GB2549799A - A multiplex assay for the sensitive and specific detection and differentiation of Clostridium difficile - Google Patents

Abstract

A diagnostic kit suitable for detecting and differentiating toxigenic Clostridium difficile comprises an oligonucleotide probe capable of binding the tcdA, tcdB or tcdC gene or mRNA. Nucleic acid probes and primers which detect the tcdA gene, the tcdB gene and/or the Δ117 deletion in the tcdC gene are provided. Diagnostic assays and methods of detecting C. difficile using such probes and primers in clinical or environmental samples are disclosed.

Description

Title A multiplex assay for the sensitive and specific detection and differentiation of Clostridium difficile.

Field of the Invention

The present invention relates to nucleic acid primers and probes to detect Clostridium difficile. More specifically the invention relates to the tcdA gene (Toxin A), tcdB gene (Toxin B) and the tcdC gene, the corresponding RNA, specific probes, primers and oligonucleotides related thereto and their use in diagnostic assays to detect and differentiate C. difficile. Back2round to the Invention

Clostridium difficile is a Gram-positive bacterium that causes severe diarrhea and other intestinal diseases when competing bacteria in the gut have been eridacated by antibiotic treatment. Clostridia are anaerobic, spore forming bacilli. Clostridium difficile is the most serious cause of antiobiotic-associated diarrhea. It is also the cause of pseudomembraneous colitis, and in rare cases this can progress to toxic megacolon, which is a life-threatening condition.

Definitions

As used herein the following terms have the following meanings:-" 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 DNA/RNA hybrid molecules that have been manufactured chemically, or synthesized enzymatically in vitro.

An "oligonucleotide" is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about 10 to about 100 nucleotides. The sugar groups of the nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may be 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 methylphosphonate linkage, or a neutral peptide linkage. Nitrogenous base analogs also may be components of oligonucleotides in accordance with the invention. A "target nucleic acid" is a nucleic acid comprising a target nucleic acid sequence. A "target nucleic acid sequence," "target nucleotide sequence" or "target sequence" is a specific deoxyribonucleotide or ribonucleotide sequence that can be hybridized to a complementary oligonucleotide.

An "oligonucleotide probe" is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a detectable hybrid proberiarget duplex under high stringency hybridization conditions. An oligonucleotide probe of the invention is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions. Non-complementary sequences, such as promoter sequences, restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes. An oligonucleotide probe of the invention 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. Particularly preferred are probes of 15 nucleotides in length, 20 nucleotides in length, 25 nucleotides in length, 30 nucleotides in length or 35 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"). "Complementarity" is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA 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 complements c>fosine (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 form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid.

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 70-100% 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 5xSSPE (43.8g/l NaCl, 6.9 g/1 NaH2P04H20 and 1.85 g/1, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt’s reagent and lOOpg/ml denatured salmon sperm DNA followed by washing in a solution comprising O.lxSSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length is used. “Medium stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of 5XSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2P04H20 and 1.85 g/1 EDIA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt’s reagent and 100 pg/ml denatured salmon sperm DNA followed by washing in a solution comprising l.OxSSPE, 1.0% SDS at 42° C, when a probe of about 500 nucleotides in length is used. ‘Low stringency’ conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of 5xSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2P04H20 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5xDenhardt’s reagent [50xDenhardt’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-vitro 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 100% to about 85%. 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%. Thus substantially similar sequences have at least 85%, or 90% or 95% homology with the sequence in question under stringent conditions. Such sequences may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher homology or identity with the sequence in question.

By "sufficiently complementary" or "substantially complementary" is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences of the invention, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same {i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection {see, e.g., 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 of the 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 alignment of sequences for comparison can be conducted, e g., by the local homology algorithm of Smith &amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &amp; Lipman, Proc. Nat 7. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection {see, e.g., Current Protocols in Molecular Biology (Ausubel etal, eds. 1987-2005, Wiley Interscience)). 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 etal., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul etal., J. Mol. Biol. 215:403-410 (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 of the invention. Software 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 of length 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 et 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 > 0) 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 alignment 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 negativescoring 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 wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLAST? program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix {see Henikoff &amp; Henikoff, Proc. Natl. Acad. Set. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-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, 2-0-methyl ribonucleotides, peptide-nucleic acids (PNAs).

By "nucleic acid hybrid" or "probe:target 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 sufficiently 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 (i.e., 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 probefarget 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 a 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 a specific organism, for 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 Clostridium difficile. Such assays may be either multiplex or singlplex assays. An object of the present invention is to provide a Clostridium difficile multiplex assay that would detect the Toxin genes associated with C. difficile and a hypervirulent marker to distinguish the hypervirulent strains. A further object is to provide an assay for the tcdB gene, which encodes the cytotoxin Toxin B, the tcdA gene which encodes the potent enterotoxin Toxin A, and the tcdC gene. A still further object of the invention is to provide a multiplex assay based on the tcdA gene, the tcdB gene and the tcdC gene. The current inventors have made use of these gene sequences to design primers that are specific to Clostridium difficile genes..

Summary of the Invention

The present invention provides for a diagnostic kit for detection and identification of Clostridium difficile, comprising an oligonucleotide probe capable of binding to at least a portion of the tcdA gene, the tcdB gene or the tcdC gene. The kit may further comprise reagents for the detection of the probe. The oligonucleotide probe may have a sequence substantially homologous to or substantially complementary to a portion of the tcdA gene, the tcdB gene or the tcdC gene or its corresponding mRNA. It will thus be capable of binding or hybridizing with a complementary DNA or RNA molecule. The nucleic acid molecule may be synthetic. Synthetic probes are unlikely to be found in nature. Furthermore synthesised oligonucleotides and primers undergo post-synthesis chemical modification or processing which would not occur in nature and which serves to clean the sequences from chemicals, truncated sequences and any disturbing salts. The sequences of the invention may further comprise a detectable label.

The oligonucleotide probe may have a sequence selected from of SEQ ID Nos. 15 to 25, 30 to 34 and 70 to 80. Particularly preferred are SEQ ID Nos. 18, 32 and 80, or a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a probe for tcdA gene, the tcdB gene or the tcdC genes. The kit may comprise a probe against two or more of tcdA gene, the tcdB gene or the tcdC gene. Preferably the kit comprises a probe against all three of tcdA gene, the tcdB gene or the tcdC gene.

The kit may comprise more than one such probe against each gene. In particular the kit may comprise a plurality of such probes. Thus the kit may comprise any combination of the probes with SEQ ID Nos. 15 to 25, 30 to 34 and 70 to 80. In addition, the kit may comprise additional probes for other organisms, such as, for example, bacterial, fungal or yeast 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.

The tcdA gene, the tcdB gene or the tcdC gene sequences allow for multi-test capability and automation in diagnostic assays. They also 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 tcdA gene, the tcdB gene or the tcdC gene. Suitably, the kit comprises a forward and a reverse primer for a portion of the tcdA gene, the tcdB gene or the tcdC gene.

The kit may also comprise additional primers or probes.

The primer may have a sequence selected from the group SEQ ID Nos. 1 to 14, 26 to 29 and 35 to 69. Particularly preferred are SEQ ID Nos. 13, 14, 68, 69, 83 and 42, and a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a primer for the ted A gene, the tcdB gene or the tcdC gene. The primers may also be a primer which preferentially hybridizes to the same nucleotide sequence as is preferentially hybridized by the primers SEQ ID Nos 13, 14, 68, 69, 83 and 42.

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 a sequence selected from the group consisting of SEQ ID Nos. 13, 68 and 83, and a sequence being substantially homologous or complementary thereto which can also act as a forward amplification primer for tcdA gene, the tcdB gene or the tcdC gene, and the reverse amplification primer having a sequence selected from the group consisting of SEQ ID Nos. 14, 44 and 69, and a sequence being substantially homologous or complementary thereto which can also act as a reverse amplification primer for the tcdA gene, the tcdB gene or the tcdC gene.

Suitably the kit may comprise a probe of SEQ ID No. 30 together with forward primers selected from SEQ Id Nos. 36 to 41 and reverse primers selected from SEQ ID Nos. 42, 43, 45 and 46. Suitably the kit may comprise a probe of SEQ ID No. 33, 34 or 35 together with a forward primer SEQ Id Nos. 41 and reverse primer SEQ ID Nos. 46. Suitably the kit may comprise a probe of SEQ ID No. 31 together with forward primers selected from SEQ ID Nos. 36 to 38 and reverse primers selected from SEQ ID Nos. 42, 43, 45 and 46. Any other combinations of primers and probes may be used in the kits of the invention.

The diagnostic kit may comprise reagents suitable for kits 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), real-time PCR, digital PCR (dPCR), Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), Branched DNA technology (bDNA) and Rolling Circle Amplification Technology (RCAT)), loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) single primer isothermal amplification (SIP A), Helicase Dependent Amplification and circular Helicase Dependent Amplification (cHDA) or other in vitro enzymatic amplification technologies. Also contemplated for use in the present invention is Whole Generation Sequencing (WGS).

The invention also provides a nucleic acid molecule selected from the group consisting of SEQ ID NO. 1 to 80. Particularly preferred are SEQ ID Nos. 13, 14, 18, 32, 42, 68, 69, and 80 and sequences substantially homologous thereto, or substantially complementary to a portion thereof and having a function in diagnostics based on the tcdA gene, the tcdB gene or the tcdC 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.13, 14,18, 32, 42, 68, 69, and 80. In a further aspect the invention provides nucleic acid sequences selected from sequence IDs numbers 1 to 80 and sequences which are substantially similar to the said sequences and also having a function in nucleic acid diagnostics. In particular the invention provides nucleic acid sequences having at least 85% sequence identity with sequence IDs numbers 1 to 80, more particularly sequences having at least 90% or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity with sequence_IDs numbers 1 to 80. The sequences may also be substantially complimentary to sequence IDs numbers 1 to 80, and having the same percentage complementarity as set out above for sequence identity.

The present invention contemplates the use of any appropriate method for amplification of target molecules using synthetic oligonucleotide primers and the detection of specific microorganisms using hydrolysis probes. Generally oligonucleotide primers and probes undergo a purification step by High Purity Salt Free (HPSF) or High-performance liquid chromatograph (HPLC) to remove chemicals and truncated sequences and to also ensure removal of any disturbing salts. This post synthesis chemical modification / processing does not occur in nature. Furthermore, these oligonucleotide primers and probes are short synthetic sequences and as such are unlikely to be found in nature. Finally modifications of oligonucleotide primers and probes can be "chemically modified or labelled" and this could include, but is not limited to, chemiluminescent, bio luminescent, fluorescent, electroactive, mass added or magnetic labels all of which are not found in nature. Real-time PCR primers may be synthesized and purified using either HPSF or HPLC and hydrolysis probes may be dual labelled synthesized with a fluorescent 5’ fluorophore and a 3’ dark quencher. Each fluorescently labelled hydrolysis probe may also be purified by HPLC after synthesis.

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 probeitarget duplex; and (iii) determining whether a probeitarget 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 Clostridium difficile, to measure Clostridium difficile titres in a patient or in a method of assessing the efficacy of a treatment regime designed to reduce Clostridium difficile titre in a patient or to measure Clostridium difficile contamination in an environment. The environment may be a hospital, or it may be a food sample, an environmental sample e.g. water, an industrial sample such as an in-process sample or an end product requiring bioburden or quality assessment.

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 ted A gene, the tcdB gene or the tcdC gene function. The disruptive agent may be selected from the group consisting of antisense RNA, PNA, and siRNA.

The oligonucleotides of the invention may be provided in a composition for detecting the nucleic acids of the target organism. 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 of the 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 Clostridium difficile comprising at least one forward in vitro amplification primer and at least one reverse in vitro amplification primer, the forward amplification primer being selected from the group consisting of one or more of 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 Clostridium difficile, comprising one or more DNA probes comprising a sequence substantially complementary to, or substantially homologous to the sequence of the tcdA gene, the tcdB gene or the tcdC gene function of the Clostridium dijficile. The present invention also provides for one or more synthetic oligonucleotides having a nucleotide sequence substantially homologous to or substantially complementary to one or more of the group consisting of the tcdA gene, the tcdB gene or the tcdC gene function gene or mRNA transcripts thereof, tcdA gene, the tcdB gene or the tcdC gene function or mRNA transcript thereof, tcdA gene, the tcdB gene or the tcdC gene function or mRNA transcript thereof, one or more of SEQ ID NO 13, 14,18, 32, 42, 68, 69, and 80.

The nucleotide may comprise DNA. The nucleotide may comprise RNA. The nucleotide may comprise a mixture of DNA, RNA and PNA. The nucleotide may comprise synthetic nucleotides. The sequences of the 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 tcdA gene, the tcdB gene or the tcdC gene function. The gene may be a gene from Clostridium difficile. The sequences of the invention are preferably sufficient so as to be able form a probe:target duplex to the portion of the sequence.

The invention also provides for a diagnostic kit for Clostridium difficile 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 15 nucleotides and about 30 nucleotides (more preferably about 15-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 &amp; structure (eg. Hybridization probe pairs for LightCycler, Taqman 5’ exonuclease probes, hairpin loop structures etc. and sequence of the oligonucleotide probe selected.

Kits and assays of the invention may also be provided wherein the oligonucleotide probe is immobilized on a surface. Such a surface may be a bead, a membrane, a column, dipstick, a 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 test sample may comprise cells of the target organism. The method may also comprise a step for releasing nucleic acid from any cells of the target 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. 13, 14, 18, 32, 42, 68, 69, and 80 in a diagnostic assay for the presence of Clostridium difficile.

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 Clostridium difficile.

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

The nucleic acid molecules, composition, kits or methods may be used in a method of assessing the efficacy of a treatment regime designed to reduce Clostridium difficile titre in a patient comprising assessing the Clostridium difficile titre in the patient (by \n vivo 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 antibacterial agent, such as a pharmaceutical drug.

The nucleic acid molecules, composition, kits or methods may be used in a diagnostic assay to measure potential Clostridium difficile 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 tcdA gene, the tcdB gene or the tcdC gene function. Suitable disruptive agents may be selected from the group consisting of antisense RNA, PNA, siRNA.

Brief Description of the Prawin2s

Figure 1: Alignment showing position of Toxin A primers and probe on the tcdA gene Figure 2: Alignment showing position of Toxin B primers and probe on the tcdB gene

Figure 3: Alignment showing position of primers and probe on the tcdC gene.

Detailed Description of the Invention Materials and Methods Sequencin2 of Gene Tar2ets

Sequencing primers were designed for each gene target, generating sequence data for the tcdA gene, two regions of the tcdB gene, the complete tcdC gene. Two regions of the tcdB gene were amplified and sequenced. Region 1 was located at the 5’ end of the gene and region 2 located at the 3’ end. Further in silica analysis confirmed the 3’ region was a suitable region to detect the Toxin B gene in all toxinogenic C. dijficile strains.

Sequence data was generated for a 521 base pair region of the tcdA gene. There is a high degree of sequence similarity between C. difficile strains with nucleotide heterogeneity at only 18 positions across the amplicon. This region was confirmed to be suitable as a target for the detection of the Toxin A gene in all toxinogenic C. difficile strains.

Sourcin2 and Assembly of Specificity Panels A Clostridium difficile inclusivity panel comprising 110 strains was assembled and is listed herein (Table 2).

Assay Development - Sin2leDlex

Singleplex assays were designed for the detection of the tcdA, tcdB and tcdC genes present in toxinogenic Clostridium difficile.

Assay Development - Multiplex

The singleplex assays were optimised and then brought forward to multiplex development.

Assay Specificity

Inclusivity

One hundred and ten C. difficile strains comprising the inclusivity panel (Table 2) were tested against the C. difficile multiplex. All strains were tested at 10^ cell equivalents per reaction in three separate experimental runs. All strains tested as expected based upon sequencing study results. The 8 non-toxinogenic strains were not detected by the multiplex assay as the toxin genes are not present in these strains. All 10 BI/NAPl/027 ribotypes were detected by the three assay. Ninety one toxinogenic strains tested positive for both tcdA and tcdB. Exclusivity

The specificity of the C. difficile multiplex assay was tested against the 18 Clostridium species on the exclusivity panel (Table 2). All species were tested at 10^ cell equivalents per reaction in three separate experimental runs. The assay did not detect any of the organisms listed on the exclusivity panel confirming the specificity of the C. difficile multiplex assay against this panel. Results are listed in Table 1.

Gastric Panel

The specificity of the C. difficile multiplex assay was tested against 92 organisms on the gastric panel (Table 2). The gastric panel comprised 67 bacteria, 10 fungi, 2 archaea, 4 protozoa and 9 DNA and RNA viruses. All species were tested at 10^ cell equivalents per reaction in three separate experimental runs. The assay did not detect any of the organisms listed on the gastric panel confirming the specificity of the C. difficile multiplex assay against this panel.

Human DNA

Human DNA (50 ng/μΐ) was tested against the C. difficile multiplex in quadruplicate. There was no cross reaction with any of the three assays.

Conclusions

The C. difficile multiplex assay of the invention can detect tcdA gene (Toxin A), tcdB gene (Toxin B) and the tcdC gene in a single reaction. All assays in the multiplex can detect less than or equal to 10 genome copies of the target organisms. The assay includes an internal control, which can be extracted, amplified, and detected along with the target C. difficile DNA. No cross reaction was observed against the Clostridia Species Panel (Exclusivity) or the Gastric Panel.

Table 2 Clostridium difficile Inclusivity Panel

Table 3 Clostridium species Exclusivity Panel

Table 4 Gastric Panel

Table 5 Primers and probes evaluated for the specific detection of the tcdC gene.

Note: ** - Probes are designed on the anti-sense strand.

Table 6 Primers and probes evaluated for the detection of the tcdB gene.

Table 7 Primers and probes evaluated for the detection of the tcdA gene.

Note: ** - Probes are designed on the anti-sense strand.

Results of Blastn analysis of C. difficile Multiplex Assay Primers showed that there is no significant similarity between the C. difficile primers and any other micro-organism sequenced.

In so far as any sequence disclosed herein differs from its counterpart in the attached sequence listing in Patentln3.3 software, the sequences within this body of text are to be considered as the correct version.

The words “comprises/comprising” and the words “having/including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. SEQ IDs

Sites of probes, oligonucleotides etc. are shown in bold and underlined. N or x= any nucleotide; w=a/t, m=a/c, r=a/g, k=g/t, s=c/g, y=c/t, h=a/t/c, v=a/g/c, d=a/g/t, b=g/t/c. In some cases, specific degeneracy options are indicated in parenthesis: e.g.: (a/g) is either A or G. SEQ ID NO 1: GGT AAC GAA TTT AGT AAT GAA RGA AAA SEQ ID NO 2:

TTC TCT ACA GGT ATY GGT GGT AT SEQ ID NO 3:

AAG GAA GCT CTA AGA AAA TA SEQ ID NO 4:

TCT TTA AGA GCA GAA AGG A SEQ ID NO 5:

CTT TAA GAG GAG AAA GCA TA SEQ ID NO 6:

AGA AGG TGG TGA GCA TA SEQ ID NO 7:

TGA TGG TCT TGA GAA GAA SEQ ID NO 8:

CTG ATG TTT GAT TAG AAA TGA SEQ ID NO 9:

AAA TTC TTT AAG AGG AGA AAG GA SEQ ID NO 10: AGA AGG TGG TGA GCA TAT ATT SEQ ID NO 11: GCTGGTGAGGATATATTGC SEQ ID NO 12: ATTCTTTAAGAGCACAAAGGA SEQ ID NO 13: AAGAGCACAAAGGATATTGC SEQ ID NO 14: ATGGTCTTCAGAACAAGCT SEQ ID NO 15:

TAG TGG CAT TTA TTT TGG TCT CTT T SEQ ID NO 16: CTG TAG TGG CAT TTA TTT TGG TCT CTT T SEQ ID NO 17: ACTGGCATTTATTTTGGTGTGTT SEQ ID NO 18: AACACACCAAAATAAATGCCAGT SEQ ID NO 19: ACACCAAAATAAATGCCAGTAGA SEQ ID NO 20:

ACACACCAAAATAAATGCCAGTAGA SEQ ID NO 21:

TGGCATTTATTTTGGTGTGTTTTTTGG SEQ ID NO 22:

ACACCAAAATAAATGCCAGTAGAGCA SEQ ID NO 23:

AACACACCAAAATAAATGCCAGTAGA SEQ ID NO 24:

AAACACACCAAAATAAATGCCAGTA SEQ ID NO 25:

TTGGTGTGTTTTTTGGCAATATATCC SEQ ID NO 26:

TAA GTT TAG GTG GAG CAA TCA A SEQ ID NO 27:

ATA TCT CCA ATA AAA GTG ACC TTC AT SEQ ID NO 28:

ATA TAG AAT TCA GAG TGG ATG GA SEQ ID NO 29:

AAC GCC TGG TAG GTT TAT ATA AA SEQ ID NO 30:

ACAGATGAATATATTGCAGCAACTGGTTCAGTTATTAT SEQ ID NO 31:

ACAGATGAATATATTGCAGCAACTGGTTCAGTTATTATTGAT SEQ ID NO 32:

TGAATATATTGCAGCAACTGGTTCAGTTATTAT SEQ ID NO 33:

AATATATTGCAGCAACTGGTTCAGTTATTAT SEQ ID NO 34:

TATATTGCAGCAACTGGTTCAGTTAT SEQ ID NO 35:

ATGCAGATTGGAGTATTTAATAC SEQ ID NO 36:

GATGAGAATTTTGAGGGAGAATCA SEQ ID NO 37:

GATGAGAATTTTGAGGGAGAATCAATAAACTATAC SEQ ID NO 38:

GATGAGAATTTTGAGGGAGAATCAATAAA SEQ ID NO 39: TTGAGGGAGAATCAATAAACTATACTGGTTG SEQ ID NO 40: GGAGAATCAATAAACTATACTGGTTG SEQ ID NO 41: TTGAGGGAGAATCAATAAACTATACTGG SEQ ID NO 42: GTATCAGGATCAAAATAATACTCCTC SEQ ID NO 43: TCACTAATTGAGCTGTATCAGGATC SEQ ID NO 44:

AGCTGTATCAGGATCAAAATAATACTCC SEQ ID NO 45:

CACTAATCACTAATTGAGCTGTATCAGGAT SEQ ID NO 46:

GAGCTGTATCAGGATCAAAATAATACTCCTC SEQ ID NO 47:

AGCTGTATCAGGATCAAAATAATACTCCTC SEQ ID NO 48:

ATA GGT GGA GAA GTC AGT GAT ATT SEQ ID NO 49:

GGT AAT AAT CTT ACT ATG TCA GAT GGT SEQ ID NO 50:

ATA GGT GGA GAA GTC AGT SEQ ID NO 51:

TAG GTC GAG AAG TCA GT SEQ ID NO 52:

CTA TCA TAG GAG AGT TTA ATA TT SEQ ID NO 52:

TCA CTA TCA TAG GAG AGT TT SEQ ID NO 54:

AGT GTC ATG TAG TAT GTC AAT SEQ ID NO 55:

ACT CTT GTT CTG TAA ACA A SEQ ID NO 56:

ATA GGT GGA GAA GTC AGT GAT SEQ ID NO 57:

GGT TCA CTA TCA TAG CAC AGT TT SEQ ID NO 58:

CTT CAC TAT CAT ACC ACA GTT TAA TAT T SEQ ID NO 59:

ATA GGT GGA GAA GTC AGT GAT ATT SEQ ID NO 60:

AGG ATG GAA TTT ATA TAT GAT AGA CAA A SEQ ID NO 61:

TCT CAA AGA ATT TGT TCT ATA TGA TTC TA SEQ ID NO 62:

TCT AGT ATC TGA ATA TAA TAG AGA TGA A SEQ ID NO 63:

ATT TAA TAA CTC TTG TTC TGT AAA CAA SEQ ID NO 64:

AGG ATG GAA TTT ATA TAT GAT AGA CAA AAA AGG TTT SEQ ID NO 65:

ACT ATT TAT TTT TCT CAA AGA ATT TGT TCT ATA TGA TTC T SEQ ID NO 66:

TGG AGA AGT CAG TGA TAT TGC SEQ ID NO 67:

GCT TCA CTA TCA TAC CAC AGT SEQ ID NO 68:

GGAGAAGTCAGTGATATTGC SEQ ID NO 69:

CTTCACTATCATACCACAGTT SEQ ID NO 70:

TTG AAT ACA TAA AAC AAT GGG CTG ATA SEQ ID NO 71:

AGA ATC ATA TAG AAC AAA TTC TTT GAG AA SEQ ID NO 72:

TCT TGA ATA CAT AAA ACA ATG GGC TGA TAT T SEQ ID NO 73:

CTT GAA TAG ATA AAA CAA TGG GCT GAT ATT AAT GCA GA SEQ ID NO 74:

TTG AAT ACA TAA AAC AAT GGG CTG ATA TTA ATG GAG A SEQ ID NO 75:

ACT CTG ATG TAG TAT CTG AAT ATA ATA GAG ATG AAA CT SEQ ID NO 76:

AGA ATG ATA TAG AAC AAA TTG TTT GAG AAA AAT AAA TAG T SEQ ID NO 77:

CAA TAG ATG ATA TTA TAA ACT CTG ATG TAG TAT CTG AAT ATA ATA GAG ATG AAA CT SEQ ID NO 78:

TAC ATA AAA CAA TGG GCT GAT ATT AAT GCA GA SEQ ID NO 79:

AAACAATGGGCTGATATTAATGCAGAA SEQ ID NO 80:

TTCTGCATTAATATCAGCCCATTGTTT

Claims (24)

Claims

1. A diagnostic kit for toxigenic Clostridium difficile comprising an oligonucleotide probe capable of binding to at least a portion of the tcdA gene, the tcdB gene or the tcdC gene or its corresponding mRNA.

2. A kit as claimed in claim 1 comprising a probe against two or more of the tcdA gene, the tcdB gene or the tcdC gene.

3. A kit as claimed in claim 1 or 2 comprising a probe against all three of tcdA gene, the tcdB gene or the tcdC gene..

4. A diagnostic kit as claimed in any preceding claim wherein the probe is selected from probes having a sequence of SEQ ID Nos 15 to 25, 30 to 34 and 70 to 80 or a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a probe for tcdA gene, the tcdB gene or the tcdC genes which can also act as a probe..

5. A diagnostic kit as claimed in claim 4 wherein the probe is selected from probes having a sequence of SEQ ID Nos 18, 32 and 80, or a sequence substantially homologous to or substantially complementary to those sequences, which can also act as a probe for tcdA gene, the tcdB gene or the tcdC genes which can also act as a probe.

6. A kit as claimed in any preceding claim further comprising a primer for amplification of at least a portion of the tcdA gene, the tcdB gene or the tcdC gene.

7. A kit as claimed in any preceding claim comprising a forward and a reverse primer for a portion of the the tcdA gene, the tcdB gene or the tcdC gene.

8. A kit as claimed in any preceding claim comprising at least one forward in vitro amplification primer and at least one reverse in vitro amplification primer, the primers being selected from the group consisting of SEQ ID Nos. 1 to 14, 26 to 29, and 35 to 69 or sequences substantially similar or complementary thereto which can also act as an amplification primer.

9. A kit as claimed in claim 8 wherein the forward amplification primer is selected from the group consisting of SEQ ID NO 13, 68 and 83, or sequences substantially similar or complementary thereto which can also act as a forward amplification primer and the reverse amplification primer being selected from the group consisting of SEQ ID NO 14, 42 and 69 or sequences substantially similar or complementary thereto which can also act as a reverse amplification primer.

10. A diagnostic kit as claimed in any preceding claim 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), real-time PCR, digital PCR (dPCR), Ligase Chain Reaction (LCR), Nucleic Acids Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), Branched DNA technology (bDNA) and Rolling Circle Amplification Technology (RCAT)), loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) single primer isothermal amplification (SIP A), Helicase Dependent Amplification and circular Helicase Dependent Amplification (cHDA) or other in vitro enzymatic amplification technologies.

11. A nucleic acid molecule selected from the group consisting of: SEQ ID NOs 1 to 80 and sequences substantially homologous or substantially complementary thereto or to a portion thereof and having a function in diagnostics based on the tcdA gene, the tcdB gene or the tcdC gene.

12. A nucleic acid molecule comprising an oligonucleotide having a sequence substantially homologous to or substantially complementary to a portion of a nucleic acid molecule as claimed in claim 9.

13. A method of detecting toxigenic Clostridium difficile in a test sample comprising the steps of (i) Mixing the test sample with at least one oligonucleotide probe capable of binding to at least a portion of the tcdA gene, the tcdB gene or the tcdC gene or its corresponding mRNA under appropriate conditions; (ii) hybridizing under a 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 toxigenic Clostridium difficile in the test sample.

14. A method as claimed in claim 13 wherein the probe is selected from the group consisting of SEQ ID NO Nos 15 to 25, 30 to 34 and 70 to 80 or sequences substantially homologous or substantially complementary thereto also capable of acting as a probe for the tcdA gene, the tcdB gene or the tcdC gene.

15. Use of a nucleic acid molecule as claimed in any one of claims 11 or 12 in a diagnostic assay to detect the presence of toxigenic Clostridium difficile in a sample.

16. Use of a kit as claimed in any one of claims 1 to 10 or a nucleic acid molecule as claimed in any one of claims 11 or 12, in a diagnostic assay to measure Clostridium difficile titres in a patient.

17. A method of assessing the efficacy of a treatment regime designed to reduce Clostridium difficile titre in a patient comprising use of a kit as claimed in any one of claims 1 to 10 or a nucleic acid molecule as claimed in any one of claims 10 or 11 at one or more key stages of the treatment regime.

18. Use of a kit as claimed in any one of claims 1 to 10 or a nucleic acid molecule as claimed in any one of claims 11 or 12, in a diagnostic assay to measure toxigenic Clostridium difficile contamination in an environment.

19. Use as claimed in claim 18, wherein the environment is a hospital, 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.

20. Use of a kit as claimed in any one of claims 1 to 10 or a nucleic acid molecule as claimed in any one of claims 11 or 12, in the identification and/or characterization of one or more disruptive agents that can be used to disrupt the the tcdA gene, the tcdB gene or the tcdC gene function.

21. Use as claimed in claim 20, wherein the disruptive agent is selected from the group consisting of antisense RNA, PNA, siRNA.

22. A kit substantially as described herein with reference to the accompanying figures.

23. A nucleic acid substantially as described herein with reference to the accompanying figures.

24. A method substantially as described herein with reference to the accompanying figures.