IES85514Y1 - Ace2 as a target gene for the molecular identification of yeast and fungal species. - Google Patents

Abstract

ABSTRACT the present invention relates to nucleic acid primers and probes for use in the idcntilictition of one or more yeast species. More specifically the invention relates to the Ace) gene. the corresponding RNA. specific probes, primers and oligonueleotides related thereto and their use in diagnostic assays to detect and I or discriminate between yeast species.

Description

Field of the Invention The present invention relates to nucleic acid primers and probes for use in the identification of one or more yeast species. More specifically the invention relates to the Ace2 gene, the corresponding RNA, specific probes, primers and oligonucleotides related thereto and their use in diagnostic assays to detect and I or discriminate between yeast species.

Background to the Invention Yeast and ftmgal infections represent a major cause of morbidity and mortality among immunocompromised patients. The number of immunocompromised patients at risk of yeast and fungal infection continues to increase each year, as does the spectrum ol‘ fungal and yeast agents causing disease. Mortality from fungal infections, particularly iiimsixe fungal infections, is 30% or greater in certain risk groups. The array oi’ available anti-fungal agents is growing; however, so too is the recognition of both intrinsic and emerging resistance to antifungal drugs. These factors are contributing to the ln<.‘[‘C'clSCCl need for cost containment in laboratory testing and have led to laboratory consolidation in testing procedures.

Invasive fungal infections are on the increase. In 2003, it was estimated that there were 9 million at risk patients of which 1.2 million developed infection. Ctmdida species now rank as the most prominent pathogens infecting immunosupressed patients. In particular, infections are common in the urinary tract, the respiratory system and the bloodstream, at the site of insertion of stents. catheters and orthopaedic joints.

Approxiinately 10% of the known Candida spp. have been implicated in human infection. Invasive candidiasis occurs when candida enters the bloodstream and it is estimated to occur at a frequency of 8/l00,000 population in the US with a mortality rate of 40%. Candida albicans is the 4'" most common cause of bloodstream infection. limergin g mycoses agents include Fusarium, Scedosporium, Zygomycetes and Trichosporon spp. (“Stalceholder Insight: Invasive fungal infections”, Datamonitor, Jan 2on4).

Immunocomprornised patients including transplant and surgical patients, neonates, cancer patients, diabetics and those with HIV/AIDS are at high risk of developing invasive fungal infections (Datamonitor report: Stakeholder opinion -Invasive fungal infections, options outweigh replacements 2004). A large number of severe cases of sepsis are reported each year. Despite improvements in its medical management, sepsis still constitutes one of the greatest challenges in intensive care medicine. Microorganisms (bacteria, fungi and yeast) responsible for causing sepsis 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 filngal infections.

Definitive diagnosis of infection caused by yeast or fungus is usually based on either, the recovery and identification of a specific agent from clinical specimens or microscopic demonstration of fungi or yeasts with distinct morphological features.

However, there are numerous cases where these methods fail to provide conclusive proof as to the infecting agent. In these instances, the detection of specific host antibody responses can be used, although again this can be affected by the immune status of the patient. ‘lime is critical in the detection and identification of bloodstream infections typically caused by bacteria. yeast or fungi. Effective treatment depends on finding the source of infection and making appropriate decisions about antibiotics or antifungals quickly and efficiently.

Fungal and yeast nucleic acid based diagnostics have focused heavily on the ribosomal RNA (rRNA) genes, RNA transcripts, and their associated DNA / RNA regions. The rRNA genes are highly conserved in all fungal species and they also contain divergent and distinctive intergenic transcribed spacer regions. Ribosomal rRNA comprises three genes: the large sub-unit gene (23S), the small sub-unit gene (1 8S) and the 5.88 gene. The 28S and 18S rRNA genes are separated by the 5.83 rRNA and two internal transcribed spacers (ITSI and ITS2). Because the ITS region contains a high number of sequence polymorphisms, numerous researchers have concentrated their efforts on these as targets (Atkins and Clark, 2004). rRNA genes are also multicopy genes with >10 copies within the fungal genome.

A number of groups are working on developing new assays for fungal and yeast infections. US2004044193 relates to, amongst a number of other aspects. the transcription factor CaTEC1 of Candida albicans; inhibitors thereof, and methods for the diagnosis and therapy of diseases which are connected with a Candida infection; and also diagnostic and pharmaceutical compositions which contain the nucleotide sequences, proteins, host cells andfor antibodies. W001 83 824 relates to hybridization assay probes and accessory oligonucleotides for detecting ribosomal nucleic acids from C 'andida albicans and/or Candida dubliniensis. U360] 7699 and US5426026 relate to a set of DNA primers, which can be used to amplify and speciate DNA from five medically important Candida species. US 6747137 discloses sequences useful for diagnosis of Candida infections. EP 0422872 and US 5658726 disclose probes based on 18S rRNA genes, and US 5958693 discloses probes based on 28S rRNA, for diagnosis of a range of yeast and fungal species. US 6017366 describes sequences based on chitin synthase gene for use in nucleic acid based diagnostics for a range of Candida species. 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 of resistant virulent strains with mutated genome sequences. Methods that enable early diagnosis ofmicrobial causes of infection enable the selection of a specific narrow spectrum antibiotic or antitungal to treat the infection (Datamonitor report: Stakeholder opinion -Invasive lungal infections, options outweigh replacements 2004; Datamonitor report: Stakeholder Opinion—Sepsis, under reaction to an overreaction, 2006). only after pathogens are correctly identified can targeted therapy using a specific antibiotic or antifungal begin. Many physicians would like to see the development of better in vitro amplification and direct detection diagnostic techniques for the early diagnosis of yeast and fungi (“Stakeholder Insight: Irzvasivefurzgul i’n_fi»c'rion.r", Datamonitor, Jan 2004‘). The present invention provides novel fungal and yeast nucleic acid targets for application in Nucleic Acid Diagnostics (NAD) tests.

These are rapid, accurate diagnostic tests for clinically significant bacterial and fungal pathogens for bioanalysis applications in the clinical sector.

Ace2 is a DNA-binding cell cycle regulated transcription factor. Ace2 functions similarly to the transcription factor SW15, yet they activate distinct genes. The translated Ace2 protein is present in the nucleus in early G1 phase of the cell cycle and thus, specifically activates the expression of genes in the G1 phase. In particular, it activates the CTSI gene. CTSI is a chitinase-encoding gene required to degrade the cell wall between parent and daughter cells during cytokinesis. There are 7 Ace2 sequences publicly available in the NCBI GenBank database including 5 Candida spp. sequences. There are no published Ace2 sequences available for Aspergillus spp The current inventors have designed PCR primers to amplify the region of Ace2 in Candida spp. equivalent to base pair position 1736 to 2197 in C. albicans. This region of the Ace2 gene has an application for the molecular identification and / or discrimination of yeast species.

Definitions "Synthetic oligonucleotide" refers to molecules of nucleic acid polymers of 2 or more nucleotide bases that are not derived directly from genomic DNA or live organisms. The term synthetic oligonucleotide is intended to encompass DNA. RNA. and DN A/ 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 10 to about I00 nucleotides. The sugar groups of the nucleotide subunits may be tibose, deoxyribosc, or modified derivatives thereof such as OMe. The nucleotide subunits may he joined 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 methylpliospltonate linkage, or a neutral peptide linkage.

Nitrogcnous base analogs also may be components of oligonucleotidcs 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 hybrid ized 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 form a detectable hybrid probeztarget 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 cndonuclease 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 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 10 to about 100 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 form a double-stranded structure ("hybrid" or "duplex"). A "fungus" or "yeast" is meant any organism of the kingdom Fungi. and preferably, is directed towards any organism of the phylum Ascomycota.

"Complementarity" is a property conferred by the base sequence of a single strand ol‘ DNA or RNA which may form a hybrid or double-stranded DNA:DNA._ RNA:RNA or DNA:R_NA through hydrogen bonding between Watson-Crick base pairs on il1C 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 form; hybrids without a sufficient degree of complementarity will not fomi. 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 dilTerence in stability between the hybrid formed with the target and the non-target nucleic acid.

With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (for example, hybridization under “high stringency" conditions, may occur between homologs with about 85-100% identity, preferably about 70-] 00% identity).

With medium stringency conditions. nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (for example, hybridization under “medium stringency" conditions may occur between homologs with about 50-70% identity). Thus, conditions of “weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.

‘High stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of SXSSPE (43.8g/l NaCl, 6.9 g/l NaH2PO4H3O and 1.85 g/l EDTA, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt’s reagent and l0Oug/ml denatured salmon sperm DNA followed by washing in a solution comprising 0. l .~ “Medium stxingency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of SXSSPE (43.8 g/l NaCl, 6.9 g/l Nal l2PO4H;O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt‘s reagent and 100 pg/ml denatured salmon sperm DNA followed by \-vnshing in a solution comprising LOXSSPE, 1.0% SDS at 42° C, when a probe ofabout 500 nucleotides in length is used.

‘Low stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of5xSSPE (43.8 g/1 NaCl, 6.9 g/l Nal-l2PO4l-l20 and 1.85 g/l l€DTA, pH adjusted to 7.4 with NaOH). 0.1% SDS, 5xDenhardt’s reagent [S0xDcnhardt’s contains per 500ml: 5g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 pg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5XSSPE, 0.1% 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 in-vitro amplification technology. For example, in the context of PCR and real-time PCR, “stringency" 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 of the target nucleic acid.

One skilled in the art will understand that substantially corresponding probes of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes of the 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 10 nucleotide target sequence to about 2 bases mismatched in an about 10 nucleotide target sequence. In preferred embodiments, the percentage is from about 85% to about 100%. In more preferred embodiments, this percentage is from about 90% to about 100%; in other preferred embodiments, this percentage is from about 95% to about 100%, e. g. 95%, 96%, 97%, 98%, 99%, or 100%.

The terms “identical“ or percent “identity," in the context of two or more nucleic acids or polypeptide sequences, reler to two or more sequences or subscquenccs that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximtun correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 20 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, eg., NCBI web site at ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.

Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segment of any one ofthe number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal aligmnent of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ol‘Needleman & Wunsch. .1. M0]. Biol. 481443 (1970), by the Search for similarity method of Pearson & Lipm21n.l’r0c. Nat ’/. Acad. Sci. USA 8553444 ( I988’). by computerized implementations ofthcse algorithms (GAP, BESTF 1'1”. FAST/\, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (sec, cg, Current Protocols in Molecular Biology (Ausubel el al., eds. 1987-"2005, Wiley lnterscicncel).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:33:89-3402 ( l 977) and /\llS(‘lll1l er al., J. M01. Biol. 2152403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins ofthe invention. Sofiware for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words oflength W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul er al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > O) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative aligmnent score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlcngth (W) of 1 1, an expectation (E) of 10, M=S, N=- 4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlcngth of 3, and expectation (E) of 10, and the BLOSUMC»? scoring matrix (see l-Ienikoff& Henikoll‘, Proc Natl. Acad. Sci L/SA 89:I09| 5 ( I 989;) alignments (B) of 50, expectation (E) of IO. M=5, N=—4, and a comparison 0|‘ both strands.

“Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or d0uble—stranded form, and complements thereof.

The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2—O—methyl ribonucleotides, peptide- nucleic acids (PNAS) 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 14 to 50 nucleotides in length, although this will depend to an extent on the overall length of the oligonucleotide probe. The structure is sufficicntly stable to be detected by means such as chemiluminescent 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 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 probe:target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probe:non-target 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 of(l‘or example Candida) and distinguish these species from other organisms. Preferential hybridization can be measured using techniques known in the art and described herein.

By "theranostics" is meant the use of diagnostic testing to diagnose the disease. choose the correct treatment regime and monitor the patient response to therapy. The theranostics of the invention may be based on the use of an NAD assay of this invention on samples, swabs or specimens collected from the patient.

Object of the Invention it is an object of the current invention to provide sequences and/or diagnostic assays to detect and identify one or more yeast species. The current inventors have used the /\cc2 gene sequence to design primers and probes that are specific to Candida Ace2 genes. Such primers not only allow the detection of yeast and fungal species but also allow distinction between Candida species. The current invention further provides for primers and probes that allow discrimination between different Candida species.

Sumgirary of thtfigventiog The present invention provides for a diagnostic kit for detection and identification of yeast species, comprising an oligonucleotide probe capable of binding to at least a portion of the Ace2 gene or its corresponding mRNA. The oligonucleotidc probe may have a sequence substantially homologous to or substantially complementary to a portion of the Ace2 gene or its corresponding mRNA. It will thus be capable of binding or hybridizing with a complementary DNA or RNA molecule. The Aee2 gene may be yeast Ace2 gene. The nucleic acid molecule may be synthetic. The kit may comprise more than one such probe. In particular, the kit may comprise a plurality of such 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. Funhennore, the sequences of the invention identified as suitable targets provide the advantages of having significant intragenie 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 of the invention to be directed towards genus-specific yeast and fungal 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 yeast and fungal diagnostics.

The Aee2 sequences allows for multi-test capability and automation in diagnostic assays.

One of the advantages of the sequences of the present invention is that the iniragenic Ace2 nucleotide sequence diversity between closely related yeast species enables specific primers and probes for use in diagnostics assays for the detection of yeast to be designed. The Aee2 nucleotide sequences, both DNA and RNA can be used with direct detection, signal amplification detection and in vitro amplification technologies in diagnostics assays. The Aee2 sequences allow for multi-test capability and automation in diagnostic assays.

The kit may further comprise a primer for amplification of at least a portion of the Ace2 gene. Suitably the kit comprises a forward and a reverse primer for a portion of the Ace2 gene.

The portion of the Ace2 gene may be equivalent to a portion of the region of the gene from base pair position 1736 to base pair position 2197 in C. albicans, Particularly preferred, are kits comprising a probe for a portion of the Ace2 Calbicans gene and / or a probe for a portion of the region of the gene equivalent to base pair position 1736 to base pair position 2197 in C. albicans Equivalent regions to base pair position 1736 to base pair position 2197 can be found in other organisms, but not necessarily in the same position.

The probe may preferentially hybridize to a portion of the Ace2 gene sequence selected from the group consisting of SEQ ID N05: 4-7 and 32-39 or their corresponding mRNA.

The kit may also comprise additional probes. The probe may have a sequence selected from the group consisting of SEQ ID Nos: 3, 30, 31, and a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a probe for the Ace? gene.

It is desirable that the probe has a sequence as defined by SEQ ID NO: 30.

The kit may comprise at least one forward in vitro amplification primer and at least one reverse in vitro amplification primer, the forward amplification primer having it sequence selected from the group consisting of SEQ ID Nos: 1, 20-24, and sequences being substantially homologous or complementary thereto which can also act as a forward amplitication primer, and the reverse amplification primer having a sequence selected from the group consisting of SEQ ID Nos: 2, 25-29, and a sequence being substantially homologous or complementary thereto which can also act as a reverse amplification primer. It is desirable that said forward primer sequence is selected from the group consisting of SEQ ID NOs:2 I, 22, and 23, and said reverse primer sequence is selected from the group consisting of SEQ ID NOs:27, 28, and 29. The diagnostic kit may be based on direct nucleic acid detection technologies, signal amplification nucleic acid detection technologies, and nucleic acid in vitro amplification technologies is selected from one or more of Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCRV), Nucleic Acids Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), Branched DNA technology (bDNA) and Rolling Circle Amplification Technology (RCAT) ), or other in vitro enzymatic amplification technologies.

The invention also provides a nucleic acid molecule selected from the group consisting of SEQ ID NO.I to SEQ ID NO. 40 and sequences substantially homologous thereto, or substantially complementary to a portion thereof and having a function in diagnostics based on the Aee2 gene.

The nucleic acid molecule may comprise an oligonucleotide having a sequence substantially homologous to or substantially complementary to a portion of a nucleic acid molecule of SEQ ID NO] to SEQ ID NO. 40. The invention also provides a method of detecting a target organism in a test sample comprising the steps of: (i) 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 of the duplex positively identifying the presence of the target organism in the test sample.

The nucleic acid molecule and kits of the present invention may be used in a diagnostic assay to detect the presence of one or more yeast and/or fungal species, to ineasurc yeast and/or fungal titres in a patient or in a method of assessing the efiicaey of a treatment regime designed to reduce yeast and/or fungal titre in a patient or to measure yeast and/or fungal contamination in an environment. The enviromnent may be u 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.

The kits and the nucleic acid molecule of the invention may be used in the identification and/or characterization of one or more disruptive agents that can be used to disrupt the Ace2 gene function. The disruptive agent may be selected from the group consisting ofantisense RNA, PNA, and siRNA.

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

The oligonueleotides of the invention may be provided in a composition for detecting the nucleic acids of yeast and fungal target organisms. Such a composition may also comprise buffers, enzymes, detergents, salts and so on, as appropriate to the intended use of the 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 yeast and/or fungal organism 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 of or a 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 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 yeast and/or fungal species, comprising one or more DNA probes comprising a sequence substantially complementary to, or substantially homologous to the sequence of the Ace2 gene of the candidate yeast and/or fungal species. The present invention also provides for one or more synthetic oligonucleotides having a nucleotide sequence suhstzintially homologous to or substantially complementary to one or more ol‘ the group consisting of the Ace2 gene or mRNA transcript thereof, the yeast Acel gene or mRNA transcript thereof, the yeast Ace2 gene or mRNA transcript thereof, one or more ofSEQ ID NO l—Sl3Q ID NO 40.

The nucleotide may comprise DNA. The nucleotide may comprise RNA. The nuclcotide may comprise a 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 of the invention) may be selected to be substantially homologous to a portion of the coding region of the Ace?! gene. The gene may be a gene from a target yeast or fungal organism. The sequences of the invention are preferably sufficient so as to be able form a probeztarget duplex to the portion of the sequence.

The invention also provides for a diagnostic kit for a target yeast or fungal organism comprising an oligonucleotide probe substantially homologous to or substantially complementary to an oligonucleotide of the 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 oligonucleotide probes: while it is preferable that amplification primers may have a complementary portion of between about l5 nucleotides and about 30 nucleotides (more preferably about l5—about 23, most preferably about 20 to about 23), oligonucleotide probes of the 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 LightCycler, Taqman 5’ exonuclease probes, hairpin loop structures etc. and sequence of the oligonueleotide probe selected.

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

The target yeast or fungal organism may be selected from the group consisting of C. albicans. C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C . duhlinicnsis, C‘. guilliermondii, C . norvegiensis, C. famata, C. haemulom’. C. kefi/r, C '. mi//'.s‘. (T l‘t.S‘l1’(‘Il1a[}1il and Aspergillus species.

The target yeast organisms may be a Candida species for the given set oi‘ primers already experimentally demonstrated. and more preferably, selected from the group consisting ol‘(.‘. albicans C. Iropicalis. C.dub!im'en.sis, C. glabrata, C. krusei, C. pamp.s'i/om‘, C. guilliermondii, C. norvcgiensis, C. famata, C. haemulom‘. (.'. kg/yr, (7. zit!/is, ('. viswanathii.

Under these circumstances, the amplification primers and oligonuclcotide 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 of the desired organisms of the target yeast organism grouping.

The test sample may comprise cells of the target yeast and/or fungal organism.

The method may also comprise a step for releasing nucleic acid from any cells of the target yeast or fungal organism that may be present in said test sample. Ideally, the test sample is a lysate of an obtained sample 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 of any one of SEQ ID NO] to SEQ ID NO. 40 in a diagnostic assay for the presence of one or more yeast species. The species may be selected from the group consisting of C. albicans C. tropicalis. C.dublim’ensis, C. glabrara, C. krusei, C. parapsilosis, C. guilliermondii, C. norvegiertsis, C. famala. C. haemuloni, C. kefi/r, C. utilis, C. viswanathii.

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 of the synthetic oligonucleotides of the invention. The kits may also comprise one or more articles selected from the group consisting of appropriate sample collecting instruments. reagent containers, buffers, labelling moieties, solutions, detergents and supplementary solutions. The invention also provides for use of the sequences, compositions, nucleotide fragments, assays, and kits of the invention in theranostics, 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 yeast species.

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

The nucleic acid molecules, composition, kits or methods may be used in a method ofassessing the efficacy of a treatment regime designed to reduce yeast and/or fungal litre in a patient comprising assessing the yeast and/or fungal titre in the patient (by in viva methods or in vitro 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 antifungal agent, such as a pharmaceutical drug.

The nucleic acid molecules, composition, kits or methods may be used in a diagnostic assay to measure potential yeast and/or fungal 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 Ace2 gene function. Suitable disruptive agents may be selected from the group consisting of antisense RNA, PNA, siRNA.

I’-J ‘Jr Brief description of the Figures Figure 1: Primers binding sites in (grey highlights) Ace2 gene in Candida albicans (XM_707274.l). The amplified region of interest is underlined (Position 1736 to 2197).

Product size= 462 base pairs Figure 2: Location of C. albicans probe Pl-CanAee2 binding site (underlined and bolded in the amplified fragment of Ace2 gene. PCR primers CanAce2-F/CanAee2-R are highlighted.

F igure 3: Real-time PCR assay for C. albicans based on the Ace2 gene with TaqMan probe P1—CanAce2. Specificity of the assay was tested using a panel of DNA from Four other Candida species and Aspergillusfumigatus. The three C. albicans strains tested were detected in the real-time PCR and there was no cross-reaction with DNA for other species.

F igurc 4: Location of indicated C. albicans primers and probes. Figure 4(a) primer ACF1 is marked in bold, while primer ACRI is marked in bold underline; Figure 4(b) primer .«\(‘l-'2 is marked in bold and primer ACR2 is marked in bold underline; Figure 4(c) primer /\(‘l"3 is marked in bold and primer ACR3 is marked in bold underline: and Figure 4(d) probe is marked in bold, ACALBI on — strand, ACALB2 in + strand.

Primer (ACF2b/ACR3b) binding sites (grey highlights) and ACALBI probe (bold text) in Ace2 of Candida albicuns (XM_707274. I ). The amplified region ol‘ interest is underlined. (Position ofthe region ofinterest: 1379-2004).

Figure 5: F igurc 6: lnclusivity panel of C. albicuns strains. Amplification plot from Real- time PC R assay for C. albicans based on the Ace2 gene with TaqMan probe ACALBI and primer set ACF2b/ACR3b. Specificity of the assay was tested using a panel of DNA from 20 C . albicans strains and 84 strains covering 19 spp. of clinically relevant and related Candida. (a) The 20 C. ulbicans strains tested were detected. (by) No cross- reaction was seen with DNA from 84 strains covering 19 other Candida species. Signal obtained only from (+) controls.

Figure 7: Exclusivity vaginitis/ vaginosis panel. Amplification plot from Real- time PCR assay for C. albicans based on the Ace2 gene with TaqMan probe ACALBI and primer set AC F2b/ACR3b. A signal was obtained only for (+) control (C. albicans) samples. No signal was obtained for the organisms in the exclusivity panel.

Figure 8: Amplification plot from Real-time PCR assay for C. albicans based on the /\ce2 gene with TaqMan probe ACALBI. Sensitivity of the assay was tested using [J in various inputs of template DNA from C. albicans. The LOD of the assay was found to be ~l cell equivalent.

Detailed Description of the Drawings EXAMPLE 1 Materials and Methods Cell Culture Candida species were cultured in Sabouraud broth (4% wt/vol glucose, 1% wt/vol peptone, 1.5 % agar) for 48 hours at 37°C in a shaking incubator.

DNA Extraction Cells from Candida spp. were pre-treated with l yticase or zymolase enzymes prior to DNA isolation. DNA was isolated from Candida spp. using either the MagNA Pure System {Roche Molecular Systems) in combination with the MagN/\ pure Yeast and Bacteria! isolation kit lll according to the manufacturers protocol, or the Qiagen l)Neusy Plant Mini kit (silica-based DNA purification in spin column tormat).

DNA sequencing of Ace2 gene rggions in Candida species The publicly available sequences of the Ace2 genes of Candida species were acquired from the NCBI database and aligned using ClustalW. The PCR Primer set, namely (.‘anAce2-F/CanAce2-R (Figure 1, Table 1) was designed and used to amplify the Acc2 gene region in Candida spp. equivalent to base pair position 1736 to base pair position 2197 in C. albicans with primer set CanAce2-F/CanAce2—R. The Acc2 gene regions were amplified in C. albicans, C.(r0picalis and C. a'ublim'en.s'i5 by conventional PCR on the iCycler BioRad PCR machine or the PT C200 Peltier thennocycler (MJ Research) using the reagents outlined in Table 2 and the themiocycling conditions described in Table 3. the PCR reaction products were purified with Roche High Pure PCR Product Purification kit or with the EXOSAP-IT kit (USB) according to the manufacturers’ protocol and subsequently sent for sequencing to Sequiserve, Germany using the forward amplification primer CanAce2-F. DNA sequence information was generated for three Candida species. (C. albicans, C. tropicalis and C. dubliniensis).

Table 1: PCR primers designed to amplify the Ace2 gene regions in Candida app.

Primer Name Primer Sequence CanAce2-F TATCACCTTTGAAAAAACAATTACC CanAce2-R CACCAACACAAATATTTCGATC Table 2: Conventional PCR reagents used to amplify the Ace2 gene regions in Candida spp.

PCR Reaction Mix SAMPLE x l x Bul'fcr('lO0 mM Tris HCl, l5 mM MgClg, 500 mM KCI pH 5 },ll .3) dNTP‘s Mix. Roche (l0mM dNTP) ill Primer" Forward CanAce2-F (l0_uMg> Tmer Reverse CanAce2-R (l0pM) pl ul Polymerase TaqPol, Roche IU/pl I pl H20 Amgen/Accugene 36-39 pl Genomic DNA Template 2-5 ul TOTAL VOLUME 50 pl Table 3: Conventional PCR reaction conditions applied to amplify the Ace2 gene regions in Candida spp.

WFCR Thermal profile Lid preheating was ON Step Temp Time I 94°C 1 min or 50°C 1 min X 35 °C 1 min °C 7 min °C Hold T 4 Table 4: TaqMan probe for C. albicans based on Ace2 gene region P1-CanAce2 CTGTCACCATTGAATGGAGTCCAG Table 5: Real-time PCR reagents Preparation of PCR Reaction Mix SAMPLE LightCycler®FastStartDNA Master HybProbe, Roche Cat. 03 003 x I 248 001 WybProb mix I0 x conc. (Red cap) 2 pl MgClg stock solution (Blue cap) (Final cone. in reaction is 3 mM) 1.6 pl Probe Pl-CariAce2 2 pl Primer Forward CanAce2-F 1 pl Primer Reverse CanAce2-R I iii mllgt) PCR-grade ”l0.4 pl lmiplatc in 2 pl mm VOLUME 20 pl Table 6: Real-time PCR thermocycling conditions PCR Thermal profile Cycle Step Temp Time Activation I 95°C 10 min Amplification 1 95°C 10 sec X 50 - 20 sec °C °C 10 sec Cooling 1 40°C Hold RESL/IXIZSI Primer and Probe Design The sequence information available for the Ace?! gene in Candida Spp. was aligned with the newly generated sequence information for the Ace2 gene in Candida spp. and analysed using bioinformatics tools. Species-specific probes were designed based on the compiled Ace2 sequence information for Candida albicans (Table 4).

Figures 2 shows the relative positions of the PCR primers and TaqMan DNA probe for the amplification and detection of C. albicans. The specificity of the TaqMan probes for the identification of C. albicans was demonstrated in a real-time PCR assay on the Ligh1Cycler using the reagents and thermocycling conditions outlined in Tables 5. For the C. albicans assay based on the Ace2 gene, PCR primers CanACc2-F/Can./\ce2-R were combined with TaqMan probe, Pl-CanAce2. The specificity of the assay for the detection of‘ (1 albicarzs was confirmed by including DNA from 21 range oi‘ closely related Candida species and A. _/imzigcitzzs species in the C. alb1'can.s‘ real—time PCR assay. The assay detected C. albicans but did not detect or cross-react with DNA from any other Candida species tested or with A. _/izmigatus DNA. Figure 3 shows the C. ii/bicuns real-time PCR assay and the specificity ofthc assay for C. albicrms.

EXAIWPLE 2 Primers and probes for specific detection and identification were designed following in silico analysis of generated sequences. Three forward and three reverse primers were generated and two probes were designed as follows. Figures 4(a) to éltd) discloses the location of these sequences in the Ace2 subsequence.

ACFI: SEQ ll) NO:20: ATCAAAGAATCATCACCA ACF2: SEQ ID N022]: AGACTTCATTGTTACCAC AC F3: SEQ II) NO:24: CACCAGGTGAATTGG ACRI : SEQ ID NO:2S: CATTGTATCGACGAGTG ACR2: SEQ ID NO:26: TGTATCGACGAGTGAAT ACR3: SEQ ID NO:27: TTCGCACATTGTATCG I) ACALBI : SEQ ID NO:30: 6FAM-ATATCTTATCCTCATCCGGTCCT--BHQl ACALB2: SEQ ID N023 l: 6FAM-AGGACCGGATGAGGATAAGATAT--Bl [Q1 The primer sets were evaluated using the following assay conditions: UNG treatment: 50°C 2min followed by 95°C lmin. The amplification included 50 cycles, 95°C 10sec, 60°C 30 sec, followed by a 2 min cooling at 40°C.

Based on initial assay performance (eg. fluorescence and efficiency), primer set ACF3/’ ACR3 was chosen for further evaluation using ACALBI probe. initial inclusivity and exclusivity experiments were performed to evaluate the potential of the assay using the chosen assay oligonucleotides. Specificity of the assay was tested using a panel of DNA from 14 C. albicans strains and 23 strains representing 19 other (‘undiu'a species. All 14 C. albicans strains tested were detected. No cross-reaction was seen with DNA from 19 the other Candida species, z'.e., signal was obtained only front the positive C‘. albicam control.

The A022 assay was shown to be specific to C. albicans, and initial performance was good under conditions tested.

Next, eight different combinations of primers were tested in order to reduce cycling times. The following conditions were tested: UNG treatment for 2min at 50°C, lmin at 95°C, followed by amplification of 50 cycles, 95°C Ssec, 60°C 10sec, followed by a cooling step of 2min at 40°C.

The combination of primer set ACF2/ACR3 and the ACALB1 probe performed the best under these conditions. The LOD of the assay is ~1- I 0 cell equivalents. This primer combination demonstrated higher fluorescence at lower template inputs, and 3 out of 3 one-cell equivalents were detected with earlier (‘p values. Slight modifications were made to the primer sequences in order to further shorten cycle times, while retaining sensitivity and specificity (Table.7).

Table 7: Modifications to primer sequences Primer name SEQ ID NO Tm ACF2 2| 53°C % ACF2b 22 57. 1°C ACF2c 23 595°C ACR3 27 527°C ACR3b 28 568°C ACR3c 29 60.2°C Further reduced cycling conditions were tested, i.e., amplification step ol‘95°C for I second and 60°C for 10 seconds. 10-fold dilutions of C. albicans DNA were prepared and inputs of 1x105 to 0.1 cell equivalents were used as template. The combination of ACF2b and ACR3b primers were selected for inelusivity and exclusivity testing due to higher fluorescence at lower template inputs and 3 out of3 one—eel| equivalents were detected with earlier C p values than other primer sets, as indicated in Table 8.

Table 8: CP values obtained for different Tm primer sets.

‘CF values for /'ACR3h Aver lane I 100000 1000 25.55 1000 . 29.80 T_ c!‘ ACR3c 1 0.1 Twenty strains of Candida alhicans were selected to demonstrate inclusivity of detection with this primer set (see Table 9, Figure 5 and Figure 6). Real-time PCR assay for C. albicans was carried out as described with TaqMan probe ACALBI and primer set ACF2b/ACR3b. All twenty C. albicans strains tested were detected, and no signal obtained from H controls (see Figure 6(a)).

Table 9: C albicans strains used in inclusivity experiments C.alMc.m: 3345 Cmlbican: 33133 C.-dbicans 178 Calbicans isolate 160 Cialbacans at vsolata Calbicau isolztn I3 C.aIbu:au isotats 765 Cnzlbicans isolate 552 C.aIlnca!a£ with [5540 C. albicans txbetculcsrs 9559 C.albx-tan: Canduderma AJDS C.a!b1caI-as infected skin at child Cialbicanc asthma Calblcans of mad C'.albicanc liom dead C.alhxcaIt-9 C albicau In order to demonstrate exclusivity of the assay, the Aee2 assay ‘was tested tor performance against 84 strains covering 1‘) spp. ofclinically relevant and related ( ‘(mdida {see Table 10 below). In order to ensure that all DNA tested is PCR znnpliliable, the Candida DNA samples were amplified with universal fungal primers HI and U2. All DNA tested was shown to be PCR amplifiable. A template input of 105 CE/rxn was used, which is higher than nonnally tested. A higher input was tested because the organisms tested are related Candida and Ace2 sequence infonnation was not confirmed for 17 species prior to testing. Each input was tested in triplicate. No cross-reactions were observed, demonstrating the specificity of the Ace2 assay for C. albicans detection (see Figure 6(h)).

Candida Specificity panel — Clinicaliy relevant and related Candida spp.

C. albicans Acez anel - Tested in PHLS NCPF Diabetes 8018 PHLS NCPF Blood 3863 PHLS NCPF Blood 90876 ATCC Blood 3959 IHEM Blood ‘ . Leuven 9656 IHEM Biood Brussels, I38 CBS 7 880 C88 man urine, of Nelherlands 381 CEIS mouth Netherlands, CBS of 63 New Zealand Maslenon PLHS NCPF ‘ UK PLHS NCPF UK IHEM Brusse|s_ IHEM Grenoble, France CBS ? CBS ' int ' ’ ,Ca-ro CBS infected hand ' CBS blood. 0f53- ear-oi USA CBS man. blood. at aut USA. New York CBS biood France, Pans

Claims (5)

Claims

1. A diagnostic kit for a yeast or fungal species comprising an oligonucleotide probe capable of binding to at least a portion of the Ace2 gene or its corresponding mRNA.

2. A kit as claimed in claim 1, wherein the portion of the Ace2 gene is a portion of the region ofthe gene from base pair position 1736 to base pair position 2197 C. albicans Ace2 gene.

3. A kit as claimed in any of claim 1 or claim 2, wherein the probe is selected from the group consisting of SEQ ID NOS: 3, 30, 31 or sequences substantially similar or complementary thereto which can also act as a probe, and/or wherein the kit further comprises a forward amplification primer being selected from the group consisting of SEQ ID Nos: 1,20-24, and sequences substantially similar or complementary thereto which can also act as a forward amplification primer and a reverse amplification primer being selected from the group consisting of SEQ ID N05: 2, 25-29. and seqilcnccs substantially similar or complementary thereto which can also act as Ll reverse amplification primer..

4. A nucleic acid molecule selected from the group consisting of: SEQ ID NO I through SEQ ID NO 40 and sequences substantially homologous or substantially complementary thereto or to a portion thereof and having a fimction in diagnostics based on the _I\ce2 gene.

5. A kit substantially as described herein with reference to the accompanying figures. l/7