IE20090470U1 - LepA/Guf1 gene sequences as a diagnostic target for the identification of bacterial species. - Google Patents

Abstract

ABSTRACT The current invention relates to a diagnostic kit for a bacterial species and/or fungal zmd/or )cu5l species comprising at least one oligonucleotide probe capable of binding to at least :1 portion of the LepA and/or Gufl genes or its corresponding mRNA.

Description

The present invention relates to nucleic acid primers and probes to detect one or more bacterial and yeast and fungal species. More specifically the invention relates to the I.epA and Gufl gene sequences, their corresponding RNAS, specific probes, primers and oligonucleotides related thereto and their use in diagnostic assays to detect and/ or discriminate bacterial. yeast and fungal species, i.e. microorganisms.

Background to the Invention Microbial infections represent a major cause of morbidity and mortality worldwide. and the spectrum of microorganisms causing disease continues to increase. Microorganisms (bacteria, fungi and yeast) responsible for causing infectious diseases are traditionally detected in hospital laboratories with the aid of microbiological culture methods with poor sensitivity (25-82% , which are very time-consuming, generally taking from two to five days to complete, and up to eight days for the diagnosis of fungal infections. Definitive diagnosis is usually based on either. the recovery and identification ofa specific microorganism from clinical specimens or microscopic demonstration of fungi with distinct morphological features. However. there are numerous cases where these methods fail to provide conclusive proof as to the infecting agent or microrganism. In these instances, the detection ofspecific host antibody responses can be used, although again this can be affected by the immune status ofthe patient.

Time is critical in the detection and identification ofinfectious microorganisms. Effective treatment depends on finding the source of infection and making appropriate decisions about antibiotics quickly and efficiently. Only after pathogens are correctly identified, can targeted therapy using a specific antibiotic begin. Many physicians would like to see the development of better in vitro amplification and direct detection diagnostic techniques for the early diagnosis of microbial infection. Recently, Roche“ launched a real time PCR based assay (Scptifast""), for the detection ofmicrobial DNA in clinical samples. Therefore, there is a clear need for the development ofnovel rapid diagnostic tests for clinically significant bacterial and fungal pathogens for bioanalysis applications in the clinical sector. This has led the current inventors to identify novel nucleic acid targets for application in Nucleic Acid Diagnostic (NAD) tests.

It is clear though. that development of faster, more accurate diagnostic methods are required, particularly in light of the selection pressure caused by modern anti-microbial treatments which give rise to increased populations ofresistant virulent strains with mutated genome sequences.

Methods that enable early diagnosis of microbial causes of infection enable the selection ofa spccifict narrow spectrurniantibioticcor a.nt,ifuij_gal to treat the infection (Datamonitor re on: Stakeholder opinion —Invasive fungal infections, options outweigh replacements 2004; Datamonitor report: Stakeholder Opinion—Sepsis, under reaction to an ovcrreaction. 2006). l,opA (leader peptidase A) has recently been assigned the function of ribosomal elongation factor (Qin at al., 2006, Cell). LepA is highly conserved and is present in all bacteria and mitochondria. There are 2444 LepA gene sequences (~ 1.8 kb in length) available in GenBank including 2229 bacterial sequences. Using Clustal W sequence alignments, the LcpA gene of Baa’!/us, Listeria, Enterobacteriaceae, Myeobacreria, Staphylococci and Slrepmcocci were compared in silico to other molecular targets including tuf/I and the .rsrA genes. In general, Le/IA seemed to have sufficient sequence heterogeneity to enable its application for microorganism species identification in nucleic acid based tests (Table l).

LepA (range of % tuj.‘-l (range of % .s‘.wa4 (range of % homology between homology between homology between species) species) species) Bacillus‘ species 72-97 8 l -99 62- l 00 L/gm-m species 39-90 99 97-99 Enlerobacteriaceae 59-99 83-99 92-99 (including Ecoli) Mvcobaclerium species 78-99 87-] 00 84- I 00 .$'/repI0coc'c'n.s' species 70-9l 76-97 62- I00 Strip/iy/ucuccus species 80-83 9 l -95 8 I -99 Table 1: Percentage range of homology between Bacillus species, Listeria species, Enterobaeteriaceae, Mycobacterium species, Streptococcus species and Staphylococcus species in the 1.epA gene compared to the tufA (equivalent commercialised mRNA) and genes (RihoSEQ technology). (jUl“l, which is similar to the E. coli elongation factor-type GTP-binding protein LepA, is a gene encoding a novel evolutionarily conserved GTPase coding protein (GTPase of Unknown Function l. Kiser GL and Weinert TA (I995) GUF1, a gene encoding a novel evolutionarily conserved GTPase in budding yeast. Yeast ll(l3): l3l I-6), which, was predicted to be the G'l‘l’ase of the elongation factor-type class. There are 94 Gufl sequences available in NCBI GeneBani< including 3 Candida and 6 Axpergillus.

Definitions "Synthetic oligonucleotide" refers to molecules ofnucleic acid polymers of2 or more nucleotide bases that are not derived directly from genomic DNA or live organisms. The term synthetic $090470 oligonucleotide is intended to encompass DNA, RNA, and DNA/RNA hybrid molecules that have been manufactured chemically, or synthesized enzymatically in vitro.

An "oligonucleotide" is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about l0 to about 100 nucleotides. The sugar groups ofthe nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may bejoined by linkages such as phosphodiester linkages. modified linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleotide sequence. Modified linkages include those in which a standard phosphodiester linkage is replaced with a different linkage. such as a phosphorothioate linkage, a methylphosphonate linkage, or a neutral peptide linkage.

Nitrogcnous base analogs also may be components ofoligonueleotides in accordance with the invention.

A "target nucleic acid" is a nucleic acid comprising a target nucleic acid sequence. A "target nucleic acid sequence," "target nucleotide sequence" or "target sequence" is a specific deoxyribonucleotide or ribonucleotide sequence that can be hybridized to a complementary oligonucleotide.

An "oligonucleotide probe" is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to fomt a detectable hybrid probe:target duplex under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions. Non-complementary sequences. such as promoter sequences‘ restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes. An oligonucleotide probe optionally may be labelled with a detectable moiety such as a radioisotope, a fluorescent moiety, a chemiluminescent, a nanopanicle moiety, an enzyme or a ligand. which can be used to detect or confirm probe hybridization to its target sequence. Oligonueleoticle probes are preferred to be in the size range of from about 10 to about I00 nucleotides in length. although it is possible for probes to be as much as and above about 500 nucleotides in length, or below 10 nucleotides in length.

A "hybrid" or a "duplex" is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairings or non—canonical base pairings between the complementary bases. "Hybridization" is the process by which two complementary strands of nucleic acid combine to fonn a double-stranded structure ("hybrid" or "dup|ex"), "Complementarity" is a property conferred by the base sequence ofa single strand of DNA or RNA which may fomt a hybrid or double-stranded DNA:DNA. RNA:RNA or DNA:RNA '4 J 9 through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) ordinarily complements thymine (T_) or uracil (U), while guanine (G) ordinarily complements cytosine (C).

The term "stringency" is used to describe the temperature, ionic strength and solvent composition existing during hybridization and the subsequent processing steps. Those skilled in the art will recognize that “stringency” conditions may be altered by varying those parameters either individually or together. Under high stringency conditions only highly complementary nucleic acid hybrids will fonn; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.

‘High stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of5xSSPE (43.8g/I NaCl. 6.9 g/l NaH;PO4H2O and 1.85 g/l EDTA. ph adjusted to 7.4 with NaOH), 0.5% SDS, 5>tDenhardt’s reagent and l00pg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.lxSSPE, l.O%SDS at 42° C. when a probe olabout 500 nucleotides in length is used.

“Medium stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of SXSSPE (43.8 g/I NaCl, 6.9 g/l NaH2PO4H2O and l.85 g/I EDTA. pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt’s reagent and I00 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising l.0xSSPE, l.O% SDS at 42"‘ C, when a probe of about 500 nucleotides in length is used.

‘Low stringency‘ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting ofSxSSPE (43.8 g/l NaCl, 6.9 g/l NaH;PO4H2O and 1.85 g/l ED'l'A, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5xDenhardt’s reagent [50xDenhardt‘s contains per 500ml: Sg liicoll (Type 400, Pharamcia), S g BSA (Fraction V; Sigma)] and 100 pg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5xSSPE, 0. I % SDS at 42° C, when a probe of about 500 nucleotides in length is used.

In the context of nucleic acid in-vitro amplification based technologies, “stringency,/“ is achieved by applying temperature conditions and ionic buffer conditions that are particular to that i'n-vitro amplification technology. For example, in the context of PCR and real-time PCR, "stringcncy“ is achieved by applying specific temperatures and ionic buffer strength for hybridisation of the oligonucleotide primers and, with regards to real-time PCR hybridisation of the probe/s, to the target nucleic acid for in-virro amplification ofthe target nucleic acid.

One skilled in the art will understand that substantially corresponding probes ofthe invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid lEv9t)-470 sequence. This variation from the nucleic acid may be stated in terms ofa percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes ofthe present invention substantially correspond to a nucleic acid sequence if these percentages are from about 100% to about 80% or from 0 base mismatches in about l0 nucleotide target seqtience to about 2 bases mismatched in an about I0 nucleotide target sequence. In preferred embodiments, the percentage is from about l00% to about 85%. In more preferred embodiments, this percentage is from about 90% to about I00‘?/o; in other preferred embodiments, this percentage is from about 95% to about 100% By "sufficiently complementary" or "substantially complementary" is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.

By "nucleic acid hybrid“ or "probeztarget duplex" is meant a structure that is a double-stranded, hydrogen-bonded structure, preferably about 10 to about 100 nucleotides in length, more preferably [4 to 50 nucleotides in length, although this will depend to an extent on the overall length ofthe oligonucleotide probe. The structure is sufficieiitly stable to be detected by means such as cliemilurninescent or fluorescent light detection, autoradiography, electrochemical analysis or gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

"RNA and DNA equivalents" refer to RNA and DNA molecules having the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar groups {ie., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents do not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree ofcomplementarity to a particular sequence.

By "preferentially hybridize" is meant that under high stringency hybridization conditions oligonucleotidc probes can hybridize their target nucleic acids to fomi stable probeztarget hybrids (thereby indicating the presence ofthe target nucleic acids) without forming stable probe:iion-target hybrids (that would indicate the presence of non-target nucleic acids from other organisins). Thus, the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one skilled in the art to accurately detect the presence oftfor example Candida) and distinguish these species from other organisms.

Preferential hybridization can be measured using techniques known in the art and described herein.

By "thcranostics" is meant the use ofdiagnostic testing to diagnose the disease, choose the correct treatment regime and monitor the patient response to therapy. The theranoslics of the lEo9o47o invention may be based on the use ofan NAD assay ofthis invention on samples, swabs or specimens collected from the patient.

Object of the Invention It is an object ofthe current invention to provide sequences and/or diagnostic assays to detect and identify one or more microorganism species (bacteria. yeast, fungi). The current inventors have made use ofthe LepA and Gufl gene sequences to design primers and probes for use in the detection and identification of bacterial and yeast and fungal species.

Summagy of the Invention the present invention provides a diagnostic kit for detection and identification of bacterial and yeast and fungal species i.e. microorganisms, comprising at least one oligonucleotide probe capable ofbinding to at least a portion ofthe LepA gene or Gufl gene or its corresponding mRNA. The oligonucleoticle probe may have a sequence substantially homologous to or substantially complementary to a portion of the LepA or Gufl gene or its corresponding mRNA.

It will thus be capable of binding or hybridizing with a complementary DNA or RNA molecule.

The nucleic acid molecule may be synthetic.

The kit may comprise more than one such probe. In particular the kit may comprise a plurality ofsuch probes. In addition the kit may comprise additional probes for other organisms. such as, for example. bacterial species or viruses.

The portion ofthe LepA gene may, for example, be equivalent to a portion ofthe region of the gene between base pair (bp) position 57 to bp 228 or bp position 522 to bp position 659 in .S'taphyloc-occus aureus. Particularly preferred are portions equivalent to a portion of the region of the gene between base pair positions 66 to 2l5, 66 to 81, 200 to 215 and I67 to 139 ofthe Group B streptoccal LepA gene, and positions 57 to 228, 57 to 74, 209 to 228 and I I 2 to I34 of the S. aureus LepA gene. The portion ofthe LepA gene may, for example, be equivalent to a portion of the region of the gene between bp position 66 to bp 21 S in Group B Streptococcus.

For Mycobacteria, the portion of LepA may be equivalent to a portion ofthe region ofthc gene between bp 6 I 8 to 772 and bp I203 to bp l8] 7 in M. tuberculosis or the equivalent regions in other Mycobaczcrium tuberculosis complex (MTC) species and non-MTC mycobacteria. ln Bordetclla. the portion ofthe Lep A may be equivalent to 3 regions bp I60 to bp 612, hp 552 to bp |08| and bp I006 to bp I638.

The portion olthe Gufl gene may be equivalent to a portion ofthe region ofthe gene from base pair position 190 to base pair position 2204 ofthe gene. Particularly preferred are portions equivalent to a portion of the region ofthe gene from base pair positions I90 to i064. 270 to 300. I90 to 212, 466 to 49], 507 to 537. 466 to 439, 740 to 762, 828 to 858, 740 to 762 and I043 to 1064 of the C. albicans Gufl gene. or from base pair position 613 to 2204, 613 to 635. lEo9o47o to 839. 1339 to I358, I573 to 1592, |95l to I973 and 2l83 to 2204 ofthe A. fumagatus Gufl gene.

The oligonucleotide probe may have a sequence selected from the group comprising SEQ ID NO I I or SEQ ID NO I2, SEQ ID N015, SEQ ID NO 20, SEQ ID NO Zl, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 30 SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 40, SEQ ll) NO 43. SEQ ID NO 46, SEQ ID NO 49, SEQ ID NO 52 or a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a probe for the LepA and or Gufl genes.

The kit may comprise more than one such probe. In particular the kit may comprise a plurality ofsuch probes. In addition the kit may comprise additional probes for other organisms, such as, for example, bacterial species or viruses.

The identified sequences are suitable not only for in vitro DNA/RNA amplification based detection systems but also for signal amplification based detection systems. Furthermore. the sequences ofthe invention identified as suitable targets provide the advantages of having significant intragenic sequence heterogeneity in some regions, which is advantageous and enables aspects of the invention to be directed towards group or species-specific targets. and also having significant sequence homogeneity in some regions, which enables aspects ofthe invention to be directed towards genus-specific microorganism primers and probes for use in direct nucleic acid detection technologies, signal amplification nucleic acid detection technologies. and nucleic acid in vitro amplification technologies for microorganism diagnostics. The LepA and Gufl sequences allow for multi-test capability and automation in diagnostic assays.

One ofthc advantages ofthe sequences ofthe present invention is that the intragenic LepA and Gull nucleotide sequence diversity between closely related microorganism species enables specific primers and probes for use in diagnostics assays for the detection of bacteria to be designed. The LepA and Gufl nucleotide sequences, both DNA and RNA can be used with direct detection, signal amplification detection and in vilro amplification technologies in diagnostics assays. The LepA and Gufl sequences allow for multi-test capability and automation in diagnostic assays.

The kit may further comprise at least one primer for amplification of at least a portion of the LepA or Gufl genes. Suitably the kit comprises a forward and a reverse primer for a portion of the LepA or Ciufl gene. The kit may also comprise additional primers or probes.

The primer may have a sequence selected from the group comprising SEQ ID NO I,2,3,4.S.6.7,8. 9, I0, I3, I4, I6. I7, I8, I9. 22, 23, 24. 25. 28, 29. 3I, 32, 33. 34, 33. 39. 41,42. 44, 45. 47. 48. 50, SI, 53.54 or a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a primers for the LepA or Gull genes.

IJ U- L.) In lEo9o47o The kit may comprise at least one forward in 1-'ll)‘0 amplification primer and/ or at least one reverse in riirr) amplification primer, the forward amplification primer having a sequence selected from the group consisting of SEQ ID NO 1,3 5,7, 9, 13, 16, 18, 22. 24. 28, 3|, 33, 38. 41, 44. 47, S0. 53, or a sequence being substantially homologous or complementary thereto which can also act as a forward amplification primer for the LepA or Gufl gene, and the reverse amplification primer having a sequence selected from the group consisting ofSEQ ID NO 2, 4, 6. 8. l0. l4. l7, l9, 23, 25, 29, 32, 34, 39, 42, 45, 48, 5], 54, or a sequence being substantially homologous or complementary thereto which can also act as a reverse amplification primer for the LepA or Gufl gene.

The diagnostic kit may be based on direct nucleic acid detection technologies, signal amplification nucleic acid detection technologies. and nucleic acid in vilm amplification technologies is selected from one or more of Polymerase Chain Reaction lPCR,). Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based Amplification (NASBA), Strand Displacement Atnplification (SDA), Transcription Mediated Amplification (TMA), Branched DNA technology (bDNA) and Rolling Circle Amplification Technology (RCAT) ). or other in virro enzymatic amplification technologies.

The invention also provides a nucleic acid molecule selected from the group consisting ofSEQ ID NO.l to SEQ ID NO. I78 and sequences substantially homologous thereto. or substantially complementary to a portion thereof and having a function in diagnostics based on the l.epA and/or (jufl genes. The nucleic acid molecule may comprise an oligonucleotide having a sequence substantially homologous to or substantially complementary to a portion ofa nucleic acid molecule of SEQ ID NO.l to SEQ ID NO. I78. The invention also provides a method of detecting a target organism in a test sample comprising the steps of: ti) mixing the test sample with at least one oligonucleotide probe as defined above under appropriate conditions; and (ii) hybridizing under high stringency conditions any nucleic acid that may be present in the test sample with the oligonucleotide to form a probe:target duplex; and (iii) determining whether a probe:target duplex is present; the presence ofthe duplex positively identifying the presence ofthe target organism in the test sample.

The nucleic acid molecule and kits ofthe present invention may be used in a diagnostic assay to detect the presence of one or more bacterial species, to measure microorganism titres in a patient or in a method of assessing the efficacy ofa treatment regime designed to reduce microorganism titre in a patient or to measure microorganism contamination in an environment.

The environment may be a hospital, or it may be a food sample, an environmental sample e.g. water, an industrial sample such as an in-process sample or an end product requiring bioburden or quality assessment.

{Q um lEo9o47o The kits and the nucleic acid molecule ofthe invention may be used in the identification and/or characterization of one or more disruptive agents that can be used to disrupt the LepA or Gufl gene function. The disruptive agent may be selected from the group consisting of antisense RNA, PNA, and siRNA.

In some embodiments ofthe invention, a nucleic acid molecule comprising a species-specific probe can be used to discriminate between species of the same genus.

The oligonucleotides ofthe invention may be provided in a composition for detecting the nucleic acids of microorganism target organisms. Such a composition may also comprise buffers, enzymes, detergents, salts and so on, as appropriate to the intended use ofthe compositions. It is also envisioned that the compositions, kits and methods ofthe invention, while described herein as comprising at least one synthetic oligonucleotide, may also comprise natural oligonucleotides with substantially the same sequences as the synthetic nucleotide fragments in place of, or alongside synthetic oligonucleotides.

The invention also provides for an in vitro amplification diagnostic kit for a target microorganism comprising at least one forward in vitro amplification primer and at least one reverse in virro amplification primer, the forward amplification primer being selected from the group consisting of one or more ofa sequence being substantially homologous or complementary thereto which can also act as a forward amplification primer, and the reverse amplification primer being selected from the group consisting of one or more ofor a sequence being substantially homologous or complementary thereto which can also act as a reverse amplification primer.

The invention also provides for a diagnostic kit for detecting the presence ofcandidate microorganism species, comprising one or more DNA probes comprising a sequence substantially complementary to, or substantially homologous to the sequence ofthe LepA or Gufl gene of the candidate microorganism species. The present invention also provides for one or more synthetic oligonucleotides having a nucleotide sequence substantially homologous to or substantially complementary to one or more of the group consisting ofthc LepA or Gufl gene or mRNA transcript thereof, the microorganism LepA gene or mRNA transcript thereof. one or more of SEQ ID NO I-SEQ ID NO I78.

The nucleotide may comprise DNA. The nucleotide may comprise RNA. The nucleotide may comprise a mixture of DNA, RNA and PNA. The nucleotide may comprise synthetic nucleoti«.lcs. The sequences ofthe invention (and the sequences relating to the methods. kits compositions and assays ofthe invention) may be selected to be substantially homologous to a portion of the coding region ofthe LepA or Gufl gene. The gene may be a gene from a target microorganism. The sequences of the invention are preferably sufficient so as to be able form a probeztargct duplex to the portion ofthe sequence, ‘P090470 The invention also provides for a diagnostic kit for a target microorganism comprising an oligonucleotide probe substantially homologous to or substantially complementary to an oligonuclcotide ofthe invention (which may be synthetic). It will be appreciated that sequences suitable for use as in vitro amplification primers may also be suitable for use as oligonucleotidc probes: while it is preferable that amplification primers may have a complementary portion of between about IS nucleotides and about 30 nucleotides (more preferably about l5-about 23, most preferably about 20 to about 23), oligonucleotide probes ofthe invention may be any suitable length. The skilled person will appreciate that different hybridization and or annealing conditions will be required depending on the length. nature & structure (eg. Hybridization probe pairs for I,ightCycler, Taqman S’ exonuclease probes, hairpin loop structures etc. and sequence ofthe oligonucleotide probe selected.

Kits and assays ofthe invention may also be provided wherein the oligonucleotide probe is immobilized on a surface. Such a surface may be a bead, a membrane, a column, dipstick, a nanoparticlc. the interior surface ofa reaction chamber such as the well ofa diagnostic plate or inside of a reaction tube. capillary or vessel or the like.

The target microorganism may be selected from the group consisting of Streptococcus, Enreroctoccus, Mt/cobacterizzm, Bacillus, Listeria, Emerobacteriaceae , Ncisscria, Clzlcnnyclra, /if}-'('Up/ Aspergil’/14.3’ Under these circumstances, the amplification primers and oligonucleotide probes of the invention may be designed to a gene specific or genus specific region so as to be able to identify one or more. or most, or substantially all ofthe desired organisms ofthe target organism grouping.

The test sample may comprise cells ofthe target microorganism. The method may also comprise a step for releasing nucleic acid from any cells ofthe target organism that may be present in said test sample. Ideally, the test sample is a biological sample obtained from a patient (such as a swab. or blood. urine. saliva, a bronchial lavage dental specimen. skin specimen. scalp specimen. transplant organ biopsy, stool. mucus. or discharge sample). The test samples may be a food sample, a water sample an environmental sample, an end product, end product or in- process industrial sample.

The invention also provides for the use ofany one of SEQ ID NO.l to SEQ ID NO. [78 in a diagnostic assay for the presence of one or more microorganism species. The species may be selected from the group consisting of Slreptococcus, Enterococcus, Mycobacrerium. Baci//us, [_i.m»riu, lzmerobacteriaceae. Netsseria, Chianzydia. Mycoplasma, Haemop/ii/ius, C l0.s'!ric/id.

Borcielel/ct and Staphylococci, Gardncrella, Candida, Aspergtilus .; The invention also provides for kits for use in clinical diagnostics, theranostics, food safety diagnostics, industrial microbiology diagnostics. environmental monitoring, veterinary diagnostics, bio-terrorism diagnostics comprising one or more ofthe synthetic oligonucleotides ofthe invention. The kits may also comprise one or more articles selected from the group consisting ofappropriate sample collecting instruments, reagent containers. buffers. labelling moieties, solutions, detergents and supplementary solutions. The invention also provides for use olthe sequences, compositions, nucleotide fragments, assays, and kits ofthe invention in lheranostics, Food safety diagnostics, Industrial microbiology diagnostics, Environmental monitoring, Veterinary diagnostics, Bio-terrorism diagnostics.

The nucleic acid molecules, composition. kits or methods may be used in a diagnostic nucleic acid based assay for the detection of microorganism species.

The nucleic acid molecules, composition, kits or methods may be used in a diagnostic assay to measure microorganism titres in a patient. The titres may be measured in vitro.

The nucleic acid molecules, composition, kits or methods may be used in a method of assessing the cflicacy ola treatment regime designed to reduce microorganism titre in a patient comprising assessing the microorganism titre in the patient (by in vivo methods or in virro methods) at one or more key stages of the treatment regime. Suitable key stages may include before treatment, during treatment and after treatment. The treatment regime may comprise an anti- microbial or anti-fungal agent, such as a pharmaceutical drug.

The nucleic acid molecules, composition, kits or methods may be used in a diagnostic assay to measure potential microorganism contamination, for example, in a hospital.

The nucleic acid molecules, composition, kits or methods may be used in the identification and/or characterization of one or more disruptive agents that can be used to disrupt the LepA and Out I gene functions. Suitable disruptive agents may be selected from the group consisting oliantiscnsc RNA, PNA, siRNA.

Brief Descriptiog of the Drawig Figure l: Real-time PCR amplification assay based on Lep/l for S. aureus demonstrating the inclusivity ofthe S. aureus Lep/l real-time PCR assay.

Figure 2: Real-time PCR amplification assay based on LepA for S. aureus demonstrating the sensitivity ofthe S. aureus Lep.4 real-time PCR assay.

Figure 3: Rcal—time PCR amplification assay based on LepA for B. pertussis demonstrating the inclusivity ofthe B. pertussis LepA real-time PCR assay.

Figure 4: Rca|~time PCR amplification assay based on LepA for B. pertzmis demonstrating the sensitivity of the B. pertussis Lap/1 real-time PCR assay. ix) ‘.1: IE090470 Figure 5: Real-time PC R amplification assay based on Lep/1 for Mycobacreritm: tzzbercwosis complex demonstrating the inclusivity of the Mycobacteriwn tuberculosis complex I.epA real- time PCR assay.

Figure 6: Real-time PCR amplification assay based on Lep/I for Mycobacrerium Iuberczi/o.s'i.s' com plex demonstrating the exclusivity of the Mycobacterizmz tuberculosis complex Le/M real- time PCR assay.

Figure 7: Real-time PCR amplification assay based on LepA for Mycobacterium Iubcrczdosis complex demonstrating the sensitivity of the Mvcobacterium tuberculosis complex Lepi-‘I real- time PCR assay.

Detailed Descriptigg of the Invention Materials and Methods Bacterial strains: DNA stocks for Mycobacteria spp. used in this study were obtained from an independent laboratory. Other bacterial species were grown in either Tryptone Soya broth, Luria broth. nutrient broth or nutrient agar overnight at 37°C‘ DNA Extraction DNA was isolated from bacterial cells using the lvlagNA Pure System (Roche Molecular Systems) in combination with the MagNA pure Yeast and Bacterial isolation kit ill or using the Edge Biosystems PurEluteTM Bacterial Genomic Kit.

LepA gene sequence analysis for diagnostics assay design The publicly available sequences ofthe LepA genes for Mycobacteria and Bordetella spp. were acquired from the Genbank database and aligned using Clustal W. PCR primers {Table 2) were designed to amplify regions of the LepA gene in a range of Mycobacterial and Bordetella species. For Bordetella, PCR primers BRIF/BRIR amplify a region equivalent to l60bp to 6I2 bp otitlie B, pertussis Tohama-a gene. PCR primers BRZF/BRZR amplify a region equivalent to 552 bp to 108! bp ofthe B. pertussis Tohama-a gene. Primer set BR3F/BR3R amplify from bp position 1006 to bp position I638 bp of B. pertussis Tohama-a gene. Primer set Myc0bSF3/Myc0bSR2 amplify a region equivalent to bp I203 to I817 bp in the M_vcobacteriimz Ill/7t’I'L'Ii/0.8’/S LepA gene. Conventional PCR amplification of these sequence regions in Bordetella and Mycobacteria species was performed using Sigma SuperPal; TM reagents on the MWG Biotech Primus using the thermocycling conditions outlined in table 3. PCR products were pnrilied for DNA sequencing using either the High Pure PCR Product Purification Kit from Roche or the ExoSAP-IT purification kit according to manufacturers instructions. Purilied PCR products were sent to the external sequence service provider for sequencing. DNA sequence information for Lep/I was generated for regions 1-3 for B. pL’!‘Il(SSi.S', B uvium, B. perrii and B. /10/messii for regions l-2 for B. pm-apertussis and B. bronclziseptica, B. hinzii and IEo9o47o region 3 ofb’. /remalum. DNA sequence information was generated for Mycobacterial species Lap ,4 region equivalent to 1203 bp to l3|7 bp in M. africarmm, M. bovis, M. bowl: BCG. M. ccmcilii, M. caprae, M. microlti, M pinipedii, M. tuberculosis, M. smegmafis, M. cc/alum, M. _/ormirzmz. M. intrace//m’are, M. ma/Ifl0I1L’fl.&‘(.’. M. pararuberculosis and M. scmfulaceuni. fOlig0nucIeotidc Name 5’-3 ’ sequence BR I F AGCGCCTTGACGTTCTC BR] R AAGATYGCCGAHATCCGC BRZF RTAYTCCTGSGGCATGAA BRZR CGTGTTCACGCCCAART 3n3r TTGATGCCSGCRATGA BR3R ACSATCAAGGCSCAGAC MycobSF3 CACTCCGCGGTAGATGTC M_vcobSR'2 AAGTTCCTAATCTGCGCCG Table 2: Sequencing primers for Bordetella and Mycobacterial spp.

Step Temperature Time Cycles I Denaturation 95°C 3-4 min I 2 Amplification a. 95°C 30 sec 30-35 Denaturation b. Annealing 50°C or 30 sec or 30-35 55°C l min c. Elongation 72°C 30 sec 30-35 3 Final 72°C 7 min I Elongation 4 d. Hold 8°C 1 Table 3: Tliermocycling conditions used for amplification of LepA gene regions in Bordetella and Mycobacteria spp.

PCR primer and DNA probe design for Lep A and GufA targets.

LcpA sequences available in GenBank for Staphylococci and closely related species were aligned and PCR primer sets SAF1/RI and SAF2/R2 and oligonucleotide probes SAP] and lEo9o47oM SAP2 were designed. Similarly, PCR primers GBSFI/RI and oligonucleotide probe GBSPI were designed based on in silico analysis of published LepA sequences for Srrcptocucci and closely related species. Sequence information generated for LepA regions I-3 in Bordelel/a spp. was aligned with available LepA sequences For Bordetella and closely related species in GenBank and 5 primer sets, 2 for region I, 2 for region 3 and I for region 2 were designed in addition to 3 oligonucleotide probes each for regions I and 3 and l probe for region 2 for B. pe/‘Izr.r.s‘i.s‘ specific identification. Sequence information in GenBank and sequence information generated in this study for Mycobacterial species was analysed to design Primer set MTC F/R and oligonueleotide probe MTCP for the detection of the MTC complex species. Additionally oligonucleotide probe MSP was designed for M. srnegrnatis LepA identification (Table 4). in silico analysis of GenBank Guf 1 sequences for Candida and Aspergillus identified 3 gene regions suitable for oligonucleotide primer and probe design. A selection of primers and probes were designed from these regions for the identification of C. albicans and Aspergillzis jirnr/‘gums.

Oligonucleotide name Oligonucleotide sequence S’-‘3 S. uureur SAFI TACCAACTGCTTTCATCT SARI CAATTTGAAGTACCTGTACA SAF2 k TTTACGTTGACATAATTCCA SAR2 CAGAAGTGACGGTTGATA SAPI TTTACG GCT TAT GTCACCGCCAT SA P2 TAGTTGCACGAACATATGGCTC Group B Srrepmcocci GBSF I AACCAATTGCTTTCAT GBSR I CAGCTATTGGACAAAA GBSPI CGTAGTGCTTTTATATCAGAACG Bonletelln pertussis BPFI A ACGAACTCGTAGTCCATCGAC BPR I A GCGCTTGTTGTTGCACAGT BPFI B GTCGAAGAAATCGAGCAC BPR] B GTATTCCTGGGGCATGAA BP I |’A CTGGCGCCCGTGGTAGCTC BPI PB TTGTTGCACAGTGTCATCACCG BP2l-‘l CGCTGTTCGACCTCATAG BPZRI CAGCTGGTCGTATTCGGA BPZI’ GACACTTCGGGCTCGAACATCA BP3Fl TCCATCTTGTTGAGCAC BI’3Rl AACGACTGACTTCGTAC BP3 F2 "l‘GCCGATCACGTCT'I‘ BP3R2 GGTGGTTCGACGCTTC BP3l’l ACTTCCATGCCCAGTTCG BP3|’3 CGAACTGGGCATGGAAG Mycobacteria M'l‘CFl AGACCGTGCGGATCTTG lE09047o M’l'CRl CATGGAGATCACCCGTGA MTCI’ TCGTCTTTGTGCACCCGATAC MSP ACGACCTTCTCGGAACCGT Table 4: Oligonucleotide primers and probes based on 1.epA for bacterial species identification Demonstration of LepA as a target for bacterial species identification in real-time PCR assays. ’| 0 demonstrate the application of LepA as a target for bacterial spp. identification real-time PCR assays were worked up for S. aurcus, GBS, Mycobacterium complex specicslMTC) and Bun/etc//ct pertussis.

S. aureus LepA real-time PCR assay: The S. uurem LepA real-time PCR assay was demonstrated using PCR primer set SAF2/R2 (0.5inM final concentration) and 5’ exonuclease probe SAP2 (0.2mM final concentration) on the l-ightCyc|er l.5 using the LightCyc|er Fast Start DNA Master llybProbe Kit and thcrmocycling conditions (Table 5). The panel of S. aureus strains listed in table 6 were tested for inclusivity and all were detected in the S. aureus LepA(Figure I) while the other staphylococci species and related species (Table 6) were not detected in the test. The limit of detection of the S. aureus LepA real-time PCR test was established to be 2-20 5‘. aurem cell equivalents ll-‘igure 2).

GBS Lep.4 real-time PCR assay: The GBS l.epA real-time PCR assay was demonstrated using PCR primer set GBSFI/RI (0.5mM final concentration) and 5’ exonuclease probe GBSPl2 (0.2mM final concentration) on the l,ightCycler 1.5 using the LightCycler Fast Start DNA Master HybProbe Kit and Iherinocycling conditions (Table 5). The panel of GBS strains listed in table 6 were tested for inclusivity and all were detected in the assay while the other streptococci species and related species (Table 7} were not detected in the test.

B. pertussis LepA real-time PCR assay: The B. pcr1zr5.w'.r LepA real-time PCR assay was demonstrated using PCR primer set BP3 F2/R2 (0.5mM Final concentration) and S‘ exonuclease probe BP3P2 (0.2mM final concentration) on the LightCycler l.5 using the LightCycler Fast Start DNA Master HybProbe Kit and ihennocycling conditions (Table 5)‘ lnclusivity testing detected 4 of4 B. pertzz.mL~: strains tested and did not detect other Bordetella spp. strains (Figure 3). The limit of detection based on IE09o47o amplification of serial dilutions of B. perrmszzs genomic DNA in the B. pertussis Le/7A real- time PCR assay was established as 20 B. pertussis cell equivalents (Figure 4).

MTC Lep.4 real-time PCR assay: A biplcx real-time PCR assay for the detection ofMTC LepA and M. smegnialix l,epA has been conligurcd on the LightCycler 2.0 instrument incorporating PCR primers M‘l‘(.‘Fl/Rl(O.SmM) for the co-amplification of MTC species and M. smegmatis and 5’exonuclease probes MTCP(0.2mM) labelled with HEX/BHQI dye quencher combination and probe MSP(O.2mM) labelled with CY5/BHQ2 dye quencher moieties. Thermocycling is performed as described in table 5. lnclusivity testing for the MTC assay showed detection of all members of the MTC while non—M'l‘C species were not detected Figures 5. 6. The LOD of the MTC assay for M. tb DNA was approximately 3 cell equivalents (Figure 7).

Step Temperature Time Cycles 1 Denaturation 95°C It} min I 2 Amplification Denaturation 95°C I0 sec 50 Annealing/elongation 60°C 30 sec 50 3 Cooling 40°C 30 sec l Table 5: Real-time PCR conditions for the S. aureus LepA real-time PCR Species panel Source/Strgi_n S. aureus DSM] 2463 S. aureus 9518 S. aureus DSM346 S. aureus 8325.4 S. aureus 252 S. aureus ATCC 9 I44 S. aureus DSM IS676 S. aureus col S. aureus NCTC 1 I963 Ecoli DSM 301 K. aerogens ATCC 43036 K. oxvroca NCTC 9528 L. monacvlugenes Serovar 7 L. monocyrogenes Food isolate S. algalacliae DSM 2134 S. epidermidis DSM 20044 S. epidermidis untyped S. haemolyticus DSM 20263 S. yaprophyticus ATCC 15305 P. mirabilis DSM 4479 B. cereus DSM 3| S. chromogenes DSM 20454 lEu9U47o [ M. caesolyticus I DSM 20597 j Table 6: S. aureus strains, Staphylococci and other species tested in the S. uureus LcpA real-time PCR assay.

Species panel GBS n=l0 Streptococcus dvsagalacticae Streptococcus prieurnorziae Streptococcus parasanguinis Streptococcus inlcrmedius Streptococcus uberis Streptococcus mitis Emerococcus /izecu/is Enterococcusfaecizmz Slreprococcus mutant‘ Streptococcus pvogenes Streptococcus sanguis Streptococcus porciruur Streptococcus bow’: Staphylococcus aureus Bacillus cereus En!eroc0ccusfaccu1i.s' Enterococcus faecium Staphylococcus epidermidis Slap/tvlococcus haemolvticus Staphylococcus‘ saproplryticus Table 7: GBS strains, Streptococci species and related species tested in the LcpA GBS real-time PCR assay. l’CR printers and TaqMan probes were designed from Lep.4 sequence information for uureus and S. aga/acliuc (Table 2). Real-time PC R assays incorporating these primers and probes were demonstrated on the LightCycler. Specificity testing was perfonned using a selection ofthe relevant closely species listed in Table 3. The S. aureus assay was 100% specific for .5‘. aureus and the S. agulactiae assays detected all S. agalacliae strains and did not cross-react with any closely related Streptococcal species.

SEQ |l)s Sites of probes. oligonucleotides etc. are shown in bold and underlined. lEo9o47o N or x: any nucleotide; w=a/t, m=a/c, r=a/g, k=g/t, s=c/g, y=c/t, h=a/L/c, v=a/g/c, d=a/g/t, b=g/1/c. In some cases, specific degeneracy options are indicated in parenthesis: c.g.: (a/g) is either A or G.

Bordetclla spa. primers and grobes: SEQ ID NO 1: E; 5: AGCGCCTTGACGTTCTC-3‘ SEQ ID NO 2: BR] R: S’-AAGATYGCCGAHATCCGC-3‘ SEQ ID NO 3: mm‘: 5‘-RT/\ YTCCTGSGGCATGAA-3’ SEQ ID NO 4: mejzizz 5'-CGTGTTCACGCCCAART-3‘ SEQ ID NO 5; BR3F: 5‘-’l'|‘GATGCCSGCRATGA—3’ SEQ ID NO 6: BRJR: 5’-ACSATCAAGGCSCAGAC-3’ SEQ ll) N0 7: urn A: 5‘-ACGAACTCGTAGTCCATCGAC-3‘ SEQ ID NO 3: BPRIA: 5‘-GCGCTTGTTGTTGCACAGT-3’ SEQ ID NO 9: BPFIB: 5’-GTCGAAGAAATCGAGCAC-3‘ SEQ ID N010: BPRI B: S’-GTATTCCTGGGGCATGAA-3’ SEQ ID NO 11: BPIPA: 5‘-CTGGCGCCCGTGGTAGCTC-3‘ SEQ 11) NO 12: BI’! PB: 5‘-TTGTTGCACAGTGTCATCACCG-3’ SEQ ll) NO 13: BPZFI: 5’—CGCTGTTCGACCTCATAG—3’ SEQ ID NO 14: man 1: 5’-CAGCTGGTCGTATTCGGA-3‘ SEQ ID NO 15: BPZI’: S’~(iACACTTCGGGCTCGAACATCA-3‘ sm ID NO 16: !Eo9o47o BP3Fl: S’-TCCATCTTGTTGAGCAC-3’ SEQ ID NO 17: BPJRI: 5’-AACGACTGACTTCGTAC-3’ SEQ ID NO 18: BP3 F 2: 5’-TGCCGATCACGTCTT-3‘ SEQ ID NO 19: Bl’3R2: 5‘-GGTGGTTCGACGCTTC-3’ SEQ ID NO 20: Bl’3Pl: S‘—ACTTCCATGCCCAGTTCG-3’ SEQ ID NO 21: B]’3l’2: 5‘—CGA ACT GGG CAT GGA AG-3‘ S. aureus primers and probes: SEQ ID NO 22: SA Fl : 5’-TACCAACTGCTTTCATCT-3‘ SEQ ID NO 23: SARI: 5ZCAATTTGAAGTACCTGTACA-3‘ SEQ ID N() 24: SA F2: 5‘-TTTACGTTGACATAATTCCA-3‘ SEQ ID NO 25: SAR2: S‘-CAGAAGTGACGGTTGATA-3‘ SEQ ll) N0 26: SAP]: 5‘-TTTACGGCTTATGTCACCGCCAT-3 ‘ SEQ ID NO 27: SAP2: 5’-TA (JTTGCACGAACATATGGCTC-3’ GBS primers and probes; SEQ ID NO 28: GBSFI: 5‘—AACCAATTGCTTTCA'l‘-3‘ SEQ ID NO 29; GBSRI: 5‘~CAGCTATTGGACAAAA-3‘ SEQ ID NO 30: (.‘BSPl: S’-CGTAGTGCTTTTATATCAGA/‘\CG—3‘ Mycobacterium primers and probes: SEQ ID NO 31: MycobSF3: S‘-CACTCCGCGGTAGATGTC-3‘ SEQ ID NO 32: MycobSR2: 5‘-AAGTTCCTAATCTGCGCCG-3’ lEo9o47o SEQ ID NO 33: MTCF1: 5‘-AGACCGTGCGGATCTTG-3’ SEQ ID no 34: MTCRI: 5’-CATGGAGATCACCCGTGA-3’ SEQ [D N 0 35: MTCP:5’- TCGTCTTTGTGCACCCGATAC-3 ‘ SEQ ID NO 36: MSP: S‘-ACGACCTTCTCGGAACCGT-3’ Crmdirla nlbicans primers and probes: SEQ ID NO 37: Gufl Region I Probe Pl-Ca|biGufI: 5’ — CGA GAG GGA AAG AGG AAT TAC‘ AGT GAA AGC C — 3’ Gufl gene nucleotide base position from 270 to 300 SEQ ID NO 38: Gufl Region I CanGufl-I l‘ Forward primer: 5‘- ATT GTO GCA CAC GTT GAC CAT GG- 3’ Gufl gene nucleotide base position from I90 to 2l2 SEQ ID NO 39: Gufl Region I CanGufl-l r Reverse primer: 5’ —TGT GCT TGA ACT CCT TGA OAT GCA TC- 3’ Gufl gene nucleotide base position from 466 to 491 SEQ ID NO 40: Gufl Region 2 Probe P2-CalbiGufl: 5’ — CTA CTT GGC ATA CAG CAT GGG ATT GAA ATT G — 3’ Gufl gene nucleotide base position from 507 to 537 SEQ ID NO 41: Gufl Region I CanGufl—2F Forward primer: 5“- GAT GCA TCT CAA GGA G’l”I‘ (‘AA GC - 3’ Gufl gene nucleotide base position from 466 to 489 SEQ ID NO 42: Gufl Region 2 CanGufl-2r Reverse primer: 5’ - ATC ATG CCA AGA ATC CAC CAA TA — 3’ Gufl gene nucleotide base position from 740 to 762 SEQ ID NO 43: Gufl Region 3 Probe P3-Ca|biGufl : 5’ — CTT GTC AGC GCA CAC AAA TAG GAC A'l'A CGA C ~ 3’ Gufl gene nucleotide base position from 828 to 858 SEQ ID NO 44: Gufl Region 3 CanGufl ~31”, Forward primer: S’- TAT TGG TGG ATT CTT GGC ATG AT - 3’ Gufl gene nucleotide base position from 740 to 762 SEQ ID NO 45: Gufl Region 3 CanGufl-3r, Reverse primer: 5’ — GGG AAT GCC CCA ACA AAT ACC A— 3’ Gufl gene nucleotide base position from 1043 to I064 Aspergillus famrigams primers and probes: SEQ ID NO 46: Gufl Region 1 probe P1-AfumiGufl 5’ —CGC AAA CCT OCT CGA TGA 'l‘A'|‘ ACA A'|‘C AC- 3‘ SEQ ID NO 47: Gufl Region I AspGufl-I f, Forward primer: 5’ —GCC CAT GTC GAT CAT GGC AAA AG- 3’ Gufl gene nucleotide base position from 6l3 to 635 SEQ ID NO 48: Gufl Region 1 AspGufl—l r Revere primer: 5’ —ACC TCT GCA CGG AAG TCC AC- 3‘ Gufl gene nucleotide base position from 820 to 839 SEQ ID NO 49: Gufl Region 2 probe P2-AfumiGut‘l 5' -CAC CAC AGA GCG TGC TCC GTG CCG GC- 3‘ SEQ ID NO 50: Gufl Region 2 ASpGufl-2f. Forward primer: 5’- GAG GTT GGC ATC ATG '|‘A'|‘ CC— 3‘ Gufl gene nucleotide base position from 1339 to I353 SEQIDN05k(hHlgbn2AwGuflQrNwmwpfimm19—AGCTGG1TGATACTG TC1”lC-3‘Guflgenenucbofidebascposnkntfionll573tol592 SEQ ID NO 52: Gufl Region 3 probe P3-AfumiGufI ‘ —CCACTC/\AGTCAAGTGGAAAGGCTCCIGAC- 3’ SEQ ID NO 53: Gufl Region 3 AspGuf]-3fForward primer: 5’ —GAG TAT TTC ACA CCA ACG CAG GT- 3’ Gufl gene nucleotide base position from I951 to I973 SEQ ID NO 54: Gufl Region 2 AspGufI-3r Reverse primer: 5’ —TTG AAT TTT GTC ACC CAT TGT C- 3‘ Gufl gene nucleotide base position from 2183 to 2204 SEQ ID 55 to 153 Gufl/LepA Sequences from public databases SEQIDNOSS The words “comprises/comprising” and the words “having/including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, lE09047o steps or components but does not preclude the presence or addition ofone or more other Features. integers, steps, components or groups thereof. lt is appreciated that certain features olithe invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.

Conversely, various Features ofthe invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub—combinalion.

Claims (5)

1. A diagnostic kit for a bacterial species and/or fungal and/or yeast species comprising at least one oligonucleotide probe capable ofbinding to at least a portion ofthc LepA and/or Gufl genes or its corresponding mRNA

2. A kit as claimed in claim I, wherein the portion ofthe LepA gene is equivalent to a portion ofthc region ofthe gene from base pair position 57 to base pair position 228, position 57 to 74, position 209 to 229, position I 12 to 134, or from position 522 to 659 ol‘Stap/iylococcus uurvu.s' LepA gene, from base pair position 66 to 215, position 66 to 81. position 200 to 215 or from position 167 to I89 in Group B Streptococcus LepA gene, from base pair position 618 to 772 in Mycobacterium species, from base pair position I203 to I817 in Myeobactcrium species. or from base pair position 16010 612, position 552 to 1081 or from position 1006 to I638 in Bordetella species and/or wherein the portion ofthe Gufl gene is equivalent to a portion ofthe region ofthe gene from base pair position 190 to base pair position 2204 ofthe gene, from base pair position 190 to 1064, position 270 to 300, position 190 to 212, position 466 to 49], position 507 to 537, position 466 to 489, position 740 to 762. position 828 to 858, position 740 to 762 or from position I043 to I064 ofthe C. albicans Gufl gene, or from base pair position 613 to 2204, position 613 to 635, position 820 to 839. position 1339 to 1358, position 1573 to 1592, position I951 to 1973 and 2183 to 2204 of the A. fumagams Gufl gene.

3. A kit as claimed in claim I or2 comprising a probe selected From the group comprising SEQ ID NO I I or SEQ ID NO 12, SEQ ID N015, SEQ ID NO 20. SEQ ID NO 21, SEQ ID NO 26. SEQ ID NO 27, SEQ ID NO 30 SEQ ID NO 35, SEQ ID NO 36. SEQ ID NO 37, SEQ ID NO 40, SEQ ID NO 43, SEQ ID NO 46, SEQ ID NO 49, SEQ ID NO 52 or sequences substantially similar or complementary thereto which can also act as a probe, and/or a forward primer selected from the group comprising of SEQ ID NO 1.3 5,7, 9, I3, I6, 18,22, 24, 28, 31, 33, 38, 41, 44, 47, 50 or 53 or sequences substantially similar or complementary thereto which can also act as a forward amplification primer and/or a reverse primer selected from the group consisting ofSEQ ID NO 2, 4, 6. 8, 10, 14, I7, 19. 23, 25. 29, 32, 34, 39, 42. 45, 48, 51 or 54, sequences substantially similar or complementary thereto which can also act as a reverse amplification primer.

4. A nucleic acid molecule selected from the group consisting of: SEQ ID NO I through SEQ ID NO 173 and sequences substantially homologous or substantially complementary thereto or to a portion thereofand having a function in diagnostics based on the LepA and or Gufl gene.

5. A kit substaniially as described herein with reference to the examples and/or the accompanying figures.