IES85513Y1 - 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

Title I full gene sequences as a diagnostic target for the identilication of bacterial species.

Field of the Invention The prcscnl 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 thc Le/M and Gufl gene seqiicnccs. their corresponding RNAS. spccilic probes, primers and oligonuclcotidcs related thereto and their use in diagnostic assays to detect and/ or discriminate bacterial. yeast and liingnl spccics. i.e. microorganisms.

Background to the Invention Microbial infections represent a major cause of morbidity and mortality worldwide. and the .~;pcctrnin of microorganisms causing diseasc continues to increase. Microorganisms (bacteria. fungi and yeast) responsible for causing intectious diseases are traditionally detected in hospital lt-tl>0rtllt)l'icS with the aid ofmicrobiological culture methods with poor sensitivity (25-32%). which are very time-consuming, generally taking from two to live days to complete. and up to eight day»; for the diagnosis of fungal infections. Definitive diagnosis is usually based on either, the recovery and identification ofa specific microorganism from clinical specimcns or l}ll'L‘l‘t)S<.'0|)tC dcmonstration of fungi with distinct morphological features. lloncver. there are numerous cases where these methods fail to provide conclusive proofas to the infecting agent or inicrorgiinism. In these instances, the detection ofspecific host antibody responses can be used. although again this can be affected by the immune status of the patient.

'I mic is critical in the detection and identification ofinfectious microorganisms. Effective treatment depends on finding the source of infection and making appropriate decisions about zuitihiutics t|lllCl\l_Y and cfiiciently. Only after pathogens are correctly identified. can targeted lllL‘r.'I|))' using a specific antibiotic begin. Many physicians would like to see the development of hcttcr in vitro amplification and direct detection diagnostic techniques for the early diagnosis of microbial infection. Recently, Roche” launched a real time PCR based assay (Septifast ). for the detection ofinicrobial 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 (N/\ D) tests.

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

Mcthods that enable early diagnosis ofmicrobial causes ofinfcction enable the selection ofa spccilic narrow spectrum antibiotic or antilungal to treat the infection (Datamonitot rcpoit: Stiikcliolder opinion -Invasive fungal infections, options outweigh replacements 2004; l)iIllill'I()I‘|lI0r report: Stakeholder Opinion-Sepsis, under reaction to an overreaetion. 2006). l_c/1/\ tlcatlcr peptidase A) has recently been assigned the function of ribosomal elongation factor (Qin er al., 2006, Cell). Lap/X is highly conserved and is present in all bacteria and mitochondria. There are 2444 LepA gene sequences (~ 1.8 kb in length) available in Genliank including 2329 bacterial sequences. Using Clustal W sequence alignments. the Lep/\ gene of litici//t/.s’. [.l.$'lt’I‘/(I. Enlerobacteriaceae, Myc0but*teria, Staphylococci and Streptococci were compared in trim: to other molecular targets including Iuf/l and the 53:24 genes. In general. Le/7,-'l sceined to have sufficient sequence heterogeneity to enable its application for microorganism species identification in nucleic acid based tests (Table I).

Lep/I {range ol'% 1uj}«t (range of % ' .s:s‘r,-'l (range o % homology between homology between homology between species) species) species) species 72-97 8l -99 62- l 00 T;.?i?-i-Ts};-¢'ii?5 ' 39-90 99 97.99 I-:iiic.~ob;.cieiiiicéfic 59-99 33.99 93.99 (including I:'.co/i) ‘ill-'/‘l':t.:t)lli3-tl_t.T!t’I'illm species 78-99 87-1 00 34- I 00 .\'II~e/2Iou»ccn.-.‘ spec ies 70-9] 76-97 62-100 Tcr;_)/i1i'liit‘r)i'c'i/.s species 80-83 9| -95 8 I -99 Table |:—_I_’crcentage range of homology between Bacillus species, Listeria ‘species, F.nti.-robacteriaceae, Mycobacterium species. Streptococcus species and Staphylococcus species in the La»! gene compared to the mfA (equivalent commercialism] mRNA) and .\‘.vr.»1 genes tRiboSEQ technology).

GUI-‘I. which is similar to the E. coil’ elongation lactor—type GTP—binding protein l.epA. is a gene encoding 2| novel evolutionarily conserved GTPase coding protein tGTPase of Unknown Function 1. Kiser GI, and Weincrt TA (I995) GUFI, a gene encoding a novel evolutionarily conserved (iTPase in budding yeast. Yeast |l{l3): l3l 1-6), which, was predicted to be the t'j'|’|’:ise of the elongation factor-type class lhcre are 04 Gufl sequences available in NCBI Licnellztnk including 3 Candida and 6 A.v/Jergi/lus.

I)cl'initions "Synthetic oligonucleotide" refers to molecules of nucleic acid polymers of2 or more nucleotide bases that are not derived directly from genomic DNA or live organisms. The term synthetic oligonuclcotide is intended to encompass DNA, RNA, and DNA/RNA hybrid molecules that have been mziitulaetured chemically. or synthesized enzymatieally in virro.

An "oligonucleolide" is a nucleotide polymer having two or more nucleotide subunits covalently joined togctltet‘. Oligonucleotides are generally about 10 to about I00 nucleotides. the sugar groups of the nucleotide subunits may be ribose. deoxyribose, or modified derivatives thereof such as OMc. The nucleotide subunits may be joined by linkages such as phosphodiestcr linkages. modilied linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonuclcotide to its complementary target nucleotide sequence. Modified linkages include those in u liiclt a standard phosphodiesler linkage is replaced with a different linkage, such as a phospliorothioate linkage, a methylphosphonate linkage. or a neutral peptide linkage.

Nilrogcnous base analogs also may be components ofoligonucleotides 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 tleoxyribonucleotide or ribonucleotide sequence that can be hybridized to a complementary oligonuelcotidc.

An "oligonuclcotide probe" is an oligonueleotide having a nucleotide sequence sufiicienlly complementary to its target nucleic acid sequence to be able to fomt a detectable hybrid probenurgct duplex under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside ofthe targeted region as long as such nucleotides do not prevent hybridization under high stringency ltybridization 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 oligonueleotide probe optionally may be labelled with a detectable moiety such as 2| radioisotope, a fluorescent moiety, a chemiluminescent, a nanoparticle moiety. an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence. Oligonucleotide probes are preferred to be in the size range of from about I0 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 It! nucleotides in length.

A "liybrid" or a "duplex" is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairings or non—eanonical base pairings between the complementary bases. "llybridization“ is the process by which two complementary strands of nucleic acid combine to form a double-stranded structure ("hybrid" or "duplcx").

"Cotnplcincntarily" is a property conferred by the base sequence ofa single strand ol‘ DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNAIRNA or DNA:RNA 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 complctnents cytosine (C). lltc term "stringency" is used to describe the temperature. ionic strength and solvent composition c.\isting during ltybridilation and the subsequent processing steps. Those skilled in the {III will recognize that “stringency" conditions may be altered by varying those parameters either individually or together. Llnder high stringency conditions only highly complementary nucleic acid hybrids will Form; hybrids without a sufficient degree olcornplemcntarity will not lorm. /\ct:oI'dittgly. the stringency ofthe 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.

‘I-Iigh stringency‘ conditions are those equivalent to binding or hybridization at 42° C. in a soltttion consisting of5xSSPl-L (43.8gfl NaCl. 6.9 g/l NaH;PO4H;O and L85 g/l l’.l)‘l”A, ph zttljustcd to 7.4 with NaOH), 0.5% SDS, SxDcnhardt‘s reagent and |00ttg/ml denatured salmon sperm DNA lollowcd by washing in a solution comprising O.lxSSPE, l.0%SDS at 42° C. when :1 probe olabottt 500 nucleotides in length is used.

"Medium 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 1,35 g/l EDTA. pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt‘s reagent and I00 pg/ml denatured salmon sperm DNA Followed by washing in a solution comprising l.0xSSPE, l.0% SDS at 42° C, when a probe otabout 500 nucleotides in length is used.

‘Low stringency‘ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of SXSSPE (43.8 g/l NaC|. 6.9 g/l NaH3PO4H;O and l.8S g/l I-jl)’l’A, pH adjusted to 7.4 with NaOH). 0.l% SDS. 5xDenhardt’s reagent [50xDenhardt‘s contains per 500ml: Sg l-‘icoll {Type 400, Pharamcia). 5 3 BSA (Fraction V: Sigma)] and I00 pg/ml dcnnturcd salmon sperm DNA followed by washing in a solution comprising 5xSSl’l:', 0. l "/0 SDS at 42° C‘. when a probe olabout 500 nucleotides in length is used.

In the context ot‘ nucleic acid in-vitro ainplification based technologies, “stringcncy“ is achieved by applying temperature conditions and ionic buffer conditions that are particular to that in-wlrn amplilicntion technology. For example, in the context of PCR and real-time PCR. "stringency“ is uclticved by applying specific temperatures and ionic buffer strength for hybridisation ofthe oligonuclcotide 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 olthe invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid DJ an sequence. Tliis variation from the nucleic acid may be stated in terms ofa percentage of identical buses within the sequence or the percentage ofperfcctly complementary bases between the probe and its target sequence. Probes ofthc present invention substantially correspond to a nucleic acid sequence if these percentages are from about I00"/o to about 80% or from 0 base mismatches in about I0 nucleotide target sequence to about 2 bases mismatched in an about I0 nucleotide target sequence. In preferred embodiments. the percentage is from about I00"/is to about 85%. In more preferred embodiments. this percentage is from about 90% to about I00"/o; in other preI‘erred embodiments. this percentage is from about 95% to about l00°/o By "sulticiciitly complementary" or "substantially complementary" is meant nucleic acids having it sulficicnt amount ofcontiguous complementary nucleotides to form. under high stringency |tyb1'idi7ation conditions. a hybrid that is stable for detection.

B) "nucleic acid hybrid" or "probeztarget duplex" is meant a structure that is a double-stranded. hydrogen-bonded structure, preferably about I0 to about I00 nucleotides in length. more pt'clL‘rubly I4 to 50 nucleotides in length. although this will depend to an extent on the overall length ofthe oligonucleotide probe. The structure is sufficiently stable to be detected by means sueli us chcinilumincscent or fluorescent light detection. autoradiography. electrochemical aiialysis or gel clcctrophorcsis. Such hybrids include RNA:RNA. RNA:DNA. or DNAIDN/\ 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 (i.e., ribose versus deoxyribosc). 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 dililiercnces in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence, By "preferentially hybridize" is meant that under high stringency hybridization conditions oligonucleotide probes can hybridize their target nucleic acids to form stable probeztarget hybrids (thereby indicating the presence ofthe target nucleic acids) without forming stable pl‘Ol'lCLllt'lll~lZll'gCl hybrids (that would indicate the presence of non-target nucleic acids from other organisms). 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 ol'( for example Candida) and distinguish these species from other organisms.

Prelcrentinl hybridization can be measured using techniques known in the an and described herein.

By "tlicran0stics" is meant the use ofdiagnostic testing to diagnose the disease. choose the correct treatment regime and monitor the patient response to therapy. The tlicranostics ofthc invention maybe based on the use of an NAD assay ofthis invention on samples. swabs or spcciniens collected from the patient.

Object ofthe Invention It is an object of the 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 ofbacterial and yeast and Fungal species.

Suninizig 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 oligonucleotidc probe capable of binding to at least a portion of the LcpA gene or Gufl gene or its corresponding inI(N/ti. the oligonuclcotide probe may have a sequence substantially homologous to or substantially complementary to a portion ofthe LepA or Gufl gene or its corresponding inRNA.

It will thus be capable ofbinding or hybridizing with a complementary DNA or RNA molecule. llic nucleic acid molecule may be synthetic.

The kit may comprise more than one such probe. In particular the kit may comprise 11 plurality ofsuch probes. In addition the kit may comprise additional probes for other organisms. sucli as. for cxtnnple. bacterial species or viruses.

The portion oltlie Lep/t gene may, for example, be equivalent to a portion otthe region of the gene between base pair (bp) position 57 to bp 228 or bp position 522 to bp position 65‘) in Snt/I/n‘/<2cm'czz.i' cmreus. Particularly preierred are portions equivalent to a portion olthe region ofthe gene between base pair positions 66 to 2| 5, 66 to 8l , 200 to 2] 5 and I67 to 189 ofthc Group E3 strcptoccal Lep/\ gene. and positions 57 to 228. 57 to 74. 209 to 228 and H2 to 134 of the S. animus l.epA gene. The portion ofthe lepA gene may. for example. be equivalent to a portion ol the region of the gene between hp position 66 to bp 2 l S in Group B SIrepI4)c'0r.'c‘mi For l\t1yc<»bncteria. the portion ofLepA may be equivalent to a portion ofthe region ofthe gene between bp 618 to 772 and hp I203 to bp l8l7 in M. tuberculosis or the equivalent regions in other MiIon/vuclcriurn tuberculo5i'.'r complex tM'|”C) species and non—MTC mycobacteria. ln Bordetella. the portion ofthe Lep A may be equivalent to 3 regions bp I60 to bp 612. bp 552 to hp I081 and lip I006 to bp I638. '|‘he portion olthe Gull gene may be equivalent to a portion ofthe region ofthe gene from base pair position 190 to base pair position 2204 of the gene. Particularly preferred are portions equivalent to a portion ofthe region ofthe gene from base pair positions I90 to 1064, 270 to 300. I90 to 2l 2. 466 to 49|, 507 to 537. 466 to 489. 740 to 762, 828 to 858, 740 to 762 and i043 to i064 olthe C. albicans Gufl gene, or from base pair position 613 to 2204. 613 to 635. to 830. I339 to I358, I573 to I592, l95l to I073 and 2I33 to 2704 ofthe A, lumagalus (iull gene. lhe oligonuclcotide probe may have a sequence selected from the group comprising SEQ ID NO I l or SI-IQ ID NO I2, SEQ ID NO I5, SEQ II) NO 20. SEQ ID NO 2]. 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. Slit) ID NO 43. SEQ ID NO 46. SEQ ID NO 49. SEQ ID NO 52 or a sequence substantially lioinologons to or substantially complementary to those sequences, which can also act as a probe tor the l.ep/\ and or Gufl genes.

‘I he kit may comprise more than one such probe. In particular the kit may comprise a plurality olsuch pro.bcs. In addition the kit may comprise additional probes for other organisms, such as. for example. bacterial species or viruses. the itlentitied sequences are suitable not only for in two DNA/RNA amplification based tleteetinn systems but also for signal amplification based detection systems. Furthermore. the sequences ol‘the invention identified as suitable targets provide the advantages of having signiliezmt intraigenic sequence heterogeneity in some regions, which is advantageous and enables aspects olthe invention to be directed towards group or species-specific targets, and also ll2t\ ing significant sequence homogeneity in sotne 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 amplilication technologies for microorganism diagnostics. The LepA and Gufl sequences allow for multi-test capability and automation in diagnostic assays.

One ot‘ the advantages of the sequences ofthe present invention is that the intragenic l.epA and (jull nucleotide sequence diversity between closely related microorganism species enables spceilie primers and probes for use in diagnostics assays for the detection of bacteria to be tlesigned. the l.ep/\ and Gull nucleotide sequences. both DNA and RNA can be used with direct detection. signal amplification detection and in vim: amplification technologies in tliagiioslics assays. The LepA and Gull sequences allow for multi-test capability and automation in diagnostic assays.

The kit may further comprise at least one primer for amplification ofat least a ponion of the I.epA or (itilil genes. Suitably the kit comprises a forward and a reverse primer for a portion of the |,ep/\ or Gull gene. The kit may also comprise additional primers or probes. lhc primer may have a sequence selected from the group comprising SEQ ID NO |.2.3.4.5.6.7.3. 9. IO. I3, 14. I6. I7. I8, 19.22. 23. 24. 25, 28, 29, 3|, 32, 3134.38. 39, 4|. 42, 44. 45. 47. 48.50.51, 53, 54 or a sequence substantially homologous to or substantially eoinplenientary to those sequences, which can also act as a primers for the Lep/\ or Gull genes.

The kit may comprise at least one forward in viiro amplification primer and/ or at least one reverse in vi/m amplification primer. the forward amplification primer having a sequence selected lroin the group consisting of SEQ ID NO l.3 5,7, 9, 13. I6‘ I8. 22. 24. 38, 3|, 138. 4 I, 44. 47. S0, 53, or a sequence being substantially homologous or complementary thereto which can also act as a forward amplilication primer for the LepA or Gull gene. and the reverse amplitication primer having a sequence selected from the group consisting of SEQ ID NO 2, 4‘ 6. 8. l0, I-‘ll. l7. I9, 23, 25, 29. 32, 34, 39, 42. 45. 48, 51, 54, or a sequence being substantially homologous or complementary thereto which can also act as a reverse ampli lication primer for the l-cpA or (iufl gene.

The diagnostic kit may be based on direct nucleic acid detection technologies, signal amplitication nucleic acid detection technologies. and nucleic acid in viiro amplification teclinologics is selected from one or more of Polymerase Chain Reaction (PCR). Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based Amplification (NASBA), Strand Displacement /\mplit'ication (SD/\). Transcription Mediated Ainplification (TMA), Branched DNA technology (hl)NA) and Rolling Circle Amplification Technology (RCAT) ). or other in l'I'l'I‘() en7.yni;itic amplilication technologies. lhc inx-‘ention also provides a nucleic acid molecule selected from the group consisting of SEQ ID N0.| to SEQ ID NO. 178 and sequences substantially homologous thereto, or substantially complctnentary to a portion thereofand having a function in diagnostics based on the [.cpA l-Illtl/0!‘ (iull genes. The nucleic acid molecule may comprise an oligonucleotide having a sequence substantially homologous to or substantially complementary to a portion olia 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 probeztarget duplex; and (iii) determining whether a probertarget duplex is present; the presence of the duplex positively identifying the presence ofthe target organism in the test sample. lltc nucleic acid molecule and kits ofthe present invention may be used in it 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. lhc 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 bioburdcn or qua|il_\- assessment.

Pu ‘J: '. J L): The kits and the nucleic acid molecule ofthc invention may be used in the identification and/or Cl]:'1l"c'lCl(.’fl'/.'cIIl0n ofone or more disruptive agents that can be used to disrupt the Lep/\ or Gufl gene function. The disruptive agent may be selected from the group consisting of antisense RNA. PNA. and siRNA.

In some embodiments olthe invention, a nucleic acid molecule comprising a species-specific probe can be used to discriminate between species of the same genus. l he oligonucleotides ofthe invention may be provided in a composition for detecting the nucleic acids ol‘ microorganism target organisms. Such a composition may also comprise butters. 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 (lcSL‘t‘il)t.'tl herein as comprising at least one synthetic oligonucleotide, may also comprise nzuurzrl oligonucleotides with substantially the same sequences as the synthetic nucleotide fragments in place of. or alongside synthetic oligonueleotides.

Ihe invention also provides for an in vilro amplilication diagnostic kit for a target microor'ga-uiisin comprising at least one forward in virro amplification printer and at least one rcverse in vnm 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 ofone or more of or 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 of candidate IIllCI‘0('vt‘g(t|1lSlTl species. comprising one or more DNA probes comprising a sequence substantially complementary to, or substantially homologous to the sequence of the Lcp/\ or (julil gene oithe 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 olithe group consisting of the Lep/\ or (lull gene or ml{NA transcript thereof, the microorganism LepA gene or mRNA transcript thereof. one or more oI‘Sl-IQ ID NO I-SEQ ID NO I78. lhc nucleotide may comprise DNA. The nucleotide may comprise RNA. The nucleotide may comprise :1 mixture of DNA, RNA and PNA. The nucleotide may comprise synthetic nucleotides. 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 ol't|ie coding region ofthe Lep/\ or Gull gene. The gene may be a gene from a target inicroorganisin. The sequences oithe invention are preferably sufficient so as to be able form a probeztargct duplex to the portion ofthe sequence.

"I he invention also provides for a diagnostic kit For a target microorganism comprising an oligonuclcotide 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 wire amplification primers may also be suitable for use as oligonucleotidc probes: while it is preferable that amplification primers may have a complementary portion ol’ between about I5 nucleotides and about 30 nucleotides (more preferably about IS-about 23. most pi‘cl‘cr2ibl_v 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 it ill be required depending on the length, nature & structure (eg. llybridizatiott probe pairs for l,ightCycler. Taqman S’ exonuclease probes. hairpin loop structures etc. and sequence olilhc oligonucleotide probe selected.

Kits and assays ofthe invention may also be provided wherein the Oligonucleotide probe is immobili/ed on a surface. Such a surface may be a bead, a membrane, a column, dipstick. 2| l‘t.’lll0[')2It‘llClC. the interior surface ofa reaction chamber such as the well ofa diagnostic plate or inside ota reaction tube, capillary or vessel or the like. lhc Iztrgct microorganism may be selected from the group consisting ofStrep/0c'(1cc'u.\’. l:'mc-mtm't'zt.s'. Mvcobacteriu/rz, Bacillus, Listeria. Enlerobucteriaceae. N(’l.S‘.\‘L’!‘l(I, C‘/t/um_vdi’u.

Mtrop/u.wmi. Huemop/rilizls, C/osiridta, Bordelella cmdS1aphyl0c0cci. Gardnerc//a. (‘am/u/u. .»l.s'pt*rgi//tI.\' Under these circumstances, the amplification primers and oligonucleotide probes ofthe 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 of the desired organisms of the target organism grouping.

Ihc test sample may comprise cells ofthe target microorganism. The method may also comprise 41 step for releasing nucleic acid from any cclls 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 Ll 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 N01 to SEQ ID NO. I78 in a diagnostic assay for the presence of one or more microorganism species. The species may be selected from the group consisting ofStrepI0c0ccus, Emerococcus, MVcor‘2ucrerit1m. Bt1L':'.//llt\', /.i.tIt»/'iu. 1;‘/1/crolvacteriaceae . Nemeriu. (.'h1antydm. Mycoplasma, Haenrop/tilius. (’/rJ.ttri'¢!m. /iordtw/lu and Staphylococci. Gardnere/la. (.'undida, A5pergillu.~: llie 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 oligonuclcotidcs o|‘the invention. The kits may also comprise one or more articles selected from the group consisting olztppropriate sample collecting instruments, reagent containers, butters. labelling moieties. solutions, detergents and supplementary solutions. The invention also provides for use olithe sequences. compositions. nucleotide fragments, assays. and kits ofthe invention in thcranostics. 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 virro.

The nucleic acid molecules, composition. kits or methods may be used in a method olassessing the efliczicy ofa treatment regime designed to reduce microorganism titre in a patient comprising assessing the microorganism titre in the patient (by in vivo methods or in vilro metltotls) at one or more key stages of the treatment regime. Suitable key stages may include bclorc 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 cliaractcrization ofone or more disruptive agents that can be used to disrupt the l.epA and (int I gene functions. Suitable disruptive agents may be selected from the group consisting otiuitisensc RNA. PN/\. siRNA. liricl‘ Description of the Drawings Figure I: Real-time PCR amplification assay based on Lep/1 for S. aureus demonstrating the inclusivity oftlie S. aureus LepA rea|—time PCR assay.

Figure 2: Real-time PCR amplification assay based on Lep/4 for S. aureus demonstrating the sensitivity of the S. aureus Le/L4 real-time PC R assay.

Figure 3: Rea|~time PCR amplification assay based on Lep/I for B. perIu.v.i'i.i~ demonstrating the tnclusiv ity olthe B. perIu.rsi.s' Lep/-l real-time PC R assay.

Figure 4: Rcaltime PCR amplification assay based on Lep/1 for B. perlmzsis demonstrating the sensitivity ofthe B. pertussis LepA real—timc PCR assay.

Figure 5: Real-time PC R amplification assay based on LepA for Mycobacrerium tzzberculosis complex tleinonstrating the inelusivity of the Mycobacteriunz tubercu/o.ri.s* complex /.epA real- timc PCR 21551:)’.

Figure 6: Real-time PC R amplification assay based on LepA for Mycobaererizmi Ill/)L’I‘t_'ll/rJ.\'/.8‘ complex tleinonstrating the exclusivity of the Mi-cobacrerizmz rzzbercu/osis complex Lep/l real- timc PCR assay.

Figure 7: Re:i|—time PC R amplification assay based on Lep/1 for Mycobac/erium Iufzerc-u!o.n‘.i complc.\ demonstrating the sensitivity of the /l/ycoliacterium tuberculosis; complex Le/7A real- tinic PCR assay.

Ilctailed Description 01‘ the Invention Materials and Methods Bacterial strains: DNA stocks Ior Mi'c0I7ac'/eria spp. used in this study were obtained from an independent laboratory. ()ther bacterial species were grown in either Tryptone Soya broth. |,uria broth. nutrient broth or nutrient agar overnight at 37"C DNA Extraction l)NA was isolated from bacterial cells using the MagNA Pure System (Roche Molecular Systeiiis) in combination with the MagNA pure Yeast and Bacterial isolation kit ill or using the Iidge l3i0S_\’>‘t(:I1'tSl)ut’l.ilutCTM Bacterial Genomic Kit.

I.epA gene sequence analysis for diagnostics assay design The publicly available sequences oflhe 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 Mycobactcrial and Bordctclla species. 1-or Bordetella, PCR primers BRIF/BRIR amplify a region equivalent to l60bp to 612 bp olthe /3. perrzmis Tohama-a gene. PCR primers BRZF/BRZR amplify a region equivalent to 552 hp to 1081 bp oflhc B. pertussis" Tohama-a gene. Primer set BR3F/BR3R antplify from hp position 1006 to bp position 1638 bp of B. pertussis Tohama-a gene. Primer set M)'eobSl‘3/Myc()bSR2 amplify a region equivalent to bp 1203 to 1817 bp in the Miico/vac-Ieriimi iii/m'cz//om l.cpA gene. Conventional PCR amplification of these sequence regions in Bordctella and Mycobacteria species was perforiiicd using Sigma SuperPak N reagents on the MW(i Biotccli Primus using the thermocycling conditions outlined in table 3. PCR products were purified for DNA sequencing using either the High Pure PCR Product Purification Kit from Roche or the l:‘xoSAP-IT purification kit according to manufacturers instructions. Purilied PCR products were sent to the external sequence service provider for Sequencing. DNA sequence information for LepA was generated for regions 1-3 for B. pertussis, 13. aviuni. /3. pi’/rii and 1)’. /n2/messii for regions 1-2 For B. pm'uperlu5.s'is and B. bror2chi.sepIicu, B. /1/‘nzii and region 2 ml‘ /3 iremamm. DNA sequence information was generated For Mycobaclerial species Lop xl region equivalent to I203 bp to I8|7 bp in M. qfricanmn, M. bowls‘. M. bowls‘ BCG. M. cm/crrii. .11. mprue, M. microlri. M. pinipcdii. M. Izlbercu/rmls‘, M. snzegrnu/iv. M. cefuIum_ .-‘II. /orlmlum. M. inrmce/lu/are, M. ma!/mmen.\‘c. M. puramherczllosis and M. .\'(.'I'()f!I/(l('c’H/I}.

(.)Ii.éoi1ii|c|cotide Name 533‘ sequence "iii<'ii'V " "‘_ " /\GCGCCTTGACGTTC'l‘C nia I R AAGATYGCCGAHATCCGC "i3_R':>i~’ ' ' "C " " RTAYTCCTGSGGCATOAA mun CGTGTTCACGCCCAART BR3|~’ TTGATGCCSGCRATGA "fi2'3‘ii' I’ ACSATCAAGGCSCAGAC " "' " Ivl)-cohSl'~‘3 CACTCCGCGGTAGATGTC M3/L'(T1»sii€2'_"’ AAGTTCCTAATCTGCGCCG uencing primers for Bordetellu and M ycobacterial spp.

Step Tcinpcrature 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 I min c. Elongation 72°C 30 sec 30-35 3 Final 72°C 7 min I Elongation 4 d. Hold 8°C I Table 3: Thermocycling conditions used for amplification of Lep/1 gene regions in Bordelvllu and Mycolmcteria spp.

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

Lop/\ sequences available in GcnBank for Staphylococci and closely related species were aligned and PCR primer sets SAFI/RI and SAF2/R2 and oligonuclcotide probes SAPI and SAP2 were designed. Similarly, PCR primers UBSFl/R] and oligonucleotide probe GBSPI were designed based on in silico analysis of published 1.epA sequences For .S‘ri-eplomcci and closely related species. Sequence information generated for LepA regions I-3 in Barrie/e//u spp. was aligited with available [.epA sequences for Bordetella and closely related species in Gcnlizmk and 5 primer sets. 2 for region I. 2 for region 3 and l for region 2 were designed in ttdilition to 2 oligonucleotide probes each for regions I and 3 and I probe for region 2 for /3. pi'I‘III.¥.s'f.\' specific identification. Sequence information in GenBank and sequence information generated in this study for Mycobacterial species was analysed to design Primer set M'|‘C F/R and oligonueleotide probe MTCP for the detection of the MTC complex species. Additionally oligonuclcotidc probe MSP was designed for M. .s'megmam' LepA identification (Table 4).

In ii"/{cu analysis of GenBank Ouf 1 sequences for Candida and Aspergillus identified 3 gene regions suitable for oligonucleotide primer and probe design. A selection ofprimcrs and probes were designed from these regions for the identification of C. albicans and A.t';)cr'gi//t.t.s‘ ’/Ill)!/‘LftIII!.$’.

W'rc'i'i< |_ g V CATGGAGATCACCCGTGA l\1_'li(_>‘_l’__» _ TCGTCTTTGTGCACCCGAT/\C Msv _ ACGACCTTCTCCiGAACC_G'l‘ Table 4: Oligonucleotide primers and probes based on LepA for bacterial species identification llcmonslrtititm ol‘LepA as a target for bacterial species identification in real-time PCR assays. in demtmstratc the application of 1.epA as a target for bacterial spp. identification reul—time PCR zissz-nys were worked up for S. aureus, OBS. Mycobacterium complex species(M‘l‘C) and B()l'(/t.’/(.’//(I /)crnr.s'.t'i'.s'.

S. (IllI’l.'l!S L0]?/4 real-time PCR assay: the .8‘. uzu't'm' 1.e’pA real-time PCR assay was demonstrated using PCR primer set SA F2/R2 t0.5mM tinal concentration) and S‘ exonuclcasc probe SA P2 (0.2mM final concentration) on the l.ig|itCyc|er l.S using the l..1ghtCyc|cr Fast Start DNA Master HybProbe Kit and tlicrtnocycliitg conditions (Table 5). The panel ofS. aurezzs strains listed in table 6 were tested For inclu::ivit)- and all were detected in the S. aureus LepA(Figure 1) while the other 51‘cipll_\ltIL'UCCl species and related species (Table 6) were not detected in the test. The limit of detection of the S. aureus LepA real-time PC R test was established to be 2-20 S. uzzrem cell cquivalcnts (tigurc 2).

CBS Le/2.4 real-time PCR assay: The CBS /.cp/\ real-time PCR assay was demonstrated using PCR primer set GBSFI/R] (0.5mM linal concentration) and 5' cxonuclcasc probe GBSPl2 t0.2mM final concentration) on the |,igltI(_‘yclci' |.5 using the LightCycler Fast Stan DNA Master Hyb|’robc Kit and lltcrmocycling conditions (Table 5). The panel of (785 strains listed in table 6 were tested For inclnsivity and all were detected in the assay while the other streptococci species and related species (Table 7) were not detected in the test.

B. /wrtm'.\‘i.s‘ I.cp.4 real-time PCR assay: "the If. [m-/1r.w'x Le/.wA real-time PCR assay was demonstrated using PC R primer set BP3 F2/R2 (t>.SmM Iinal concentration) and 5‘ exonuclease probe BP3P2 (0.2mM final concentration) on the l.ig|it(‘yc|cr l.S using the LightCyclcr Fast Start DNA Master HybPrr>be Kit and ll1C‘I'lTl0CyClll‘lg conditions (Table 5). lnclusivity testing detected 4 of4 B. perrzu;sz.s' strains tested and did not detect other Bordelelta spp. strains (Figure 3). The limit of detection based on amplilication of serial dilutions of B. perIus.«*m~ genomic DNA in the B. per/2/sxis l.epA real- time PCR assay was established as 20 B. pu'tz«.s:s-is cell equivalents (Figure 4).

M'I‘(,‘ LcpA real-time PCR assay: A biplcx real-time PCR assay for the detection of'MTC LepA and M. sniegzimxrix l,epA has been configured on the LightCycler 2.0 instrument incorporating PCR primers MT(.‘Fl/Rl(0.5mM) for’ the co-amplification of MTC species and M. smegmaiis and S‘e.\'onuclease probes M I Cl’(0.2mM) labelled with I-lF.X/BHQI dye quencher combination and probe MSl’(0.2inM) labelled with CV5/BHQ2 dye quencher moieties. Thermocycling is performed as described in table 5. lnelusivity testing for the MTC assay showed detection of all members 0|‘ the MIC while non-M‘|‘C species were not detected Figures 5. 6. The LOD ofthe MTC assay for M. lb DNA \\.'1s approximately 3 cell equivalents (Figure 7). _‘ Step Temperature Time Cycles 1 Denaturation 95°C I0 min I 2 Amplification Denaturation 95°C I0 sec 50 H Annealing,/elongation 60°C 30 sec 50 ’i 3 Cooling 40°C 30 see I ‘Fable 5: Real-time PCR conditions for the S. aureus LepA real-time PCR Species ganel Source/Strain S. aureus DSMl2463 S. aureus 9518 S. aureus DSM346 S. azzrem 8325.4 S. aureus 252 S. aureus ATCC 9144 '. uureus DSM l5676 S. aureus col . aureux NCTC I 1963 Ecol)’ DSM 30] K, aerogem‘ ATCC 43086 K. oxyloca NCTC 9528 L. monocvtogencs Serovar 7 L. monocyrogenes Food isolate S. alga/acliae DSM ZI34 S‘. epic/errnidix DSM 20044 S. epiderlrridis unryped S. /7aem0Ivn‘cu.s' DSM 20263 S. saprnphylicus ATCC 15305 P inirubi/i.€ DSM 4479 B. cercus DSM 3 l S. clvrornogenex DSM 20454 I M. caesoiyticus I DSM 20597 ] Table 6: S. aureus strains, Staphylococci and other species tested in the S. aurem‘ LcpA rea|—timc PCR assay.

Species panel OBS IF] 0 Sir-epr0cm"‘c-u.s' dlmagulac-Iicae .$'trep!oc0cc*ux pncmrmniuc Slrgflococcus purusanguimls Streptococcus in!€I'!I7e(/ills Streptococcus zuberis Streptococcus milis Enterococcus /éwcalm Enterococcus faecium Streptococcus mulans Slreprococcuspyogenes StrepI0coccu.s- .s'(ingm's Strcpmcocgu.sporc'!r2us SIrepI0c0cc'u.s' /)oi'i.s Slap/IV/0(‘0€ClI.\‘ am-ous Bacillus ccrcus Enlerococcus_faccali.v Enlerococcus /iiecimn Slaphy10c0cc:i.v cpiderrniciis Sraphvlococcus haemo/yn‘cu.i Stcgaliyiococcus .s'apr0ph_vu'cus Table 7: (IBS strains. Streptococci species and related species tested in the LcpA GBS rcal-lime PCR assay.

|’('R primers and laqlvlan probes were designed from Lep/I sequence informalion for S‘ aureuv and X. ug¢:/uczicw (Table 2). Real-time PCR assays incorporating these primers and probes were Llcinonslruled on the LightCycler. Specificity testing was performed using 3 seleclion ofllic relevant closely species listed in Table 3. The S. aurcus assay was l00% specific for S. uzrrcus and I|1c.S'. ugaluctirzc assays detected all S. agalactiae strains and did not cross-react with any closely related Streptococcal species.

The words “comprises/comprising" and the words “having/including" when used herein with I‘clL'rCr:cc to the present invention are used to specify the presence ofstated features. integers. steps or cmiipttttcnls but does not preclude the presence or addition of one or more other lbttttircs. ititegcrs. steps. components or groups thereof.

It is appreciated that certain features ofthe invention, which are, for clarity, described in the context ofseparate 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 :1 single embodiment, may also be provided separately or in any suitable sub-combination.

Sequence listing S|iQ ID»; Silcs ol‘prob<.~s. oligonuclcoiidcs etc. are shown in bold and underlined.

N or x- any nucleotide; w=a/I. m=a/C. r=a/g. k=g/I. s=c/g, y=c/I. h=a/t/c. v=a/g/C. d=a/g/L b=g/1/c. In some cases, specific degeneracy options are indicated in parenthesis: c.g.: (a/g)

Claims (5)

Claims

1. A tli:-ignostic 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 ofthe LcpA and/or (Juli! genes or its corresponding mRNA

2. A kit as claimed in claim I, wherein the portion ofthe LepA gene is equivalent to a portion of the region ollhe gene from base pair position 57 to base pair position 228. position 57 to 74. position 209 to 229, position I I2 to I34. or from position 522 to 659 oI‘Slap/Iyioco(‘aux uimwx l.cp/\ gene, from base pair position 66 to 215. position 66 to 81. position 200 to Z I 5 or from position I67 to I89 in Group B .S'IrepIoc0Ccu.s LcpA gene, from base pair position ol8 to 772 in Mycobacterium species. from base pair position I203 to l8l7 in M)‘CL)i)tICI€l‘iUIII species, or from base pair position I60 to 612, position 552 to I08l or from position 1006 to I638 in Bordetella species and/or wherein the portion ofthe Gull gene is equivalent to a portion ofthe region ofthe gene from base pair position I90 to base pair position 2204 ofthe gene, from base pair position I90 to I064, position 270 to 300, position I90 to 2 I 2. position 466 to 49l, position 507 to 537, position 466 to 489, position 740 to 763. position 828 to 858. position 740 to 762 or from position I043 to 1064 ofthe C. zilbiczins (iull gene, or from base pair position 6|3 to 2204, position 6l3 to 635. position 820 to 839. position I339 [0 I358. position I573 to I592. position I9S| to I973 and 2183 to 2104 olthe A. fumagatus Gufl gene.

3. A kit as claimed in claim I or 2 comprising a probe selected from the group comprising SIZQ ID NO II or SEQ ID N012, SEQ ID NO I5. SEQ ID NO 20, SEQ ID NO 2|. 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 scqticnces 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. I8. 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 of SEQ ID NO 2, 4. 6. 8. I0. I4. l7. I9. 23. 25. 29. 32, 34, 39, 42, 45, 48. SI 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 I78 and sequences substantially homologous or substantially complementary thereto or to a portion Ihereofand having a Function in diagnostics based on the l.epA and or Gull gem.

5. A kit substantially as described herein with reference to the examples and/or the uccon1panying figures