay when available. Unlike the MTBDRsl platform, the assay can be performed in a closed-loop real-time PCR system and is easily adapted for high-throughput analysis since all assay steps are performed in 384-well plates. The SMB assay also avoids potential problems associated with alternative mutation detection methods. High-resolution melt curve analysis requires the ability to detect small differences in melting curves (Yadav, RS, et al., 2012, J Appl Microbiol 113:856-862). Other post-PCR melting molecular assays must detect mutations by decoding complex fluorescence patterns (Rice, J.E., et al., 2012. Nucleic Acids Res 40:e164.) In contrast, SMB analysis provides clear and easily distinguishable peaks and characteristic Tm shifts to identify mutations of interest. Individual Tm values can also be used to form clusters of samples that share the same genotype.

As described herein, the assay was tested on a panel of 603 clinical specimens representing both new and untreated retreatment TB cases and the association of target mutations with the susceptibility pattern of clinical isolates was assessed. It was noted that 100% of isolates with the A1401G mutation in the rrs gene were strongly correlated with high-level resistance to both AMK and KAN. However, mutations in the eis promoter resulted in only moderate to weak resistance to KAN and no resistance to AMK, consistent with previous studies (Campbell, PJ, et al, 2011, Antimicrob Agents Chemother 55: 2032-2041, Zaunbrecher, MA, et al., 2009, Proc Natl Acad Sci USA 106: 20004-20009). The present study also demonstrated that the LJ absolute concentration method for susceptibility testing does not adequately detect moderate or weak resistance to KAN. In fact, nearly two-thirds of the samples with eis promoter mutations were found to be susceptible to KAN using the LJ medium. However, all but one of the eis promoter mutations were found to be resistant to KAN using the MGIT method. Two of these isolates contained the C(-12)T mutation of eis . These mutants were also resistant to KAN when tested using MYCOTB, which showed a KAN MIC of 5 μg/mL. Previous studies have shown that clinical isolates with C(-12)T mutations do not (Zaunbrecher (2009)) or correlate poorly (Campbell, 2011; Hoshide, M., L. et al., 2014, J Clin Microbiol 52: 1322–1329) with resistance to KAN. These studies may have missed the association between this mutation and low-level KAN resistance due to the testing method used to establish phenotypic susceptibility. These results suggest that the MGIT or MYCOTB assays should be preferred when testing phenotypic resistance to KAN. They also highlight the effectiveness of genotypic resistance tests, such as those described here, in detecting mutations that confer low-level resistance that may be missed by phenotypic tests alone (Rigouts, L., M. et al., 2013, J Clin Microbiol 51: 2641-2645, Sirgel, FA, et al., 2012, Microb Drug Resist 18: 193-197, and Van Deun, A., et al., 2013, J Clin Microbiol 51: 2633-2640).

The study reported here included one sample that was a mixture of wild-type rrs and C1402T rrs mutants. This sample was susceptible to both AMK and KAN using LJ medium. Isolates with C1402T mutations have been reported to be susceptible to AMK but resistant to KAN (Maus, CE, et al., 2005, Antimicrob Agents Chemother 49: 3192-3197). In this particular case, repeated susceptibility tests using LJ medium demonstrated susceptibility to KAN, presumably due to heterogeneous resistance in the sample. In the present invention, molecular analysis was a better predictor of potential resistance than phenotypic analysis, since SMB analysis was able to accurately detect the presence of both wild-type and mutant DNA.

The frequency of 1484 rrs mutations in clinical isolates with resistance to AMK or KAN was very low (Georghiou, SB, et al., 2012, PLoS One 7: e33275), making its clinical significance controversial. A separate version of the assay targeting codon 1484 rrs found no mutations in any of the 603 isolates tested, as well as an additional 259 isolates from the New Jersey-New York area, which included 33 isolates resistant to AMK and KAN. The absence of 1484 rrs mutations in this expanded study set was confirmed by Sanger sequencing (data not shown). In light of the extremely low prevalence of 1484 rrs mutations, it is unlikely that this codon has much value in predicting aminoglycoside resistance. Thus, in molecular assays of aminoglycoside resistance, it is recommended to use only codons 1401-1402 of the rrs gene.

Twenty-two AMK- or KAN-resistant strains were found to contain wild-type sequences of the rrs gene and eis promoter region. A recent study demonstrated a possible link between whiB7 5'UTR mutations and KAN resistance by identifying a whiB7 5'UTR mutation in one clinical strain with unexplained KAN resistance (Reeves, A-Z, et al., 2013, Antimicrob Agents Chemother 57: 1857-1865). In addition, several novel 5'UTR mutations as well as a deletion in whiB7 were described that appear to be associated with KAN resistance. No suitable universal biomarkers that could be responsible for KAN and AMK resistance in the remaining 15-20% of clinical strains with wild-type rrs and eis promoter region have been identified. Samples containing wild-type rrs gene DNA and eis promoter regions mixed with trace amounts of mutant targets from the KAN- or AMK-resistant subpopulation may also explain the remaining discrepancies between the phenotypic resistance tests and the SMB assay described herein. However, costly heterogeneous resistance testing is beyond the scope of the present study. Some recent studies have suggested that the PPE60 and Rv3168 genes may be involved in the mechanisms of unexplained KAN resistance (Farhat, MR, et al., 2013, Nat Genet 45: 1183-1189; Zhang, H., et al., 2013, Nat Genet 45: 1255-1260), although this remains to be tested in a clinical setting.

In summary, a sensitive and specific assay for the detection of AMK and KAN resistance in M. tb was developed and validated in clinical isolates with a high prevalence of MDR and XDR TB. The results show that A1401G mutations in rrs encode high-level cross-resistance to both AMK and KAN and that eis promoter mutations encode moderate to weak resistance to KAN, consistent with previous functional genomic studies (Zaunbrecher 2009). When the performance of the assay described herein was compared with three different solid- and liquid-based phenotypic susceptibility testing methods, it was found that LJ-based susceptibility assays essentially missed weak to moderate resistance to KAN caused by eis promoter mutations. These results provide compelling evidence for the value of genotypic testing for detecting aminoglycoside resistance and also demonstrate the potential for specific application of the SMB-based assays described herein.

EXAMPLES

Example 1

This example describes the materials and methods used in Examples 2-7 below.

DNA samples

M. tb test specimens consisted of DNA extracted from 603 consecutive M. tb isolates obtained from 503 patients enrolled in a natural history study of MDR-TB (NCT00341601 at cliniatialials.gov) at Masan National Hospital in Changwon, Republic of Korea. Two cohorts were tested. Cohort A consisted of treatment-naïve new TB cases (158 specimens) and cohort B consisted of retreatment TB cases (445 specimens). Fresh sputum specimens were collected from each patient at baseline and cultured for M. tb . A subset of patients had repeat sputum specimens collected at months 1, 4, and 6 of treatment and also cultured for M. tb . Test samples of non-tuberculous mycobacteria (NTM), gram-positive, and gram-negative bacteria were obtained from the New Jersey Medical School (NJMS) DNA repository as described previously (Chakravorty, S., 2012. J Clin Microbiol 50:2194-2202).

Analysis of phenotypic sensitivity to drugs

Phenotypic drug susceptibility testing was performed for all 603 isolates using the absolute concentration method on LJ medium to determine susceptibility to AMK and KAN using critical concentrations of 40 μg/mL (the standard concentration used in 2012 when these isolates were analyzed) of both antibiotics (Jnawali, HN, 2013, Diagn Microbiol Infect Dis 76: 187-196) at the International Tuberculosis Research Center (ITRC), South Korea. MICs of AMK and KAN for 173/603 samples were also assessed using the TREK Sensititre® MYCOTB MIC Plates (MYCOTB, TREK Diagnostic Systems, Cleveland, OH, USA) as described previously (Lee, J., 2014, Antimicrob Agents Chemother 58: 11-18). KAN resistance was also assessed for 560/603 samples using the Mycobacterial Growth Indicator Tube (MGIT) system (Becton Dickinson, Franklin Lakes, NJ, USA) at a cutoff concentration of 2.5 μg/mL. For samples with phenotypic susceptibility assay results that were discordant with Sanger target gene sequencing results, the phenotypic susceptibility assay was repeated to confirm the initial results. In cases where MGIT and LJ-based susceptibility assay results were discordant, both assays were repeated to confirm or correct the initial results.

DNA preparation and sequencing

DNA for SMB analysis and Sanger sequencing was prepared from cultured isolates by boiling one loop of culture in 200 μl of Instagene Matrix resin (Bio-Rad Laboratories, Hercules, CA, USA) in the presence of 0.1% Triton X100 for 10-15 min. The supernatant was recovered after centrifugation and quantified using a Nanodrop microvolume sample spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For Sanger sequencing, two different fragments of the rrs gene (nucleotides 420–980 and 1293–1537) and a fragment upstream of the eis coding region with the entire eis promoter region were amplified using 0.5 μM forward and reverse primers, IX PCR buffer, 250 mM dNTPs, 2.5 mM MgCl ₂ and 0.03 U/μl AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) according to the following parameters: initial denaturation at 95°C for 10 min, then 40 cycles of 95°C for 10 s, 58–60°C for 30 s and 72°C for 10–30 s depending on the amplicon size. The eis promoter region and rrs gene fragments were amplified as described previously (10, 33). For a subset of samples, a 538 bp fragment of the whiB7 gene containing 412 bp of the 5′ untranslated region and 126 bp of the ORF was amplified and sequenced using primers whiB7 F 5′-aaacgcgcaggtcagaaaat-3′ and whiB7 R 5′-cagtgtcttggctacctcga-3′ (SEQ ID NOs: 70 and 71). In addition, a 275 bp fragment of the whiB7 gene containing almost the entire whiB7 ORF was amplified using the primers whiB7 -ingene-F 5′-GTCGGTACTGACAGTCCCC-3′ and whiB7 -ingene-R 5′-ATGCAACAGCATCCTTGCG-3′ (SEQ ID NOs: 72 and 73). The PCR products were subjected to bidirectional sequencing using gene-specific forward and reverse primers on a 3130XL Genetic Analyzer (Applied Bio-Systems, Foster City, CA, USA) using the BigDye Terminator Cycle Sequencing Kit version 3.1 (Applied Biosystems) according to the manufacturer's instructions.

Molecular beacons and primers for analysis

The targets of SMB analyses were M.tb mutations in codons 1401 and 1402 of the rrs gene and mutations in the promoter region of the eis gene. A 113 bp fragment (nucleotides 1335-1451) of the rrs gene was amplified using primers AMG-F (5'-GCTAGTAATCGCAGATCAGCAACGCTGC-3', SEQ ID No: 51) and AMG-R (5'-CCTCCCGAGGGTTAGGCCACT-3', SEQ ID No: 52), and a 98 bp fragment spanning the promoter region and the initial five codons of the eis gene (from -81 to 17 nucleotides) was amplified using primers eis -F (5'-CACAGGGTCACAGTCACAGAATC-3', SEQ ID No: 18) and eis -R (5'-GCATCGCGTGATCCTTTGCCAGAC-3', SEQ ID No: 53). The rrs primers were designed to be specific to the genus Mycobacterium, and the eis primers were designed to be specific to the M. tb complex. The targets of one SMB probe rrs -1400 (5'-6-carboxyfluorescein-cacgaccgcccgtcacgtcatgaaagtcggtcgtg-BHQ1-3', SEQ ID No: 59) and two SMB probes eis -1 (5'-cyanine-5-caggcggtcgtaatattcacgtgcacctggccgccgcctg-BHQ2-3' SEQ ID No: 16) and eis -2 (5'-TexasRed-ctcgcggcatatgccacagtcggattctctgacgcgag-BHQ2-3', SEQ ID No: 61) (where the underlined sequences represent the main part of SMB and BHQ represents the Black Hole Quencher) were the rrs gene and the eis promoter region, respectively. The rrs probe was complementary to the antisense strand, and the eis probes to the SMB sense strand were designed using the in silico DNA folding program at http://mfold.rna.albany.edu/?q_mfold/dna-folding-form, and the probe-target hybrid folding programs at http://mfold.rna.albany.edu/?q_DINAMelt/Two-state-melting were used to predict possible structures and melting temperatures (Tm) of the probe-target hybrids. The probes were designed to maximize the Tm difference between the wild-type and mutant sequences in the corresponding target regions to unambiguously detect mutations. Primers were purchased from Sigma Aldrich (St. Louis, MO, USA), and SMB probes were from Biosearch Technologies (Novato, CA, USA).

Analysis procedure

All samples were independently coded and randomly assigned to ensure blinded validation of the assay. The assay was tested at ITRC in Masan, South Korea, and New Jersey Medical School (NJMS), Rutgers University, Newark, NJ, USA. After testing of the entire set of 603 samples, the samples were decoded and the PCR results were compared with the corresponding sequencing and phenotypic drug susceptibility assay results. Results were also compared across sites. Assay results were not shared with treating physicians or used in treatment decisions. PCR was performed in 384-well plates using a Roche Light Cycler 480 II Real-Time PCR System (Roche Diagnostics Co., Indianapolis, IN, USA) in a 20-μl reaction volume containing 100 nM forward primer and 1 μM reverse primer for the rrs gene and 1 μM forward primer and 50 nM reverse primer for the eis promoter region, 1 ng/μl rrs -1400 and eis -1 and 0.8 ng/μl eis - ₂ , 4 mM MgCl2, 250 mM deoxynucleoside triphosphates (dNTPs), 1X PCR buffer, 8% glycerol, 0.06 U/μl Platinum® TfiExo(-) DNA polymerase (Life Technologies, Grand Island, NY, USA) and 2 to 5 ng of sample DNA or an equivalent volume of water. PCR was performed with the following steps: enzyme activation for 2 min at 95°C, then 50 cycles of denaturation at 95°C for 10 s and combined annealing and extension at 67°C for 30 s. After PCR cycles, post-PCR Tm analysis was performed by denaturation at 95°C for 2 min, followed by cooling to 45°C and then gradual heating to 85°C while continuously monitoring fluorescence at a rate of 1 data point per degree Celsius. Tm values were determined at the end of the reaction using Tm analysis software (Light Cycler 480). However, each Tm value was also checked by a trained observer, after which a final Tm determination was made. Distinct double peaks in samples for any of the probes corresponding to the Tm for the wild type and mutants were considered indicative of heterogeneous resistance. No-template controls in which sterile water was used as a template instead of DNA were used as DNA-negative controls, and a DNA-positive control in which 1 g of M. tb H37Rv genomic DNA was used as a template was included in each assay plate.

Coordination of participation of human subjects

This study was approved by the institutional review boards of Masan National Hospital, NIAID, and Rutgers University (formerly UMDNJ), and all subjects provided written informed consent (Rutgers University ESU protocol #0120090104).

Example 2. Determination of Tm values associated with wild-type and mutant sequences

The SMB-based assay described herein detects resistance to AMK and KAN by searching for mutations in the rrs gene and eis promoter of M. tb known to be associated with resistance. The assay consisted of a PCR step followed by Tm analysis in the presence of SMB probes complementary to fragments of the rrs and eis target amplicons. We first assessed the ability of the assay to detect target mutations in artificial oligonucleotides and sequenced DNA templates from selected wild-type and mutant M. tb strains (data not shown). Wild-type sequences were detected by the presence of Tm values within 1°C of known mean values for the wild-type targets.

Mutant sequences were identified by shifts in Tm values of at least five standard deviations from the mean wild-type Tm values. The ability of the assay to detect the most common mutations associated with resistance to AMK and KAN in clinical DNA samples was then assessed.

The assays were performed on a panel of 603 clinical specimens consisting of 487 specimens with wild-type sequences and 116 specimens with mutations in the assay targets. Five of these specimens contained mixtures of both wild-type and mutant DNA as detected by Sanger sequencing. The SMB assay correctly identified 115/116 (99%) mutant or mixed specimens (heterogeneous specimens containing both mutant and wild-type DNA) as mutant or mixed specimens and 487/487 (100%) pure wild-type specimens as wild-type. The assay described here identified one mixed specimen (according to Sanger sequencing) as wild-type. The Tm values obtained for each SMB probe and the wild-type or mutant targets were well reproducible. For the wild-type targets, the rrs -1400, eis -1, and eis -2 probes demonstrated average Tm values of 70.1±0.15°C, 63.9±0.19°C, and 69±0.23°C, respectively (Table 3). The A1401G and C1402T mutations in the targets resulted in a decrease in Tm values for the rrs -1400 probe by 3.9°C (±0.17) and 5.6°C (±0.21), respectively (Table 3). Similarly, the eis -1 and eis -2 probes reliably detected a number of mutations in the eis promoter region as mutants by a decrease in Tm values by 4.3–6.5°C compared to the expected wild-type Tm values (Table 3). PCR analyses performed in two different laboratories at Rutgers University and ITRC were in complete agreement for all samples identified as wild-type and mutant, as well as mixtures.

The analysis results allowed clear separation of 603 samples into wild-type and mutant Tm clusters based on individual three-point Tm patterns (Fig. 1). The analysis correctly identified mutations in all 75 samples that contained only the A1401G mutation (Table 3, Fig. 2, plot A). Three of the four samples containing mixtures of A1401G and wild-type sequences were also detected as mixed wild-type/mutant samples based on the presence of a double Tm peak. One sample containing a mixture of C1402T and wild-type DNA was also detected by the presence of double Tm peaks in the mutant Tm sample specific for the C1402T mutation (Table 3, Fig. 2, plot A). The 32 samples with mutations in the eis promoter region contained five different polymorphisms (at positions -8, -10, -12, -14, and -37). All of these mutations were successfully detected by one of the eis SMBs (Table 3, Fig. 2, panels B and C). Four samples containing mutations in both the rrs gene and the eis promoter region were also correctly identified as double mutants (Table 3). Sequencing of the rrs gene did not reveal any samples with the codon 1484 mutation, regardless of the drug susceptibility pattern.

Example 3. Detection of resistance to amikacin

In this example, assays were performed to evaluate the performance of the molecular assay compared to the results of the phenotypic drug susceptibility assay. The apparent performance of the genotypic drug susceptibility assay may vary depending on the mutations chosen for inclusion in the assay and the phenotypic assay used as the “gold standard” (Kim, SJ 2005 Eur Respir J 25: 564-569; Rigouts, L., Et al., 2013, J Clin Microbiol 51: 2641-2645 and Van Deun, A., et al., 2013, J Clin Microbiol 51: 2633-2640). Taking the LJ-based drug susceptibility assay as the “gold standard” (for all 603 samples tested), the SMB rrs Tm specific for the A1401G mutation classified 82/90 AMK-resistant samples as resistant (sensitivity 91.1%; 95% CI 82.8% to 96.8%). The wild-type specific Tm classified 512/513 AMK-susceptible samples as susceptible. Using the SMB assay described herein, one isolate among the 513 AMK-susceptible isolates was identified as a mixture of wild-type and C1402T mutant DNA due to the presence of a distinct double peak formed by the rrs probe corresponding to the wild-type Tm and the C1402T mutant-specific Tm (Fig. 2, plot A). This result was confirmed by Sanger sequencing. Since previous studies have shown that the C1402T mutation does not encode AMK resistance (24), the specific Tm corresponding to the C1402T mutation cannot be considered as an indicator of AMK susceptibility. This consideration led to the conclusion that the assay described herein correctly identified all 513/513 AMK-susceptible samples, resulting in 100% specificity (95% CI 99 to 100%). Inclusion of Tm values specific for mutations in the eis promoter region in the assay did not increase the sensitivity for detecting AMK resistance, but decreased the specificity from 100% to 93.8% (95% CI 91.2 to 95.6%). These results are consistent with previous reports showing that eis promoter mutations are not associated with AMK resistance according to LJ drug susceptibility analysis (Campbell 2011 and Zaunbrecher 2009).

Example 4. Detection of resistance to kanamycin

The performance of the assay in detecting resistance to KAN was also assessed using the LJ-based drug susceptibility assay as the “gold standard” for all 603 samples. Using the Tm values obtained for the SMB rrs described herein, typical of the A1401G or C1402T mutation, to determine resistance, the assay identified 82/106 samples as resistant to KAN (sensitivity 77.4%, 95% CI 68.0 to 84.7%). Conversely, using the Tm of the SMB rrs specific for the wild-type target to determine susceptibility, 496/497 KAN-susceptible samples were determined to be susceptible (Table 4) (specificity 99.8%, 95% CI 98.7 to 100%). Adding the Tm values for the two eis SMBs specific for eis promoter mutations to determine resistance increased the sensitivity for detecting KAN resistance from 77.4% to 87.7% (95% CI 79.5 to 93%), as 11 additional KAN-resistant samples were classified as resistant. However, the specificity decreased from 99.8% to 95.6% (95% CI 93.3 to 97.1%), as 21 KAN-susceptible samples with eis promoter mutations were then “falsely” detected as KAN-resistant (Table 4).

A similar analysis was then performed using the MGIT-based DSA results as the gold standard for the 560 samples that yielded an MGIT result. This subset included all samples containing only eis promoter mutations. Comparison of the assay results with the MGIT-based gold standard identified eis mutants with discordant KAN resistance assay results on LJ medium. Using MGIT as the gold standard and Tm SMB rrs values specific for only the A1401G or C1402T mutations to determine KAN resistance, only 63/113 KAN-resistant samples were detected as resistant by the SMB assay (sensitivity 55.8%, 95% CI 46.1 to 65%). Conversely, when using the Tm of the wild-type target SMB rrs to determine susceptibility, 445/447 samples were detected as susceptible to KAN (specificity 99.8%; 95% CI 98.5 to 100%; Table 4). In contrast to the case with the LJ-based susceptibility assay, including the Tm values specific to the eis promoter mutations here increased the sensitivity for the resistance assay from 55.8% to 82.3%, while the specificity of the KAN resistance assay remained very high at 99.5% (95% CI 98.2 to 100%). Thus, when using the MGIT-based susceptibility assay, the eis assay detected 29 additional samples resistant to KAN without affecting the specificity (Table 4).

Example 5. Relationship between mutations detected by the assay and MIC

The discrepancy between resistance determined by the assay described herein and resistance determined by the two phenotypic susceptibility assays described herein mainly relates to isolates with eis promoter mutations. Previous studies have shown that eis promoter mutations result in relatively low-level resistance to KAN, whereas rrs gene mutations result in high-level resistance to AMK, KAN, and CAP (Campbell 2011; Du, Q., et al., 2013, Diagn Microbiol Infect Dis 77: 138–142, Georghiou 2012, and Zaunbrecher 2009). An additional finding was a discrepancy between the LJ- and MGIT-based susceptibility assays. To further investigate the relationship between mutations in the rrs gene and eis promoter and their differential susceptibility patterns in the LJ- and MGIT-based systems, MIC analysis was performed. Samples susceptible to both AMK and KAN (and containing both wild-type target regions) or selected as typical representatives of the most common mutation patterns in the two target genes (A1401G in rrs and G(-10)A, C(-14)T and G(-37)T) in eis were assayed by the MYCOTB assay to determine their MICs. Additional isolates known to contain both wild-type targets were also assayed as controls. The AMK MICs for isolates containing only eis promoter mutations (without rrs mutations) were found to range from 0.25 μg/mL to 2 μg/mL, with most samples having MICs between 0.5 μg/mL and 1 μg/mL (Fig. 3). Only one eis promoter mutant had an AMK MIC of 4 μg/mL. Control isolates without eis or rrs promoter mutations had MICs between 0.25 μg/mL and 0.5 μg/mL. Thus, the AMK MICs for isolates with the mutant or wild-type eis promoter essentially overlapped. In contrast, the KAN MICs for most eis promoter mutants ranged from 5 μg/ml to 20 μg/ml, with one isolate having an MIC of 40 μg/ml (Fig. 3). Only two eis promoter mutants had low MICs of 2.5 μg/ml. Isolates with wild-type eis promoter sequences exhibited MICs ranging from 0.6 μg/ml to 2.5 μg/ml, which is 2- to 30-fold lower than the average MIC of eis promoter mutants (Fig. 3). Thus, in contrast to the situation with AMK, the KAN MICs for wild-type isolates overlapped very little with the KAN MICs for eis promoter mutants. These results clearly indicate that eis promoter mutants should be considered to have weak to moderate resistance to KAN, even if resistance is not detected in LJ-based or even MGIT-based susceptibility assays.

Example 6. Specificity of the assay for bacteria other than M. tb .

The sensitivity of the assay was tested against a panel of 18 non-tuberculosis Mycobacteria (NTM) species obtained from the ATCC repository (Manassas, VA, USA), 121 clinical NTM strains representing 26 species, and 18 Gram-positive and Gram-negative bacterial species. The rrs region, the target of the assay described herein, is highly conserved among the different NTM species. Thus, the rrs assay yielded a Tm of 70°C (identical to the Tm obtained in the presence of wild-type M.tb DNA) for all NTM tested, as expected based on sequence homology, except for M. xenopi, for which no Tm value was obtained. Obtaining Tm values identical to those for aminoglycoside-susceptible M. tb for NTM species was not expected, except for false resistance results. When DNA from AMK- and KAN-resistant rrs mutant M. tb strains was mixed with a 10- to 20-fold excess of NTM DNA, the assay yielded a clear double Tm peak corresponding to the mutant Tm value for the M. tb target and the wild-type Tm value for the NTM sequence (data not shown), indicating that resistance-associated rrs mutations in M. tb can be detected by the assay described here even in the presence of large amounts of background NTM DNA. No visible melting curve was obtained for the eis probes in the presence of any of the NTM species tested, even when ¹⁰⁷ genomic equivalents of DNA were added to the PCR assay. No Tm values were obtained for any of the Gram-positive or Gram-negative bacteria using either the rrs or eis SMBs; thus, they do not result in false resistance results with this assay.

Example 7. Additional genetic causes of resistance to AMK and KAN

The study in this example included 22 samples resistant to AMK and/or KAN but containing wild-type rrs gene and eis promoter sequences. Recent studies have shown that mutations in the 5′ untranslated region (UTR) of the whiB7 gene can confer aminoglycoside resistance in M. tb (27). To determine whether mutations in whiB7 could confer phenotypic resistance to the susceptibility detected by this assay in some isolates, a 412-bp region upstream of the whiB7 start site and including a fragment of the whiB7 open reading frame was sequenced for all 22 samples. A random set of 30 fully susceptible isolates were also sequenced as a control. Six of the 22 discordant isolates from three patients demonstrated mutations in the 5′ UTR of whiB7 . One sample contained a cytosine deletion at position +138 in the 5' UTR, two samples from one patient contained an A to G mutation at position +237, and the remaining three samples from one patient demonstrated an A to C mutation at position +273 (Table 5), suggesting that the transcription initiation site is at position +1 (Reeves, AZ, 2013, 57:1857-1865). Three samples from one patient failed to amplify from the 5' UTR after repeated PCR attempts despite a working positive PCR control. This indicated the presence of a large deletion in the 5' UTR region, as a 275 bp fragment was readily amplified from sites within the whiB7 ORF for all three samples. All whiB7 mutation-positive samples were resistant to KAN only, consistent with the proposed mechanism of whiB7 action through eis gene activation (Reeves 2013). The KAN MICs for these isolates were also low at 5 μg/mL, similar to the MICs observed for the eis promoter mutants. None of the 30 aminoglycoside-susceptible control samples contained whiB7 5′ UTR mutations. Further studies are needed to confirm the association of these mutations and whiB7 5′ UTR deletions with aminoglycoside resistance. However, the absence of such mutations in susceptible isolates implies that they may play a role in aminoglycoside resistance, and these mutations may be targets for future assays to improve sensitivity in detecting weak KAN resistance.

SD represents the +/- standard deviation of the Tm value for each probe and different clinical samples, and dTm represents the difference in Tm for mutant sequences and for wild-type sequences using each probe.

SD: standard deviation, dTM: delta Tm

Probes No. 1, 2 and 3 correspond to zones rrs -1400, eis -1 and eis -2, respectively.

LJ and MGIT stand for sensitivity analysis by LJ proportions and MGIT method, respectively.

LJ and MGIT stand for sensitivity analysis by LJ proportions and MGIT method, respectively.

BUT: not determined, B/M: no mutation, U: resistant, S: sensitive.

The above examples and description of preferred embodiments should be taken as illustrative and not as limiting the present invention as defined by the claims. It should be appreciated that numerous variations and combinations of the above functions can be used without departing from the scope of the present invention as defined by the claims. Such variations should not be considered as a departure from the scope of the present invention, and all such variations should be within the scope of the following claims. All references cited herein are incorporated herein by reference in their entirety.

Claims (19)

1. A set of oligonucleotides for amplifying a fragment of a region of M. tuberculosis , comprising at least one pair of primers, wherein the forward primer is selected from a group of oligonucleotide sequences comprising a sequence that is at least 85% identical to SEQ ID NO: 2, 3, 4, 7, 8, 9, 15, 18, 19, 20, 21, 23, 25, 29, 30, 31, 32, 35, 37, 38, 39, 42, 43, 46, 49 or 51; and the reverse primer is selected from a group of oligonucleotide sequences comprising a sequence that is at least 85% identical to 4, 5, 10, 11, 12, 13, 14, 16, 17, 22, 24, 26, 27, 28, 33, 34, 36, 40, 41, 44, 45, 47, 48, 50, 52 or 53. 2. A set of oligonucleotides according to claim 1, characterized in that said region of M. tuberculosis is selected from the group consisting of the rpoB gene, the gyrA gene, the gyrB gene, the inhA promoter, the rrs gene, the eis promoter, the embB gene, the katG gene, the dosR gene, the IS6110 gene, and the IS1081 gene. 3. A kit for detecting drug resistance in M. tuberculosis , comprising a set of oligonucleotides according to any one of claims 1, 2. 4. A method for detecting drug resistance in M. tuberculosis , comprising amplifying a first target nucleotide sequence using a first pair of primers to obtain a first amplicon, wherein said first pair of primers is a pair of primers from the set of oligonucleotides according to claim 1, and detection of a mutation in the specified first amplicon, Moreover, the presence of a mutation indicates drug resistance. 5. The method according to item 4, characterized in that said detection step is carried out by means of sequencing. 6. The method according to item 4, characterized in that said detection step is carried out by means of a method including contacting said first amplicon with a first probe specific for said mutation under conditions favorable for hybridization to form a probe-target hybrid; performing a melting temperature (Tm) analysis to determine a test Tm value for said probe-target hybrid; and comparing the specified test value Tm with a predetermined reference value Tm, wherein a test Tm value that differs from a predetermined Tm value indicates the presence of a mutation. 7. The method according to claim 6, characterized in that said test value Tm, which is lower than said predetermined reference value Tm, indicates the presence of a mutation. 8. The method according to any one of claims 4 to 7, further comprising amplifying the second target nucleotide sequence using a second pair of primers to obtain a second amplicon, wherein said second pair of primers is specific for a fragment of a second region selected from the group consisting of the rpoB gene, the gyrA gene, the gyrB gene, the inhA promoter, the rrs gene, the eis promoter, the embB gene and the katG gene. 9. The method according to claim 8, characterized in that said first region is the rrs gene or the eis promoter. 10. The method according to claim 9, characterized in that said mutation is a mutation A1401G or C1402T in the rrs gene. 11. The method according to any one of claims 4-10, characterized in that said resistance is resistance to a drug selected from the group consisting of isoniazid, rifampicin, drugs of the fluoroquinolone class, amikacin, kanamycin, capreomycin and ethambutol.