Methods and compositions for amplifying and / or detecting clostridioides difficile nucleic acids

Abstract

Disclosed are nucleic acid oligomers, including amplification oligomers and detection probes, for detection of Clostridium difficile target nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding formulations, reaction mixtures, and kits. Methods of synthesizing the nucleic acid oligomers are also disclosed.

Description

METHODS AND COMPOSITIONS FOR AMPLIFYING AND / OR DETECTING CLOSTRIDIOIDES DIFFICILE NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No.63 / 747,873, filed January 21, 2025, which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Copy, created on December 11, 2024, is named “GPR_9810PC_Seq_Listing_ST26.xml” and is 124,499 bytes in size.BACKGROUND

[0003] Clostridioides difficile is an anaerobic toxigenic bacterium that causes severe infectious colitis that leads to significant morbidity and mortality worldwide, mainly occurring after or during antimicrobial therapy. It is responsible for 15–25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudomembranous colitis (PMC). A rise in multidrug resistant clinical isolates to multiple antibiotics and their reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of Clostridioides difficile infection (CDI) more complicated, allowing the persistence of C. difficile in the intestinal environment. See Burke and Lamont, Gut Liver. 8:1-6, 2014; Vuotto et al., Adv. Exp. Med. Biol. 1050:97-115, 2018; Barbut and Petit, Clin. Microbiol. Infect. 7:405-410, 2001.

[0004] If the strain is toxigenic, toxins A and B are produced simultaneously in almost all cases, causing fluid secretion, inflammation and mucosal damage, leading to diarrhea or PMC. Both enhanced bacterial toxins and diminished host immune response contribute to symptomatic disease. See Burke and Lamont, supra., Barbut and Petit, supra.

[0005] In mild-to-moderate cases, oral metronidazole remains adequate first-line therapy, but in the absence of a good clinical response, switching to vancomycin may be necessary. Oral vancomycin should be used as initial therapy in severely ill patients or patients who cannot tolerate metronidazole. Rectal administration of vancomycin may be used as adjunctive therapy for severely ill patients. See Shen and Surawicz, Gastroenterol. Hepatol. (N Y) 4:134-139, 2008.

[0006] There is a need to efficiently and sensitively detect the presence of C. difficile in samples, particularly toxigenic C. difficile strains and including detection in biological specimens to provide diagnostic and prognostic information to physicians treating patients suffering from, or suspected of suffering from, infectious colitis or related disorders.SUMMARY

[0007] In some aspects, the present disclosure provides a composition or kit for determining the presence or absence of a toxigenic C. difficile strain in a sample. The composition or kit generally includes a set of oligonucleotides comprising each of (a)-(d) as follows: (a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid; (b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid; (c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid; and (d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid. In another, non-mutually exclusive aspect, the composition or kit generally includes at least one detection probe oligomer capable of hybridizing to a target region of a tcdA, tcdB, tcdC, or cdtB target nucleic acid or to an amplicon of said target region.

[0008] In another aspect, the present disclosure provides an oligonucleotide for determining the presence or absence of a toxigenic C. difficile strain in a sample, whereinsaid oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0-19 nucleotide analogs.

[0009] In other aspects, the present invention provides a reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample. In one such aspect, composition or kit generally includes a set of oligonucleotides comprising each of (a)-(d) as follows: (a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid; (b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid; (c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid; and (d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid. In another, non-mutually exclusive aspect, the reaction mixture generally includes at least one detection probe oligomer capable of hybridizing to a target region of a tcdA, tcdB, tcdC, or cdtB target nucleic acid or to an amplicon of said target region. In yet another non-mutually exclusive aspect, the reaction mixture comprises an oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0 to 19 nucleotide analogs.

[0010] In another aspect, the present invention provides a method for determining the presence or absence of a toxigenic C. difficile strain in a sample. The method generally includes performing an in vitro nucleic acid amplification reaction, utilizing an oligomer combination comprising (a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid, (b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid, (c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid, and (d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB)target nucleic acid, to generate one or more amplification products corresponding to the tcdA, tcdB, tcdC, and cdtB target regions, and detecting the presence or absence of the one or more amplification products, whereby the combined presence of the tcdA, tcdB, ctdB, and tcdC amplification products indicates the presence of the toxigenic C. difficile strain in the sample.

[0011] In another aspect, the present invention provides a method for synthesizing an oligonucleotide, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0 to 19 nucleotide analogs (e.g., a nucleotide sequence selected from the group consisting of SEQ ID NOs:41-58). In a related aspect, the present invention provides a method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide target sequences flanking a target region of a tcdA, tcdB, tcdC, or cdtB target nucleic acid, and wherein the first oligonucleotide and the second oligonucleotide each comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0 to 19 nucleotide analogs.

[0012] Representative embodiments of these aspects are further set forth below.Embodiments

[0013] Embodiment 1. A composition or kit for determining the presence or absence of a toxigenic C. difficile strain in a sample, said composition or kit comprising a set of oligonucleotides comprising each of (a)-(d):(a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid, wherein the first amplification oligomer set comprises first and second tcdA-specific amplification oligomers respectively comprising first and second tcdA-specific target-hybridizing sequences, wherein(i) the first tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:12, including from 0 to 19 nucleotide analogs; and(ii) the second tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs;(b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid, wherein the second amplification oligomer set comprises first and second tcdB- specific amplification oligomers respectively comprising first and second aB-specific target-hybridizing sequences, wherein(i) the first tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the second tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs;(c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid, wherein the third amplification oligomer set comprises first and second tcdC- specific amplification oligomers respectively comprising first and second fct / C-specific target-hybridizing sequences, wherein(i) the first tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO: 65 and that includes at least the sequence of SEQ ID NO:64, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 66, including from 0 to 19 nucleotide analogs; and(ii) the second tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs; a sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO: 68 and that includes at least the sequence of SEQ ID NO:67, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:70 and that includes at least the sequence of SEQ ID NO:69, including from 0 to 19 nucleotide analogs; and(d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid, wherein the third amplification oligomer set comprises first and second cdtB-specific amplification oligomers respectively comprising first and second cdtB-specific target-hybridizing sequences, wherein(i) the first cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, including from 0 to 19 nucleotide analogs; and(ii) the second cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs.

[0014] Embodiment 2. The composition or kit of Embodiment 1, wherein the first tcdA-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:12, including from 0 to 19 nucleotide analogs.

[0015] Embodiment 3. The composition or kit of Embodiment 1 or 2, wherein the second tcdA-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs.

[0016] Embodiment 4. The composition or kit of any one of Embodiments 1 to 3, wherein the first tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

[0017] Embodiment 5. The composition or kit of any one of Embodiments 1 to 4, wherein the second tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38, including from 0 to 19 nucleotide analogs.

[0018] Embodiment 6. The composition or kit of any one of Embodiments 1 to 3, wherein the second amplification oligomer set further comprises third and fourth tcdB-specific amplification oligomers respectively comprising third and fourth tcdB-specific target-hybridizing sequences, wherein(i) the third aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the fourth aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs.

[0019] Embodiment 7. The composition or kit of Embodiment 6, wherein the third tcdB-specific target-hybridizing sequence consists of the nucleotide sequenceshown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs.

[0020] Embodiment 8. The composition or kit of Embodiment 6 or 7, wherein the fourth tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38, including from 0 to 19 nucleotide analogs.

[0021] Embodiment 9. The composition or kit of Embodiment 6, wherein the first aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 18, including from 0 to 19 nucleotide analogs,the second TS-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, including from 0 to 19 nucleotide analogs,the third tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:33, including from 0 to 19 nucleotide analogs, andthe fourth aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 38, including from 0 to 19 nucleotide analogs.

[0022] Embodiment 10. The composition or kit of any one of Embodiments 1 to 9, wherein the first tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

[0023] Embodiment 11. The composition or kit of Embodiment 10, wherein the first tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:8, including from 0 to 19 nucleotide analogs.

[0024] Embodiment 12. The composition or kit of any one of Embodiments 1 to 11, wherein the second tcdC-specific target-hybridizing sequence consists of thenucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs.

[0025] Embodiment 13. The composition or kit of Embodiment 12, wherein the second tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:19, including from 0 to 19 nucleotide analogs.

[0026] Embodiment 14. The composition or kit of any one of Embodiments 1 to 13, wherein the first cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:14, including from 0 to 19 nucleotide analogs.

[0027] Embodiment 15. The composition or kit of any one of Embodiments 1 to 14, wherein the second cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs.

[0028] Embodiment 16. The composition or kit of any one of Embodiments 1 to 15, further comprising at least one tcdA-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a tcdA target sequence amplifiable by the first and second tcdA-specific amplification oligomers.

[0029] Embodiment 17. The composition or kit of Embodiment 16, wherein the tcdA-specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:10, including from 0 to 19 nucleotide analogs.

[0030] Embodiment 18. The composition or kit of Embodiment 17, wherein the tcdA-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:10, including from 0 to 19 nucleotide analogs.

[0031] Embodiment 19. The composition or kit of Embodiment 18, wherein the tcdA-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:54.

[0032] Embodiment 20. The composition or kit of any one of Embodiments 1 to 19, further comprising at least one tcdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a tcdB target sequence amplifiable by the first and second aB-specific amplification oligomers.

[0033] Embodiment 21. The composition or kit of Embodiment 20, wherein the tcdB-specific detection probe target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

[0034] Embodiment 22. The composition or kit of Embodiment 21, wherein the aB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:55, or SEQ ID NO:56

[0035] Embodiment 23. The composition or kit of Embodiment 21, wherein the aB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4, including from 0 to 19 nucleotide analogs.

[0036] Embodiments 24. The composition or kit of Embodiment 23, wherein the aB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:55, or SEQ ID NO:56.

[0037] Embodiment 25. The composition or kit of Embodiment 20, wherein the composition or kit comprises at least two aB-specific detection probes, wherein a first aB-specific detection probe comprises a first aB-specific detection probe targethybridizing sequence and a second aB-specific detection comprises a second tcdB-specific detection probe target-hybridizing sequence, and wherein each of the first andsecond aB-specific detection probe target hybridizing sequences is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

[0038] Embodiment 26. The composition or kit of Embodiment 25, wherein the first aB-specific detection probe target hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 2, including from 0 to 19 nucleotide analogs, andthe second tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:4, including from 0 to 19 nucleotide analogs.

[0039] Embodiment 27. The composition or kit of Embodiment 26, wherein the first aB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:55, and / orthe second tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:45, SEQ ID NO:49, or SEQ IDNO:56.

[0040] Embodiment 28. The composition or kit of any one of Embodiments 1 to 27, further comprising at least one fct / C-specific detection probe comprising a targethybridizing sequence configured to specifically hybridize to a tcdC target sequence amplifiable by the first and second fct / C-specific amplification oligomers, wherein the fct / C-specific detection probe target-hybridizing sequence is capable of detecting a single base pair deletion at nucleotide 117 of the tcdC gene.

[0041] Embodiment 29. The composition or kit of Embodiment 28, wherein the fct / C-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:23 or SEQ ID NO:24, including from 0 to 19 nucleotide analogs.

[0042] Embodiment 30. The composition or kit of Embodiment 29, wherein the fct / C-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or SEQ ID NO:57.

[0043] Embodiment 31. The composition or kit of any one of Embodiments 1 to 30, further comprising at least one ctaB-specific detection probe comprising a targethybridizing sequence configured to specifically hybridize to a ctdB target sequence amplifiable by the first and second ctaB-specific amplification oligomers.

[0044] Embodiment 32. The composition or kit of Embodiment 31, wherein the ctdB-specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

[0045] Embodiment 33. The composition or kit of Embodiment 32, wherein the ctdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

[0046] Embodiment 34. The composition or kit of Embodiment 33, wherein the ctaB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:47 or SEQ ID NO:58.

[0047] Embodiment 35. The composition or kit of any one of Embodiments 16 to 34, wherein one or more of the detection probes comprises a detectable label.

[0048] Embodiment 36. The composition or kit of Embodiment 35, wherein the detectable label is a fluorescent or chemiluminescent label.

[0049] Embodiment 37. The composition or kit of Embodiment 35, wherein the detectable label is a fluorescent label and each of the one or more detection probes further comprises a non-fluorescent quencher.

[0050] Embodiment 38. The composition or kit of any one of Embodiments 1 to 37, wherein the set of oligonucleotides are contained in a formulation comprising at least one of (a) a non-linear surfactant, (b) a lyoprotectant, (c) a-cyclodextrin, and (d) a chelating agent.

[0051] Embodiment 39. The composition or kit of Embodiment 38, wherein the formulation is a lyophilized formulation.

[0052] Embodiment 40. An oligonucleotide for determining the presence or absence of a toxigenic C. difficile strain in a sample, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0-20 nucleotide analogs.

[0053] Embodiment 41. The oligonucleotide of Embodiment 40, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs:41-58.

[0054] Embodiment 42. The oligonucleotide of Embodiment 40 or 41, wherein the 3’ end of said oligonucleotide is attached to a solid support.

[0055] Embodiment 43. The oligonucleotide of Embodiment 42, wherein the solid support is a controlled pore glass.

[0056] Embodiment 44. A reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample, said reaction mixture comprising a set of oligonucleotides as specified in any one of Embodiments 1 to 37.

[0057] Embodiment 45. A reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample, said reaction mixture comprising the oligonucleotide of Embodiment 40 or 41.

[0058] Embodiment 46. The reaction mixture of Embodiment 44 or 45, further comprising at least one of (a) a non-linear surfactant, (b) a lyoprotectant, (c) a-cyclodextrin, and (d) a chelating agent.

[0059] Embodiment 47. A method for determining the presence or absence of a toxigenic C. difficile strain in a sample, the method comprising:(1) contacting a sample, said sample suspected of containing the toxigenic C. difficile strain, with an oligomer combination comprising(a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid, wherein the first amplification oligomer set comprises first and second / ct / d -specific amplification oligomers respectively comprising first and second / ct / d -specific target-hybridizing sequences, wherein(i) the first tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, including from 0 to 19 nucleotide analogs; and(ii) the second / ct / d -specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs;(b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid, wherein the second amplification oligomer set comprises first and second tcdB-specific amplification oligomers respectively comprising first and second tcdB-specific target-hybridizing sequences, wherein(i) the first tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; and a sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 60 and thatincludes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the second tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs;(c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid, wherein the third amplification oligomer set comprises first and second tcdC-specific amplification oligomers respectively comprising first and second tcdC-specific target-hybridizing sequences, wherein(i) the first tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO: 65 and that includes at least the sequence of SEQ ID NO: 64, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 66, including from 0 to 19 nucleotide analogs; and(ii) the second tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO: 68 and that includes at least the sequence of SEQ ID NO: 67, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:70 and that includes at least the sequence of SEQ ID NO: 69, including from 0 to 19 nucleotide analogs; and(d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid, wherein the third amplification oligomer set comprises first and second cdtB-specific amplification oligomers respectively comprising first and second cdtB-specific target-hybridizing sequences, wherein(i) the first cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, including from 0 to 19 nucleotide analogs; and(ii) the second cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs;(2) performing an in vitro nucleic acid amplification reaction, wherein any C. difficile led A., tcdB, tcdC, and ctdB target nucleic acid present in the sample is used as a template for generating one or more amplification products corresponding to the C. difficile tcdA, tcdB, tcdC, and ctdB target regions;(3) detecting the presence or absence of one or more tcdA, tcdB, tcdC, and ctdB amplification products respectively corresponding to the C. difficile tcdA, tcdB, tcdC, and ctdB target regions, wherein the tcdC amplification product contains a single base pair deletion corresponding to nucleotide 117 of the tcdC gene, and wherein detecting the tcdC amplification product comprises detection of said single base pair deletion,whereby the combined presence of the tcdA, tcdB, ctdB, and tcdC amplification products indicates the presence of the toxigenic C. difficile strain in the sample.

[0060] Embodiment 48. The method of Embodiment 47, wherein the first / ct / d-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 12, including from 0 to 19 nucleotide analogs.

[0061] Embodiment 49. The method of Embodiment 47 or 48, wherein the second fct / 4-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs.

[0062] Embodiment 50. The method of any one of Embodiments 47 to 49, wherein the first tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

[0063] Embodiment Sl. The method of any one of Embodiments 47 to 50, wherein the second aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs.

[0064] Embodiment 52. The method of any one of Embodiments 47 to 49, wherein the second amplification oligomer set further comprises third and fourth tcdB-specific amplification oligomers respectively comprising third and fourth tcdB-specific target-hybridizing sequences, wherein(i) the third aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the fourth aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs.

[0065] Embodiment 53. The method of Embodiment 52, wherein the third tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs.

[0066] Embodiment 54. The method of Embodiment 52 or 53, wherein the fourth aB-specific target-hybridizing sequence consists of the nucleotide sequence shownin SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs.

[0067] Embodiment 55. The method of Embodiment 52, whereinthe first aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 18, including from 0 to 19 nucleotide analogs,the second TS-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, including from 0 to 19 nucleotide analogs,the third tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:33, including from 0 to 19 nucleotide analogs, andthe fourth aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 38, including from 0 to 19 nucleotide analogs.

[0068] Embodiment 56. The method of any one of Embodiments 47 to 55, wherein the first tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

[0069] Embodiment 57. The method of Embodiment 56, wherein the first fct / C-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:8, including from 0 to 19 nucleotide analogs.

[0070] Embodiment 58. The method of any one of Embodiments 47 to 57, wherein the second tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs.

[0071] Embodiment 59. The method of Embodiment 58, wherein the second tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 19, including from 0 to 19 nucleotide analogs.

[0072] Embodiment 60. The method of any one of Embodiments 47 to 59, wherein the first cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:14, including from 0 to 19 nucleotide analogs.

[0073] Embodiment 61. The method of any one of Embodiments 47 to 60, wherein the second cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs.

[0074] Embodiment 62. The method of any one of Embodiments 47 to 61, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one / ct / 4 -specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdA amplification product.

[0075] Embodiment 63. The method of Embodiment 62, wherein the tcdA-specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10, including from 0 to 19 nucleotide analogs.

[0076] Embodiment 64. The method of Embodiment 63, wherein the tcdA-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 10, including from 0 to 19 nucleotide analogs.

[0077] Embodiment 65. The method of Embodiment 64, wherein the tcdA-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:46, SEQ ID NO:50, or SEQ ID NO:54.

[0078] Embodiment 66. The method of any one of Embodiments 47 to 65, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one aB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdB amplification product.

[0079] Embodiment 67. The method of Embodiment 66, wherein the tcdB-specific detection probe target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

[0080] Embodiment 68. The method of Embodiment 67, wherein the tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:55, or SEQ ID NO:56

[0081] Embodiment69. The method of Embodiment 67, wherein the tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4, including from 0 to 19 nucleotide analogs.

[0082] Embodiments 70. The method of Embodiment 69, wherein the tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:55, or SEQ ID NO:56.

[0083] Embodiment 71. The method of Embodiment 66, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least two fctZS-specific detection probes, wherein a first aB-specific detection probe comprises a first aB-specific detection probe target-hybridizing sequence and a second aB-specific detection comprises a second aB-specific detection probe targethybridizing sequence, and wherein each of the first and second tcdB-specific detection probe target hybridizing sequences is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

[0084] Embodiment 72. The method of Embodiment 71, whereinthe first aB-specific detection probe target hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 2, including from 0 to 19 nucleotide analogs, andthe second tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:4, including from 0 to 19 nucleotide analogs.

[0085] Embodiment 73. The method of Embodiment 72, whereinthe first aB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:55, and / orthe second tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:45, SEQ ID NO:49, or SEQ IDNO:56.

[0086] Embodiment 74. The method of any one of Embodiments 47 to 73, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one fct / C-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdC amplification product, wherein the fct / C-specific detection probe target-hybridizing sequence is capable of detecting a single base pair deletion at nucleotide 117 of the tcdC gene.

[0087] Embodiment 75. The method of Embodiment 74, wherein the tcdC-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:23 or SEQ ID NO:24, including from 0 to 19 nucleotide analogs.

[0088] Embodiment 76. The method of Embodiment 75, wherein the tcdC-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:51, SEQ ID NO:52, or SEQ IDNO:53, or SEQ ID NO:57.

[0089] Embodiment 77. The method of any one of Embodiments 47 to 76, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction with at least one ctdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the ctdB amplification product.

[0090] Embodiment 78. The method of Embodiment 77, wherein the ctdB-specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

[0091] Embodiment 79. The method of Embodiment 78, wherein the ctdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

[0092] Embodiment 80. The method of Embodiment 79, wherein the ctdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:47 or SEQ ID NO:58.

[0093] Embodiment 81. The method of any one of Embodiments 62 to 80, wherein one or more of the detection probes comprises a detectable label.

[0094] Embodiment 82. The method of Embodiment 81, wherein the detectable label is a fluorescent or chemiluminescent label.

[0095] Embodiment 83. The method or kit of Embodiment 81, wherein the detectable label is a fluorescent label and each of the one or more detection probes further comprises a non-fluorescent quencher.

[0096] Embodiment 84. The method of any one of Embodiments 47 to 83, wherein the sample is a human sample.

[0097] Embodiment 85. The method of any one of Embodiments 47 to 84, wherein the sample is a stool sample or a blood sample.

[0098] Embodiment 86. The method of any one of Embodiments 47 to 85, wherein the method is a multiplex method in which the first, second, third, and fourth amplification oligomer sets are used for amplification of any C. difficile ted A., tcdB, tcdC, and ctdB target nucleic acid in the same in vitro nucleic acid amplification reaction.

[0099] Embodiment 87. A method for synthesizing an oligonucleotide, comprising the steps of:(a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 19 additional times, thereby generating at least 20 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 20 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-10, 12, 14-20, 22-24, 26- 34, and 36-40, including from 0 to 19 nucleotide analogs.

[0100] Embodiment 88. The method of Embodiment 87, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs:41-58.

[0101] Embodiment 89. A method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, wherein each of the synthesizing the first oligonucleotide and the synthesizing the second oligonucleotide comprises the steps of:(a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 19 additional times, thereby generating at least 20 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 20 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, and wherein the first oligonucleotide and the second oligonucleotide respectively comprise the nucleotide sequences of any one of SEQ ID NO: 12 and SEQ ID NO:32, including from 0 to 19 nucleotide analogs;SEQ ID NO: 18 and SEQ ID NO: 36, including from 0 to 19 nucleotide analogs;SEQ ID NO: 18 and SEQ ID NO: 38, including from 0 to 19 nucleotide analogs;SEQ ID NO:33 and SEQ ID NO: 36, including from 0 to 19 nucleotide analogs;SEQ ID NO:33 and SEQ ID NO: 38, including from 0 to 19 nucleotide analogs;SEQ ID NO:14 and SEQ ID NO:15, including from 0 to 19 nucleotide analogs;SEQ ID NO:8 and SEQ ID NO: 19, including from 0 to 19 nucleotide analogs; orSEQ ID NO:29 and SEQ ID NO:40, including from 0 to 19 nucleotide analogs.

[0102] These and other aspects and embodiments will become evident upon reference to the following detailed description.DEFINITIONS

[0103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art pertinent to the methods and compositions described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0104] The terms “a,” “an,” and “the” include plural referents, unless the context clearly indicates otherwise. For example, “a nucleic acid” as used herein is understood to represent one or more nucleic acids. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0105] When a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to ±10%.

[0106] All ranges are to be interpreted as encompassing the endpoints in the absence of express exclusions such as “not including the endpoints”; thus, for example, “from 0 to 19” includes the values 0 and 20.

[0107] “Sample” includes any specimen that may contain C. difficile, including components thereof, such as nucleic acids or fragments of nucleic acids. Samples include “biological samples” which include any tissue or material derived from a living or dead human, including, for example, stool, blood, plasma, serum, blood cells, saliva, mucous, and cerebrospinal fluid. The biological sample may be treated to physically or mechanically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like, which are used to prepare a biological sample for analysis. Also, samples may include processed samples such as samples in which one or more components have been concentrated or purified. Processed samples include, e.g., those obtained from passing samples over or through a filtering device, or following centrifugation, or by adherence to a medium, matrix, or support.

[0108] A “nucleotide” as used herein is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar, and a nitrogenous base (also referred to herein as “nucleobase”). The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2’ -deoxyribose.

[0109] “Nucleic acid” and “polynucleotide” refer to a multimeric compound comprising nucleotides and / or nucleotide analogs linked together to form a biopolymer.The biopolymers include conventional RNA, conventional DNA, mixed RNA-DNA, and nucleotide-analog-containing versions thereof. A nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid may be ribose, deoxyribose, or similar compounds with substitutions, e.g., analogs with a methoxy, fluoro or halide group at the 2’ position of the ribose (also referred to herein as “2’-0-Me” or “2’-methoxy” or 2’-fluoro, or “2’-halide”). Nitrogenous bases may be conventional bases, adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), and analogs thereof (e.g., inosine, 5 methyl 2’ deoxy cytosine (“5-methyl cytosine”) (5mC), isoguanine, 5-(l-propynyl)-2’-deoxycytidine (pdC), or 5-(l-propynyl)-2’ -deoxyuridine (pdU)). As used in the present disclosure, pdC is considered a cytosine analogue; and pdU is considered a thymine analogue. Nucleic acids may include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer.

[0110] The phrase “including from 0 to 19 nucleotide analogs,” as used herein following reference to one or more nucleotide sequences by SEQ ID NO, means that the referenced sequence(s) include equivalents of each sequence having from 0 to 19 nucleotide analogs (also referred to herein as “modified nucleotides”). By “equivalents having from 0 to 19 nucleotide analogs” is meant oligonucleotides that (i) have from 0 to 19 nucleotide analogs substituting conventional nucleotides within the reference sequence and (ii) have essentially the same complementary base pair hybridization properties as the reference sequence. Exemplary modified nucleotides are shown in Table 36, infra.

[0111] “Oligomer,” “oligonucleotide,” or “oligo” refers to a nucleic acid of generally less than 1,000 nucleotides (nt), including those in a size range having a lower limit of about 5 nt and an upper limit of about 500 to 900 nt. Some particular embodiments are oligonucleotides in a size range with a lower limit of about 5 to 15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50 to 600 nt, and other particular embodiments are in a size range with a lower limit of about 10 to 20 nt and an upper limit of about 22 to 100 nt. Oligonucleotides may be purified from naturally occurring sources but may be synthesized by using any well-known enzymatic or chemical method. Oligomers may be referred to by a functional name (e.g., detection probe, primer, or promoter primer) but those skilled in the art will understand that such terms refer to oligomers.

[0112] A “target nucleic acid” as used herein is a nucleic acid comprising a target sequence to be amplified. Target nucleic acids may be DNA or RNA and may be either single-stranded or double-stranded. The target nucleic acid may include other sequences besides the target sequence, which may not be amplified.

[0113] The term “target region” or “target nucleic acid region” as used herein refers to the particular nucleotide sequence of the target nucleic acid that is to be amplified and / or detected. The “target region” includes the complexing sequences to which oligonucleotides (e.g., priming oligonucleotides and / or promoter oligonucleotides) complex during an amplification processes (e.g., PCR, TMA). Unless the context clearly dictates otherwise, where the target nucleic acid is originally single-stranded, the term “target region” will also refer to the sequence complementary to the “target region” as present in the target nucleic acid, and where the target nucleic acid is originally doublestranded, the term “target region” refers to both the sense (+) and antisense (-) strands.

[0114] The term “target sequence” or “target nucleic acid sequence” as used herein refers to the particular nucleotide sequence of the target nucleic acid to which oligonucleotides (e.g., priming oligonucleotides, detection probes, or capture probes) complex during amplification and / or detection of the target nucleic acid.

[0115] “Target-hybridizing sequence” or “target-specific sequence” is used herein to refer to the portion of an oligomer that is configured to hybridize with a target nucleic acid sequence. Preferably, the target-hybridizing sequences are configured to specifically hybridize with a target nucleic acid sequence. Target-hybridizing sequences may be 100% complementary to the portion of the target sequence to which they are configured to hybridize, but not necessarily. Target-hybridizing sequences may also include inserted, deleted and / or substituted nucleotide residues relative to a target sequence.

[0116] “Non-target-specific sequence” or “non-target-hybridizing sequence” as used herein refers to a region of an oligomer sequence, wherein said region does not stably hybridize with a target sequence under standard hybridization conditions. Oligomers with non-target-specific sequences include, but are not limited to, promoter primers, promoter providers, target capture oligomers, torches, and molecular beacons.

[0117] The term “target a sequence,” as used herein in reference to a region of a C. difficile nucleic acid, refers to a process whereby an oligonucleotide hybridizes to a target region in a manner that allows for amplification and detection as described herein.In one embodiment, the oligonucleotide is complementary with the targeted C. difficile nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted C. difficile nucleic acid sequence.

[0118] The term “configured to” denotes an actual arrangement of the polynucleotide sequence configuration of a referenced oligonucleotide target-hybridizing sequence. For example, amplification oligomers that are configured to generate a specified amplicon from a target nucleic acid region have polynucleotide sequences that hybridize to the target region and can be used in an amplification reaction to generate the amplicon. Also, as an example, oligonucleotides that are configured to specifically hybridize to a target region have a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0119] The term “configured to specifically hybridize to” as used herein means that the target-hybridizing region of an amplification oligonucleotide, detection probe, or other oligonucleotide is designed to have a polynucleotide sequence that could target a sequence of the referenced C. difficile target region. The oligonucleotide is designed to function as a component of an assay for amplification and detection of C. difficile target nucleic acid from a sample, and therefore is designed to target C. difficile nucleic acid in the presence of other nucleic acids commonly found in testing samples. “Specifically hybridize to” does not mean exclusively hybridize to, as some small level of hybridization to non-target nucleic acids may occur, as is understood in the art. Rather, “specifically hybridize to” means that the oligonucleotide is configured to function in an assay to primarily hybridize the target so that an accurate detection of target nucleic acid in a sample can be determined.

[0120] An “amplification oligonucleotide” or “amplification oligomer” is an oligonucleotide that hybridizes to a target nucleic acid and participates in a nucleic acid amplification reaction, e.g., serving as a primer. Amplification oligomers can have 3’ ends that are extended by polymerization as part of the nucleic acid amplification reaction. Amplification oligomers can alternatively have 3’ ends that are not extended by polymerization, but provide a component that facilitates nucleic acid amplification, e.g., a promoter sequence joined 5’ to the target hybridizing sequence of the amplification oligomer. Such an amplification oligomer is referred to as a promoter provider.Amplification oligomers that provide both a 3’ target hybridizing region that is extendable by polymerization and a 5’ promoter sequence are referred to as promoter primers. Amplification oligomers may be optionally modified to include 5’ non-target hybridizing regions such as tags, promoters (as mentioned), or other sequences used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0121] “Nucleic acid amplification” refers to any in vitro procedure that produces multiple copies of a target nucleic acid sequence, or its complementary sequence, or fragments thereof ( / .<., an amplified sequence containing less than the complete target nucleic acid). Examples of nucleic acid amplification procedures include transcription associated methods, such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) and others (e.g., U. S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), and polymerase chain reaction (PCR) (e.g., U. S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159).

[0122] By “amplicon” or “amplification product” is meant a nucleic acid molecule generated in a nucleic acid amplification reaction and which is derived from a target nucleic acid. An amplicon or amplification product contains a target nucleic acid region that may be of the same or opposite sense as the target nucleic acid.

[0123] “Detection probe oligomer,” “detection probe,” or “probe” refers to an oligomer that hybridizes specifically to a target nucleic acid region, including an amplified product, under conditions that promote nucleic acid hybridization, for detection of the target nucleic acid. Detection may either be direct ( / .<., probe hybridized directly to the target) or indirect ( / .<., a probe hybridized to an intermediate structure that links the probe to the target). A probe’s target sequence generally refers to the specific sequence within a larger sequence which the probe hybridizes specifically. A detection probe may include target-specific sequence(s) and non-target-specific sequence(s). Such non-target-specific sequences can include sequences which will confer a desired secondary or tertiary structure, such as a hairpin structure, which can be used to facilitate detection and / or amplification.

[0124] By “stringent hybridization conditions,” or “stringent conditions” is meant conditions permitting an oligomer to preferentially hybridize to a target nucleic acid region and not to nucleic acid derived from a closely related non-target nucleic acid ( / .<., conditions permitting an oligomer to hybridize to its target sequence to form a stableoligomer:target hybrid, but not form a sufficient number of stable oligomer:non-target hybrids, so as to allow for amplification and / or detection of target nucleic acids but not non-targeted organisms). While the definition of stringent hybridization conditions does not vary, the actual reaction environment that can be used for stringent hybridization may vary depending upon factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and sequences of non-target nucleic acids that may be present in the test sample, and the target sequence. Hybridization conditions include the temperature and the composition of the hybridization reagents or solutions. Stringent hybridization conditions are readily ascertained by those having ordinary skill in the art.

[0125] “Label” or “detectable label” refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal. Direct joining may use covalent bonds or non-covalent interactions (e.g, hydrogen bonding, hydrophobic or ionic interactions, and chelate or coordination complex formation) whereas indirect joining may use a bridging moiety or linker (e.g, via an antibody or additional oligonucleotide(s), which may amplify a detectable signal). Any detectable moiety may be used, e.g., radionuclide, ligand such as biotin or avidin, enzyme, enzyme substrate, reactive group, chromophore such as a dye or particle (e.g., latex or metal bead) that imparts a detectable color, luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent compound such as an acridinium ester (“AE”) compound), and fluorescent compound (z.e., fluorophore). Embodiments of fluorophores include those that absorb light in the range of about 495 to 690 nm and emit light in the range of about 520 to 705 nm, which include those known as HEX, FAM™, TET™, CAL FLUOR™ (Orange or Red), and QUASAR™ compounds. Fluorophores may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to diminish background fluorescence. Such quenchers are well known in the art and include, e.g., BLACK HOLE QUENCHER™ (or BHQ™) or TAMRA™ compounds. Particular embodiments include a “homogeneous detectable label” that is detectable in a homogeneous system in which bound labeled probe in a mixture exhibits a detectable change compared to unbound labeled probe, which allows the label to be detected without physically removing hybridized from unhybridized labeled probe (e.g., US Pat. Nos.5,283,174, 5,656,207, and 5,658,737). Particular homogeneous detectable labels includechemiluminescent compounds, including acridinium ester (“AE”) compounds, such as standard AE or AE derivatives, which are well known (US Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Methods of synthesizing labels, attaching labels to nucleic acid, and detecting signals from labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Ch. 10, and US Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333, and EP Pat. App. 0 747 706). Particular methods of linking an AE compound to a nucleic acid are known (e.g., US Pat. No. 5,585,481 and US Pat. No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example 8). Particular AE labeling positions are a probe’s central region and near a region of A / T base pairs, at a probe’s 3’ or 5’ terminus, or at or near a mismatch site with a known sequence that is the probe should not detect compared to the desired target sequence. Other detectably labeled probes include, e.g., TaqMan™ probes, molecular torches, and molecular beacons. TaqMan™ probes include a donor and acceptor label wherein fluorescence is detected upon enzymatically degrading the probe during amplification in order to release the fluorophore from the presence of the quencher. Molecular torches and beacons exist in open and closed configurations wherein the closed configuration quenches the fluorophore and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to target opens the otherwise closed probes.

[0126] A “non-extendable” oligomer includes a blocking moiety at or near its 3’-terminus to prevent extension. A blocking group near the 3’ end is in some embodiments within five residues of the 3’ end and is sufficiently large to limit binding of a polymerase to the oligomer. In other embodiments, a blocking group is covalently attached to the 3’ terminus. Suitable blocking groups include, e.g., alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues, cordycepin, 3 ’-deoxy nucleotides, 3’-phosphorylated nucleotides, inverted nucleotides, proteins, peptides, and labels such as fluorophores or quenchers.

[0127] References, particularly in the embodiments, to “the sequence of SEQ ID N0: X” refer to the sequence of nucleotides and / or nucleotide analogs linked together to form a biopolymer. Reference to a sequence by SEQ ID NO does not connote the identity of the backbone (e.g., RNA, 2’-0-Me RNA, or DNA) or any nucleobase modifications (e.g., methylation of cytosine residues (“5MeC”)) unless the context clearly dictatesotherwise. In some instances, the sequence of a SEQ ID NO is followed by the statement “including from [x-y] nucleotide analogs”; it is understood that the nucleotide analogs may be substitutions within the sequence of the SEQ ID NO. Unless the context clearly dictates otherwise, reference to a sequence by SEQ ID NO includes reference to its complementary sequence (e.g., reference to the sequence 5’-ttagc-3’ includes reference to the sequence 5’-gctaa-3’).

[0128] “Separating” or “purifying” means that one or more components of a sample are removed or separated from other sample components. Sample components include target nucleic acids usually in a generally aqueous solution phase, which may also include cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids. “Separating” or “purifying” does not connote any degree of purification. Typically, separating or purifying removes at least 70%, or at least 80%, or at least 95% of the target nucleic acid from other sample components.

[0129] The term “non-linear surfactant,” as used herein, means a surfactant having a branched chain structure. A non-linear surfactant may include one or more ring structures, which may be, for example, in a principal chain and / or in one or more branched chains. Exemplary non-linear surfactants include polysorbate 20, polysorbate 40, polysorbate 60, and digitonin. In certain variations, the non-linear surfactant is non-ionic.

[0130] The term “specificity,” in the context of an amplification and / or detection system, is used herein to refer to the characteristic of the system which describes its ability to distinguish between target and non-target sequences dependent on sequence and assay conditions. In terms of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of side-products (e.g., the signal -to-noise ratio). In terms of detection, specificity generally refers to the ratio of signal produced from target nucleic acids to signal produced from non-target nucleic acids.

[0131] The term “sensitivity” is used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantitated. The sensitivity of an amplification reaction is generally a measure of the smallest copy number of the target nucleic acid that can be reliably detected in the amplification system, and will depend, for example, on the detection assay being employed, and the specificity of the amplification reaction, e.g., the ratio of specific amplicons to side-products.DETAILED DESCRIPTION

[0132] Provided herein are compositions, kits, and methods for amplifying and / or detecting target nucleic acid from toxigenic C. difficile strains. Preferably, the samples are biological samples. The compositions, kits, and methods provide oligonucleotide sequences that target C. difficile gene sequences or their complementary sequences. Such oligonucleotides may be used as amplification oligonucleotides, which may include primers, promoter primers, blocked oligonucleotides, and promoter provider oligonucleotides, whose functions have been described previously (see, e.g., US Patent Nos. 4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and 7,374,885; each incorporated by reference herein). Other oligonucleotides may be used as probes for detecting amplified sequences or for capture of a C. difficile target nucleic acid.

[0133] The methods provide for the sensitive and specific detection of toxigenic C. difficile strains. The methods include performing nucleic acid amplification of one or more target regions of C. difficile and detecting one or more amplified products by, for example, specifically hybridizing the amplified product(s) with one or more nucleic acid detection probes that provide a signal to indicate the presence of C. difficile in the sample. The amplification step includes contacting the sample with one or more C. difficile-specific amplification oligomers specific for a target sequence in C. difficile target nucleic acid. Target nucleic acids include, e.g., regions within the C. difficile toxin A gene sequence (tcdA) and the toxin B gene sequence (tcdB). In some variations, the disclosed methods also target the identification of presumptive BI / NAP 1 / 027 hypervirulent C. difficile strain via the additional detection of the single base pair deletion at nucleotide 117 in the tcdC gene sequence and the subunit B of the binary toxin gene sequence (cdtB). Nucleic acid amplification is performed to produce an amplification product corresponding to one or more of the C. difficile target nucleic acids, if present in the sample, wherein the amplification reaction synthesizes additional copies of the target sequence or its complement by using at least one nucleic acid polymerase and the one or more amplification oligomers to produce the copies from a template strand e.g., by extending the sequence from a primer using the template strand). One embodiment for detecting an amplification product uses a hybridizing step that includes contacting the amplified product with at least one detection probe oligomer specific for a sequence amplified by the selectedamplification oligomers, e.g., a sequence contained in the target sequence flanked by a pair of selected amplification oligomers.

[0134] In some aspects, oligonucleotides are provided, e.g., in a kit or composition. Oligonucleotides generally comprise a target-hybridizing region, e.g., configured to hybridize specifically to a target nucleic acid of C. difficile. While oligonucleotides of different lengths and base composition may be used for amplifying target nucleic acids, in some embodiments, oligonucleotides in this disclosure have targethybridizing regions from about 10 to about 60 bases in length, from about 14 to about 50 bases in length, from about 14 to about 40 bases in length, from about 14 to about 35 bases in length, from about 15 to about 30 bases in length, or from about 16 to about 30 bases in length. In some embodiments, an oligonucleotide comprises a second region of sequence in addition to the target-hybridizing region, such as a promoter, which can be located 5’ of the target-hybridizing region. In some embodiments, an oligonucleotide does not comprise a second region of sequence.

[0135] In some embodiments, a set of oligonucleotides comprising a combination of two or more oligonucleotides are provided, e.g., in a kit or composition, such as an amplification oligomer (e.g., primer) pair or an amplification oligomer pair and a third oligonucleotide that is optionally labeled (e.g., for use as a probe), wherein the oligonucleotides are configured to hybridize to a target nucleic acid of C. difficile. In some embodiments, a set of oligonucleotides includes a combination of two or more (e.g., three or four) amplification oligomer pairs configured to hybridize to two or more target nucleic acids of C. difficile, or a combination of two or more amplification oligomer pairs and two or more additional oligonucleotides that are optionally labeled.

[0136] In some embodiments, one or more oligonucleotides comprise a nonWatson Crick (NWC) position. In some embodiments, a C. difficile amplification oligomer, a C. difficile amplification oligomer pair, and / or a C. difficile probe comprises a NWC position, such as a position that includes inosine.

[0137] In some embodiments, one or more oligonucleotides comprise a position comprising 5-methylcytosine. In some embodiments, a C. difficile amplification oligomer, a r C. difficile amplification oligomer pair, and / or a C. difficile probe comprises a position comprising 5-methylcytosine.

[0138] In some embodiments, one or more oligonucleotides comprise a position comprising propyne dU. In some embodiments, a C. difficile amplification oligomer, a C. difficile amplification oligomer pair, and / or a C. difficile probe comprises a position comprising propyne dU.

[0139] Exemplary oligomers targeting C. difficile target nucleic acid in accordance with the present disclosure are shown in Table 35. Exemplary amplification oligomer pairs and optional third oligomers (e.g., detection probe) targeting different C. difficile gene sequences are set forth (by SEQ ID NO) in the following Table 1.Table 1. Exemplary Oligonucleotide SetsTarget Gene Oligonucleotide 1 Oligonucleotide 2 Oligonucleotide 3(e.g., forward (e.g., reverse (e.g., probe, primer) primer) optionally labeled) led A 12 32 1018 36 2, 418 38 2, 433 36 2, 433 38 2, 418 37 2, 4 tcdB33 37 2, 417 36 2, 417 38 2, 417 37 2, 426 38 1, 331 38 1, 38 19 23, 246 13 11, 23, 24 29 40 23, 2430 40 23, 2430 19 23, 249 40 23, 249 19 23, 2430 34 23, 2430 28 23, 2430 39 23, 24 tcdC30 20 23, 24Target Gene Oligonucleotide 1 Oligonucleotide 2 Oligonucleotide 3 (e.g., forward (e.g., reverse (e.g., probe, primer) primer) optionally labeled) 30 22 23, 24 30 5 23, 24 30 27 23, 24 30 7 23, 24 9 34 23, 24 9 28 23, 24 9 39 23, 24 9 20 23, 24 9 22 23, 24 9 5 23, 24 9 27 23, 24 9 7 23, 24 cdtB 14 15 1635 21 25

[0140] Exemplary oligomers containing one or more nucleotide analogues (“modified oligomers”) are set forth by SEQ ID NO in the following Table 2 below.Table 2. Exemplary Modified OligomersModified Oligomer Corresponds toSEQ ID NO SEQ ID NO*41 142 343 2544 245 446 1047 1648 249 450 1051 2452 2453 24Modified Oligomer Corresponds toSEQ ID NO SEQ ID NO*54 1055 256 457 2358 16* “Corresponds to” means that the modified oligomeris an example of an oligomer containing one or morenucleotide analogues relative to this SEQ ID NO.

[0141] In some embodiments, an oligonucleotide is provided that comprises a label. Such an oligonucleotide can be used as a detection probe. In some embodiments, the labeled oligonucleotide has a sequence corresponding to a SEQ ID NO listed in the Oligonucleotide 3 column of Table 1. In some embodiments, the label is a non-nucleotide label. Suitable labels include compounds that emit a detectable light signal, e.g, fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label, may be present on a particular probe, or detection may rely on using a mixture of probes, in which each probe is labeled with a compound that produces a detectable signal (see. e.g., US Pat. Nos. 6,180,340 and 6,350,579, each incorporated by reference herein). Labels may be attached to a probe by various means including covalent linkages, chelation, and ionic interactions, but in some embodiments the label is covalently attached. For example, in some embodiments, a detection probe has an attached chemiluminescent label such as, e.g, an acridinium ester (AE) compound (see, e.g., US Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744). A label, such as a fluorescent or chemiluminescent label, can be attached to the probe by a non-nucleotide linker (see, e.g., US Pat. Nos. 5,585,481; 5,656,744; and 5,639,604). In some embodiments, the label may include one or more of Quasar670, CalRed610, CalOrange560, fluorescein, ROX, FAM, and HEX.

[0142] In some embodiments, a detection probe (e.g., comprising a fluorescent label) further comprises a second label that interacts with the first label. For example, the second label can be a quencher. In some embodiments, the second label may include one or both of BHQ-1 and BHQ-2. Such probes can be used, e.g., in TaqMan™ assays, where hybridization of the probe to a target or amplicon followed by nucleolysis by a polymerase comprising 5’-3’ exonuclease activity results in liberation of the fluorescent label andthereby increased fluorescence, or fluorescence independent of the interaction with the second label.

[0143] In some applications, one or more detection probes exhibiting at least some degree of self-complementarity are used to facilitate detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection. Specific embodiments of such detection probes include, for example, probes that form conformations held by intramolecular hybridization, such as conformations generally referred to as hairpins. Suitable hairpin probes include a “molecular torch” (see, e.g., US Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945) and a “molecular beacon” (see, e.g., US Pat. No. 5,118,801 and U. S. Pat. No. 5,312,728). Molecular torches include distinct regions of self-complementarity (coined “the target-binding domain” and “the target-closing domain”) which are connected by a joining region (e.g., a -(CH₂CH₂O)₃-linker) and which hybridize to one another under predetermined hybridization assay conditions. When exposed to an appropriate target or denaturing conditions, the two complementary regions (which may be fully or partially complementary) of the molecular torch melt, leaving the target-binding domain available for hybridization to a target sequence when the predetermined hybridization assay conditions are restored. Molecular torches are designed so that the target-binding domain favors hybridization to the target sequence over the target-closing domain. The target-binding domain and the target-closing domain of a molecular torch include interacting labels (e.g., fluorescent / quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized as opposed to when the molecular torch is hybridized to a target nucleic acid, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized probe having a viable label associated therewith.

[0144] Examples of interacting donor / acceptor label pairs that may be used in connection with the disclosure, making no attempt to distinguish FRET from non-FRET pairs, include fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, lucifer yellow / DABCYL, BODIPY / DABCYL, eosine / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BHQ-1, CY5 / BHQ-2, CY3 / BHQ-1, CY3 / BHQ-2 and fluorescein / QSY7 dye. Those having an ordinary level of skill in the art will understandthat when donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. Non-fluorescent acceptors such as DABCYL and the QSY7 dyes advantageously eliminate the potential problem of background fluorescence resulting from direct ( / .<., non-sensitized) acceptor excitation. Exemplary fluorophore moi eties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, the ATTO dyes, the DY dyes, and the CY dyes. Exemplary quencher moieties that can be used as another member of a donor-acceptor pair include DABCYL, BlackBerry Quencher, and the Black Hole Quencher moieties.

[0145] In some embodiments, a labeled oligonucleotide (e.g., probe) is non-extendable. For example, the labeled oligomer can be rendered non-extendable by 3’-phosphorylation, having a 3 ’-terminal 3 ’-deoxynucleotide (e.g., a terminal 2’,3’-dideoxynucleotide), having a 3 ’-terminal inverted nucleotide (e.g., in which the last nucleotide is inverted such that it is joined to the penultimate nucleotide by a 3’ to 3’ phosphodiester linkage or analog thereof, such as a phosphorothioate), or having an attached fluorophore, quencher, or other label that interferes with extension (possibly but not necessarily attached via the 3’ position of the terminal nucleotide). In some embodiments, the 3 ’-terminal nucleotide is not methylated.

[0146] Also provided by the disclosure is a reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample in accordance with the methods as described herein. A reaction mixture in accordance with the present disclosure comprises at least one or more of the following: an oligonucleotide as described herein for amplification of a target nucleic acid; and an oligonucleotide (e.g., probe) as described herein for determining the presence or absence of an amplification product of the target nucleic acid. For a reaction mixture that includes a detection probe together with an amplification oligonucleotide combination, the amplification oligonucleotides and detection probe oligonucleotides for a reaction mixture are linked by a common target region ( / .<., the reaction mixture will include a probe that binds to a sequence amplifiable by an amplification oligonucleotides combination of the reaction mixture). In some embodiments, a reaction mixture is for performing a multiplex amplification and / or detection reaction, wherein the mixture comprises at least one of (a) and (b): (a) at least two or more (e.g., three or four) amplification oligonucleotide sets for amplifying two ormore target regions of C. difficile target nucleic acid (e.g., first, second, third, and fourth oligonucleotide sets for amplifying target regions of C. difficile tcdA, tcdB, tcdC, and cdtB target nucleic acid), and (b) at least two (e.g., three or four) detection probe oligonucleotides for detecting two or more target regions, or amplicons thereof, of C. difficile target nucleic acid (e.g., first, second, third, and fourth detection probe oligonucleotides for detecting target regions, or amplicons thereof, of C. difficile tcdA, tcdB, tcdC, and cdtB target nucleic acid).

[0147] A reaction mixture may further include a number of optional components such as, for example, capture probes, e.g., poly-(k) capture probes as described in US 2013 / 0209992, which is incorporated herein by reference, and / or poly-(R) capture probes as described in US 2020 / 0165599, which is incorporated herein by reference. For an amplification reaction mixture, the reaction mixture will typically include other reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, and dTTP; and / or ATP, CTP, GTP and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or reverse transcriptase and / or RNA polymerase), and will typically include test sample components, in which a target nucleic acid may or may not be present. Suitable reagents include, for example, formulations containing lithium lauryl sulfate (LLS), sodium lauryl sulfate (SLS), NaH2PO4, Na2HPO4, EDTA, EGTA, LiOH, NaCl, KC1, MgCl2, NaOH, ethanol, methylparaben, propylparaben, trehalose, Tris Buffer, Triton X-100, paramagnetic particles, target capture oligonucleotides, HEPES, succinic acid, polymerases (e.g., DNA polymerases, reverse transcriptases), and / or RNasin.

[0148] In some embodiments, a reaction mixture comprises KC1. In some embodiments, the KC1 concentration is about 50 mM. In some embodiments, the KC1 concentration is greater than about 50 mM, e.g., about 60-150 mM, about 75-125 mM, about 80-120 mM, about 85-115 mM, or about 90-110 mM. In some embodiments, the KC1 concentration is about 55-65 mM, about 65-75 mM, about 75-85 mM, about 85-95 mM, about 95-105 mM, about 105-115 mM, about 115-125 mM, about 125-135 mM, or about 135-145 mM. In some embodiments, a composition according to the disclosure comprises KC1, e.g., at any of the foregoing concentrations. In some embodiments, a method according to the disclosure comprises performing an amplification reaction in the presence of KC1, e.g., at any of the foregoing concentrations.

[0149] In some embodiments, a reaction mixture comprises a non-linear surfactant such as, for example, polysorbate 20. In certain variations, the non-linear surfactant (e.g., polysorbate 20) is present in the reaction mixture at a concentration of from about 0.001% to about 0.025% (v / v) or from about 0.0015% to about 0.015% (v / v). In certain embodiments, a reaction mixture comprises a-cyclodextrin. In certain variations, the a-cyclodextrin is present at a concentration from about 1.0 mg / mL to about 10 mg / mL, from about 3.0 mg / mL to about 9.0 mg / mL, or from about 2.0 mg / mL to about 7.0 mg / mL.

[0150] In some embodiments, a reaction mixture comprises a lyoprotectant. Exemplary lyoprotectants include glycerol; non-reducing sugars such as, e.g., sucrose, raffinose, or trehalose; and amino acids such as, e.g., glycine, arginine, or methionine. In certain variations wherein the lyoprotectant is trehalose, trehalose is present at a concentration of from about 0.1 M to about 0.2 M (e.g., about 0.15 M).

[0151] In some embodiments, a reaction mixture comprises a chelating agent. Suitable chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(P-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA). In some embodiments comprising EDTA as the chelating agent, EDTA is present in the reaction mixture at a concentration of from about 0.025 mM to about 0.25 mM (e.g., at a concentration of about 0.08 mM).

[0152] Also provided by the subject disclosure are kits for practicing the methods as described herein. A kit in accordance with the present disclosure comprises at least one or more of the following: an oligonucleotide as described herein for amplification of a target nucleic acid; and an oligonucleotide (e.g., probe) as described herein for determining the presence or absence of an amplification product of the target nucleic acid. In some embodiments, any oligonucleotide combination described herein is present in the kit. The kits may further include a number of optional components such as, for example, capture probes, e.g., poly-(k) capture probes as described in US 2013 / 0209992 and / or poly-(R) capture probes as described in US 2020 / 0165599. Other reagents that may be present in the kits include reagents suitable for performing in vitro amplification such as, e.g., buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP; and / or ATP, CTP, GTP and UTP), and / or enzymes (e.g., a thermostable DNA polymerase, or a reverse transcriptase and / or RNA polymerase). Oligonucleotides as described herein may be packaged in a variety of different embodiments, and those skilled in the art willappreciate that the disclosure embraces many different kit configurations. In addition, for a kit that includes a detection probe together with an amplification oligomer combination, the amplification oligonucleotides and detection probe oligonucleotides for a kit are linked by a common target region ( / .<., the kit will include a probe that binds to a sequence amplifiable by an amplification oligonucleotides combination of the kit). In some embodiments, a kit is for performing a multiplex amplification and / or detection reaction, wherein the kit comprises at least one of (a) and (b): (a) at least two or more (e.g., three or four) amplification oligonucleotide sets for amplifying two or more target regions of C. difficile target nucleic acid (e.g., first, second, third, and fourth oligonucleotide sets for amplifying target regions of C. difficile tcdA, tcdB, tcdC, and cdtB target nucleic acid), and (b) at least two (e.g., three or four) detection probe oligonucleotides for detecting two or more target regions, or amplicons thereof, of C. difficile target nucleic acid (e.g., first, second, third, and fourth detection probe oligonucleotides for detecting target regions, or amplicons thereof, of C. difficile tcdA, tcdB, tcdC, and cdtB target nucleic acid). In certain embodiments, the kit further includes a set of instructions for practicing methods in accordance with the present disclosure, where the instructions may be associated with a package insert and / or the packaging of the kit or the components thereof.

[0153] In some embodiments of a kit as described herein, the oligonucleotides are contained in a formulation comprising at least one of a non-linear surfactant (e.g., polysorbate 20), a-cyclodextrin, a lyoprotectant (e.g., a non-reducing sugar such as sucrose, raffinose, or trehalose, or an amino acid such as glycine, arginine, or methionine), and a chelating agent (e.g., EDTA or EGTA). In some such embodiments, polysorbate 20 is present in the formulation at a concentration of from about 0.002% to about 0.05% (v / v) or about 0.003% to about 0.03% (v / v), a-cyclodextrin is present at a concentration from about 1.0 mg / mL to about 10 mg / mL or about 3.0 mg / mL to about 9 mg / mL, trehalose is present in the formulation at a concentration of from about 0.2 M to about 0.4 M (e.g., about 0.26 M or about 0.3 M), and / or EDTA is present in the formulation at a concentration of from about 0.05 mM to about 0.5 mM (e.g., about 0.16 mM or about 0.14 mM). In certain variations, the formulation is a lyophilized formulation. In some embodiments of a lyophilized formulation, the formulation is for reconstitution into an aqueous formulation containing polysorbate 20, trehalose, and / or EDTA at concentration(s) as specified above.

[0154] Also provided by the subject disclosure are methods for determining the presence or absence of a toxigenic C. difficile strain in a sample by, for example, using one or more of the oligonucleotides disclosed herein. Any method disclosed herein is also to be understood as a disclosure of corresponding uses of materials involved in the method directed to the purpose of the method. Any of the oligonucleotides and any combinations (e.g., kits and compositions) comprising such an oligonucleotide are to be understood as also disclosed for use in detecting C. difficile target nucleic acid and for use in the preparation of a composition for detecting C. difficile target nucleic acid.

[0155] Broadly speaking, methods can comprise one or more of the following components: target capture, in which a target nucleic acid (e.g., from a sample, such as a clinical sample) is annealed to a capture oligomer; isolation, e.g., washing, to remove material not associated with a capture oligomer; amplification; and amplicon detection, e.g., amplicon quantification, which may be performed in real time with amplification. Certain embodiments involve each of the foregoing steps. Certain embodiments involve exponential amplification, optionally with a preceding linear amplification step. Certain embodiments involve exponential amplification and amplicon detection. Certain embodiments involve any two of the components listed above. Certain embodiments involve any two components listed adjacently above, e.g., washing and amplification, or amplification and detection.

[0156] Amplifying a C. difficile target nucleic acid region utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified (e.g., one or more oriented in the sense direction and one or more oriented in the antisense direction for exponential amplification). Particularly suitable oligomer combinations for amplification of C. difficile target regions are described herein. Exemplary amplification oligomers for amplifying C. difficile target regions (including exemplary target-hybridizing core sequences and regions) are listed in Table 35, infra (see also exemplary modified oligomers in Table 2, supra, and Table 36, infra), and particular combinations of first and second amplification oligomers for different C. difficile target genes are set forth herein (see, e.g., Table 1, supra, Embodiments section, supra, and Examples 1-7, infra (including Tables 3, 4, 9, 10, 13, 14, 16, 18, and 23)).

[0157] A detection method in accordance with the present disclosure can further include the step of obtaining the sample to be subjected to subsequent steps of the method.In certain embodiments, “obtaining” a sample to be used includes, for example, receiving the sample at a testing facility or other location where one or more steps of the method are performed, and / or retrieving the sample from a location (e.g., from storage or other depository) within a facility where one or more steps of the method are performed.

[0158] In certain embodiments, the method further includes purifying the C. difficile target nucleic acid from other components in the sample, e.g, before an amplification, such as before a capture step. Such purification may include methods of separating and / or concentrating C. difficile contained in a sample from other sample components, or removing or degrading non-nucleic acid sample components, e.g, protein, carbohydrate, salt, lipid, etc. In some embodiments, purifying the target nucleic acid includes degrading nucleic acid in the sample, e.g., with DNase, and optionally removing or inactivating the DNase or removing degraded nucleic acid.

[0159] In particular embodiments comprising a target purification step, a target nucleic acid is captured specifically or non-specifically and separated from other sample components. Non-specific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adherence of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture that contains C. difficile nucleic acid and other sample components.

[0160] Target capture typically occurs in a solution phase mixture that contains one or more capture probe oligomers that hybridize to the C. difficile target nucleic acid under hybridizing conditions. For embodiments comprising a capture probe tail, the target: capture-probe complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to an immobilized probe. Certain embodiments use a particulate solid support, such as paramagnetic beads. Selective and non-specific target capture methods are also described, e.g., in US Patent No. 6,110,678 and International Patent Application Pub. No. WO 2008 / 016988, each incorporated by reference herein.

[0161] Isolation can follow capture, where, for example, the complex on the solid support is separated from other sample components. Isolation can be accomplished by any appropriate technique, e.g., washing a support associated with the C. difficile target nucleic acid one or more times (e.g., two or three times) to remove other sample components and / or unbound oligomer. In embodiments using a particulate solid support, such as paramagneticbeads, particles associated with the C. difficile target may be suspended in a washing solution and retrieved from the washing solution, in some embodiments by using magnetic attraction. To limit the number of handling steps, the C. difficile target nucleic acid may be amplified by simply mixing the target region in the complex on the support with amplification oligomers and proceeding with amplification steps.

[0162] Exponentially amplifying a target sequence utilizes an in vitro amplification reaction using at least two amplification oligomers that flank a target region to be amplified. In some embodiments, at least one oligonucleotide as described above is provided. In some embodiments, at least two oligonucleotides as described above are provided. In other embodiments, at least three, at least four, at least, five, at least six, at least seven, or at least eight oligonucleotides are provided. In some preferred variations, at least eight amplification oligonucleotides are provided for amplification of target regions of each of C. difficile tcdA, tcdB, tcdC, and cdtB target genes. The amplification reaction can be cycled or isothermal. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA).

[0163] A detection step may be performed using any of a variety of known techniques to detect a signal specifically associated with the amplified target region, such as, e.g., by hybridizing the amplification product with a labeled detection probe and detecting a signal resulting from the labeled probe (including from label released from the probe following hybridization in some embodiments), performing electrophoresis on the sample and / or the amplification product, or determining the sequence of the amplification product. In some embodiments, the labeled probe comprises a second moiety, such as a quencher or other moiety that interacts with the first label, as discussed above. The detection step may also provide additional information on the amplified sequence, such as, e.g., all or a portion of its nucleic acid base sequence. Detection may be performed after the amplification reaction is completed or may be performed simultaneously with amplifying the target region, e.g., in real time. In one embodiment, the detection step allows homogeneous detection, e.g., detection of the hybridized probe without removal of unhybridized probe from the mixture (see, e.g., US Pat. Nos. 5,639,604 and 5,283,174). In some embodiments, the nucleic acids are associated with a surface that results in a physicalchange, such as a detectable electrical change. Amplified nucleic acids may be detected by concentrating them in or on a matrix and detecting the nucleic acids or dyes associated with them (e.g., an intercalating agent such as ethidium bromide or cyber green) or detecting an increase in dye associated with nucleic acid in solution phase. Other methods of detection may use nucleic acid detection probes that are configured to specifically hybridize to a sequence in the amplified product and detecting the presence of the probe: product complex, or by using a complex of probes that may amplify the detectable signal associated with the amplified products (see, e.g., US Pat. Nos. 5,424,413; 5,451,503; and 5,849,481; each incorporated by reference herein). Directly or indirectly labeled probes that specifically associate with the amplified product provide a detectable signal that indicates the presence of the target nucleic acid in the sample. In particular, the amplified product will contain a target sequence in or complementary to a target nucleic sequence of C. difficile, and a probe will bind directly or indirectly to a sequence contained in the amplified product to indicate the presence or absence of C. difficile in the tested sample.

[0164] In embodiments that detect the amplified product near or at the end of the amplification step, a linear detection probe may be used to provide a signal to indicate hybridization of the probe to the amplified product. One example of such detection uses a luminescently labeled probe that hybridizes to target nucleic acid. The luminescent label is then hydrolyzed from non-hybridized probe. Detection is performed by chemiluminescence using a luminometer. (See, e.g., International Patent Application Pub. No. WO 89 / 002476, incorporated by reference herein). In other embodiments that use realtime detection, the detection probe may be a hairpin probe such as, for example, a molecular beacon, molecular torch, or hybridization switch probe that is labeled with a reporter moiety that is detected when the probe binds to amplified product (e.g, a dual -labeled hairpin probe comprising both a fluorescent label and a quenching moiety). In other embodiments for real-time detection, the detection probe is a linear oligomer such as, e.g, an oligomer labeled with both a fluorophore and a quenching moiety (e.g., a TaqMan probe). Such probes may comprise target-hybridizing sequences and non-targethybridizing sequences. Various forms of such probes have been described previously (see, e.g., US Patent Nos. 5,210,015; 5,487,972; 5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. Nos.20060068417A1 and 20060194240A1; each incorporated by reference herein). ExemplaryC. difficile-specific detection probe oligomers are listed in Tables 1 and 2, supra, and Tables 25 and 26, infra, and are also set forth in the Embodiments section, supra, and Examples, infra (including, e.g., their use in combination with at least two C. difficile-specific amplification oligomers for detection of a C. difficile target nucleic acid).

[0165] Assays for detection of a C. difficile nucleic acid may optionally include a non-C. difficile internal control (IC) nucleic acid that is amplified and detected in the same assay reaction mixtures by using amplification and detection oligomers specific for the IC sequence. IC nucleic acid sequences can be, e.g., a DNA plasmid, an RNA template sequence (e.g., an in vitro transcript), or a synthetic nucleic acid that is spiked into a sample. Alternatively, the IC nucleic acid sequence may be a cellular component, which may be from exogenous cellular sources or endogenous cellular sources relative to the specimen. In these instances, an internal control nucleic acid is co-amplified with the C. difficile nucleic acid in the amplification reaction mixtures. The internal control amplification product and the C. difficile target region amplification product can be detected independently.

[0166] In certain embodiments, amplification and detection of a signal from an amplified IC sequence demonstrates that the assay reagents, conditions, and performance of assay steps were properly used in the assay if no signal is obtained for an intended target C. difficile nucleic acid (e.g., samples that test negative for C. difficile). An IC may also be used as an internal calibrator for the assay when a quantitative result is desired, i.e., the signal obtained from the IC amplification and detection is used to set a parameter used in an algorithm for quantitating the amount of C. difficile nucleic acid in a sample based on the signal obtained for an amplified C. difficile target region. ICs are also useful for monitoring the integrity of one or more steps in an assay. The primers and probe for the IC target sequence are configured and synthesized by using any well-known method provided that the primers and probe function for amplification of the IC target sequence and detection of the amplified IC sequence using substantially the same assay conditions used to amplify and detect the C. difficile target region(s). In certain embodiments that include a target capture-based purification step, it is preferred that a target capture probe specific for the IC target be included in the assay in the target capture step so that the IC is treated in the assay in a manner analogous to that for the intended C. difficile analyte(s) in all of the assay steps.

[0167] Methods for determining the presence or absence of C. difficile as described herein may have a detection sensitivity of, for example, from 10 to 5,000 cp / mL, from 50 to 5,000 cp / mL, from 100 to 5,000 cp / mL, from 250 to 5,000 cp / mL, from 500 to 5,000 cp / mL, from 750 to 5,000 cp / mL, from 10 to 2,500 cp / mL, from 50 to 2,500 cp / mL, from 100 to 2,500 cp / mL, from 250 to 2,500 cp / mL, from 500 to 2,500 cp / mL, from 750 to 2,500 cp / mL, from 10 to 1,500 cp / mL, from 50 to 1,500 cp / mL, from 100 to 1,500 cp / mL, from 250 to 1,500 cp / mL, from 500 to 1,500 cp / mL, or from 750 to 1,500 cp / mL (e.g., copies of IVT in Aptima® tube).

[0168] Also provided by the subject disclosure are methods for synthesizing one or more (e.g., one or more pairs) of the oligonucleotides disclosed herein, the oligonucleotides useful for determining the presence or absence of a toxigenic C. difficile strain. The method may, for example, include the steps of (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is bound (e.g., covalently bound) at a 3’ position to the solid support; (b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue; (c) repeating step (b) at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, or at least 28 additional times, thereby generating at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleobase residues coupled to the solid support; and (d) cleaving the at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide or oligonucleotides. In some embodiments, the oligonucleotide has a length of from 18 to 32 or from 18 to 30 contiguous nucleobase residues.

[0169] A method for synthesizing one or more of the oligonucleotides disclosed herein may be a solid phase method. For example, phosphorami di te solid-phase chemistry for joining nucleotides by phosphodiester linkages is disclosed in Caruthers et al., “Chemical Synthesis of Deoxynucleotides by the Phosphoramidite Method,” Methods EnzymoL 154:287 (1987). As another example, automated solid-phase chemical synthesisusing cyanoethyl phosphoramidite precursors has been described in Barone et al., “In Situ Activation of bis-dialkylaminephosphines - a New Method for Synthesizing Deoxy oligonucleotides on Polymer Supports,” Nucleic Acids Res. 12(10):4051 (1984). As another example, U. S. Patent No. 5,449,769, titled “Method and Reagent for Sulfurization of Organophosphorous Compounds,” discloses a procedure for synthesizing oligonucleotides containing phosphorothioate linkages. In addition, U. S. Patent No.5,811,538, titled “Process for the Purification of Oligomers,” discloses the synthesis of oligonucleotides having different linkages, including methylphosphonate linkages. Moreover, methods for the organic synthesis of oligonucleotides are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at Ch. 10.

[0170] Following synthesis and purification of a particular oligonucleotide, several different procedures may be utilized to purify and control the quality of the oligonucleotide. Suitable procedures include electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high pressure liquid chromatography).

[0171] The present disclosure is further illustrated by the following non-limiting examples.Example 1: Evaluation of tcdA and tcdC Oligonucleotides

[0172] Primer and probe combinations targeting the C. difficile tcdA and tcdC genes were evaluated for the ability to recognize tcdA and tcdC plasmids in singleplex PCR reactions. The primers and probes used (without reference to combinations) are shown in Table 3 below.Table 3. tcdA and tcdC Primer and Probe SequencesTarget Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C diff tcdA FP03 Primer 12C diff tcdA RP03 Primer 32tcdA C diff tcdA PR02 Probe 10 5mC at residues 9, 14, 23, 5’ FAM 24, and 25 3’ BHQ1 C diff tcdA PR02B Probe 10 5mC at residues 9, 14, 18, 5’ FAM 23, 24, and 25 3’ BHQ1 pdU at residues 6, 7, 10,and 20tcdC FP01 Primer 6tcdC RP01 Primer 13tcdC117delA Probe 24 Propyne C at residue 1 5’ FAM PR05-FAM pdU at residues 3, 4, 5, 7, 3’ BHQ18, 9, 10, 13, 15, 17, 18,tcdC and 19tcdC117delA Probe 24 pdU at residues 3, 4, 5, 7, 5’ CalFluor PR05-HEX 8, 9, 10, 13, 15, 17, 18, Orange 560 and 19 3’ BHQ1 tcdC117delA Probe 24 5mC at residue 1 5’ Cal Red 610 PR05-ROX pdU at residues 3, 4, 5, 7, 3’ BHQ28, 9, 10, 13, 15, 17, 18,and 19* Listed SEQ ID NOs indicated unmodified sequences. Modifications, if any, are indicated in the “Modifications” column.

[0173] The primer and probe combinations tested are shown in Table 4 below.Table 4. Primer and Probe Combinations for TestingCombination Forward primer Reverse primer ProbeNo.1 C diff tcdA FP03 C diff tcdA RP03 C diff tcdA PR022 C diff tcdA FP03 C diff tcdA RP03 C diff tcdA PR02B3 tcdC FP01 tcdC RP01 tcdC117delA PR05-FAM4 tcdC FP01 tcdC RP01 tcdC117delA PR05-HEX5 tcdC FP01 tcdC RP01 tcdC117delA PR05-ROXExperiment 1

[0174] In a first experiment, oligo combinations 1 and 2 were tested with tcdA plasmid, and combination 3 was tested with five different tcdC plasmids (tcdC WT, tcdCsc-1 Al 17A, tcdC 117T, tcdC 117G, tcdCl 17T-120T 183 & 184T) (having the target insert sequences shown in SEQ ID NOs:76-80, respectively; see Table 38, infra). Target plasmids were diluted to concentrations of 1, 10, 100, and 1,000 copies / pL. Target plasmids were tested at 5, 50, 500, and 5,000 copies / rxn. Results are shown in Tables 5-7 below.Table 5. Results for Oligo Combination 1 with tcdA PlasmidConcentration 5000 500 50 5 0(C / PCR)25.46 28.93 32.09 36.42 - Ct 25.33 28.93 32.29 39.41 - replicates25.61 29.30 32.41 - - Ct 25.46 29.05 32.26 37.91 - MeanSD 0.14 0.21 0.16 2.11 - PCR 76%efficiencyRn 1,900,000- 2,000,000Table 6. Result s for Oligo Combination 2 wit i tcdA PlasmidConcentration 5000 500 50 5 0(C / PCR)25.35 29.23 32.35 35.88 - Ct 25.77 29.32 32.68 35.44 - replicates25.78 29.28 32.27 35.26 - Ct 25.63 29.28 32.43 35.53 - MeanSD 0.25 0.04 0.22 0.32 - PCR 102%efficiencyRn 1,600,000- 1,800,000Table 7. Results for Combination 3 with tcdC sc-1 A117A Plasmid Concentration 5000 500 50 5 0(C / PCR)27.75 30.93 35.45 38.76 - Ct 27.46 31.06 34.90 39.00 - replicates27.53 31.20 35.08 36.77 - Ct 27.58 31.07 35.14 38.18 - MeanSD 0.15 0.13 0.28 1.23 - PCR 90%efficiencyRn 250,000-425,000

[0175] Experiment 1 Conclusion: Combination 1 (using probe C diff tcdA PR02) gave a higher fluorescence signal, but less efficiency, than Combination 2 (using probe C diff tcdA PR02B) because the sensitivity with probe C diff tcdA PR02B at the lower concentration was better. For tcdC plasmids, only tcdC sc-1 A117A was detected; tcdC WT, tcdC 117T, tcdC 117G, and tcdC117T-120T 183 & 184T were negative, with tcdC117T-120T 183 & 184T showing a high background. These results demonstrate that the oligo combination is specific for the detection of the tcdC plasmid with the nucleotide deletion in position 117 (tcdC sc-1 A117A).Experiment 2

[0176] In a second experiment, oligo combinations 3, 4, and 5 were tested with tcdC plasmid tcdC sc-1 A117A in singleplex PCR to analyze results using three different probes. Target plasmid was diluted to concentrations of 1, 10, 100, and 1,000 copies / pL. Target plasmid was tested at 5, 50, 500, and 5,000 copies / rxn. Results are summarized in Table 8 below.Table 8. Results for Combinations 3, 4, and 5 with tcdC sc-1 A117A Plasmid Oligo 3 4 5 CombinationRn 550,000-880,000 400,000-600,000 180,000-280,000PCR efficiency 91% 85% 87%

[0177] Experiment 2 Conclusion: Oligo combination 3 (using probe tcdCl 17delA PR05-FAM) showed the highest PCR efficiency and fluorescence.Experiment 3

[0178] In a third experiment, because of the high background observed for tcdC plasmid tcdC117T-120T 183 & 184T in Experiment 1, oligo combination 3 was retested with this plasmid at high concentration of the target. Target plasmid was tested at 50,000 and 500,000 copies / rxn. The results of this experiment again showed tcdCl 17T-120T 183 & 184T as negative with high background, but not higher with the higher concentrations.Example 2: Evaluation of tcdB and cdtB Oligonucleotides

[0179] Primer and probe combinations targeting the C. difficile tcdB and cdtB genes were evaluated for the ability to recognize tcdB and cdtB plasmids in singleplex PCR reactions. The primers and probes used (without reference to combinations) are shown in Table 9 below.Table 9. tcdB and cdtB Primer and Probe SequencesTarget Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C diff tcdB FP03 Primer 17C diff tcdB FP05 Primer 18C diff tcdB FP06 Primer 33C diff tcdB RP01 Primer 38C diff tcdB RP02 Primer 37tcdB C diff tcdB RP03 Primer 36C diff tcdB PR03 Probe 2 5mC at residues 4, 13, 15, 5’ FAM and 16 3’ BHQ1 C diff tcdB PR04 Probe 4 5mC at residues 13, 15, and 5’ FAM 16 3’ BHQ1 C diff tcdB Probe 2 5mC at residues 4, 13, 15, 5’ FAM PR03B and 16 3’ BHQ1 pdU at residues 9, 20, 21,and 22C diff tcdB Probe 4 5mC at resides 13, 15, and 5’ FAM PR04B 16 3’ BHQ1 pdU at residues 9, 20, and21C diff cdtB FP03 Primer 14C diff cdtB RP07 Primer 15Target Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*cdtB C diff cdtB PR03 Probe 16 5mC at residues 3, 9, 10, 5’ BHQ115, 18, 21, 24, 25, and 26 3’ ROX* Listed SEQ ID NOs indicated unmodified sequences. Modifications, if any, are indicated in the “Modifications” column.

[0180] The primer and probe combinations tested are shown in Table 10. below.Table 10. Primer and Probe Combinations for TestingCombination Forward primer (s) Reverse primer(s) Probe(s)No.1 C diff tcdB FP03 C diff tcdB RP01 C diff tcdB PR03C diff tcdB FP05 C diff tcdB RP02 C diff tcdB PR04C diff tcdB FP06 C diff tcdB RP032 C diff tcdB FP03 C diff tcdB RP01 C diff tcdB PR03BC diff tcdB FP05 C diff tcdB RP02 C diff tcdB PR04BC diff tcdB FP06 C diff tcdB RP033 C diff cdtB FP03 C diff cdtB RP07 C diff cdtB PR03

[0181] Oligo combinations 1 and 2 were tested with four different tcdB plasmids (tcdB 3905-5038, tcdB 3215-3730, tcdB 2503-3099, and tcdB 7236-7517; having the target insert sequences shown in SEQ ID NOs: 81-84, respectively), and combination 3 was tested with two different cdtB plasmids (cdtB AM478 and cdtB A765; having the target insert sequence shown in SEQ ID NOs:85 and 86, respectively) (see Table 38, infra). Target plasmids were diluted to concentrations of 1, 10, 100, and 1,000 copies / p. L. Target plasmids were tested at 5, 50, 500, and 5,000 copies / rxn. Results are summarized in Tables 11 and 12 below.Table 11. Results for Combinations 1 and 2 with tcdB 3905-5038 Plasmid Oligo 1 2CombinationRn ~1, 800, 000-3, 400, 000 ~1, 900, 000-2, 800, 000PCR efficiency 104% 93%Threshold 250,000Table 12. Results for Combination 3 with cdtB AM478 & A765 Plasmids Plasmid cdtB AM478 cdtB A765Rn ~ 2,500,000-3,000,000 ~2, 625, 000-3, 000, 000PCR efficiency 93% 101%Threshold 100,000

[0182] Conclusion: For tcdB plasmids, only tcdB 3905-5038 was consistently amplified with the tested tcdB oligo combinations. Combination 1 (using probes C diff tcdB PR03 and C diff tcdB PR04) had a higher PCR efficiency compared to Combination 2 (using probes C diff tcdB PR03B and C diff tcdB PR04B). tcdB 3215-3730 had amplification with a high Ct on one replicate, while the other two plasmids were not amplified.

[0183] For cdtB plasmids, both cdtB AM478 and cdtB A765 were amplified using the cdtB primer and probe design of Combination 3.Example 3: tcdA and tcdB Specificity

[0184] The specificity of tcdA and tcdB oligo combinations were evaluated due to the homology between the tcdA and tcdB genes. The oligo combinations tested were tcdA Combination 2 from Example 1 and tcdB Combination 2 from Example 2, as shown in Table 13 below.Table 13. Primer and Probe Combinations for TestingTarget Forward primer(s) Reverse primer(s) Probe(s)tcdA C diff tcdA FP03 C diff tcdA RP03 C diff tcdA PR02B tcdB C diff tcdB FP03 C diff tcdB RP01 C diff tcdB PR03BC diff tcdB FP05 C diff tcdB RP02 C diff tcdB PR04BC diff tcdB FP06 C diff tcdB RP03

[0185] Oligo combinations were tested with both tcdA and tcdB plasmids (tcdA 4086 and tcdB 3905-5038, respectively) (having the target insert sequences shown in SEQ ID NOs:87 and 81, respectively; see Table 38, infra). Target plasmids were tested at 500 and 5,000 copies / rxn. The results of this experiment showed that the tcdA oligos are specific for tcdA plasmid and the tcdB oligos are specific for the tcdB plasmid ( / .<., nosignal was observed for tcdA oligos with tcdB plasmid or for tcdB oligos with tcdA plasmid).Example 4: Evaluation of tcdB Oligonucleotides

[0186] A primer and probe combination targeting the C. difficile tcdB gene was evaluated in a singleplex PCR reaction using genomic DNA of C. difficile strain 630 (ATCC, BAA-1382D-5) as template. The primers and probes used are shown in Table 14 below.Table 14. tcdB Primer and Probe SequencesOligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C diff tcdB PR01 Probe 1 5mC at residues 4, 13, 15, and 16 5’ FAM 3’ BHQ1 C diff tcdB FP01 Primer 26C diff tcdB RP01 Primer 38C diff tcdB FP02 Primer 31C diff tcdB PR02 Probe 3 5mC at residues 13, 15, and 16 5’ FAM 3’ BHQ1* Listed SEQ ID NOs indicated unmodified sequences. Modifications, if any, are indicated in the “Modifications” column.

[0187] C. difficile genomic DNA was tested at 1, 10, 100, and 1,000 copies / rxn. Results are shown in Table 15 below.Table 15. C. difficile Genomic DNA Detection with tcdB Primers and Probes cp / rxn LOG (cp / rxn) Count Ct Avg Ct SD Ct Avg RFU 1 x 10° 0 - - - 36601 1 x 1011 3 38.5 1.6 1603507 1 x 1022 3 34.1 0.4 1860385 1 x 1033 3 30.6 0.2 1965040 1 x 1044 3 27.3 0.1 1991836- - - - -(blank)

[0188] Conclusion: The tcdB primers and probes detect down to 10 copies per reaction of genomic DNA from strain 630. The replicates are more spread (SD of Ct = 1.6) at 10 copies per reaction. The efficiency of this assay is 97%.Example 5: Evaluation of Analytical Sensitivity on Different C. difficile Strains

[0189] Primers and probes targeting the C. difficile led A., tcdB, and cdtB genes were evaluated in a multiplex PCR assay for analytical sensitivity on five different C. difficile strains. The primers and probes used are shown in Table 16 below.Table 16. tcdA, tcdB, and cdtB Primer and Probe SequencesTarget Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C diff tcdA FP03 Primer 12tcdA C diff tcdA RP03 Primer 32C diff tcdA PR02 Probe 10 5mC at residues 9, 14, 23, 5’ FAM 24, and 25 3’ BHQ1 C diff tcdB FP03 Primer 17C diff tcdB FP05 Primer 18C diff tcdB FP06 Primer 33C diff tcdB RP01 Primer 38tcdB C diff tcdB RP02 Primer 37C diff tcdB RP03 Primer 36C diff tcdB PR03 Probe 2 5mC at residues 4, 13, 15, 5’ FAM and 16 3’ BHQ1 C diff tcdB PR04 Probe 4 5mC at residues 13, 15, and 5’ FAM 16 3’ BHQ1 C diff cdtB FP03 Primer 14C diff cdtB RP07 Primer 15cdtB C diff cdtB PR03 Probe 16 5mC at residues 3, 9, 10, 15, 5’ BHQ118, 21, 24, 25, and 26 3’ ROX* Listed SEQ ID NOs indicated unmodified sequences. Modifications, if any, are indicated in the “Modifications” column.

[0190] C. difficile strains were diluted to concentrations of 3.33, 33.33, 333.33, and 1,666.67 CFU / mL. Strains and control (Zeptometrix Control) were tested at 0.10, 1.00, 10.00, and 50.00 CFU / rxn. Results are shown in Table 17 below.Table 17. C. difficile Limit of Detection (LoD) Using tcdA, tcdB, and cdtB Oligos Strain LoD (CFU / ml 3 replicates) Positive tcdA / tcdB cdtB Targets Zepto Control 33.33 (3 / 3) 33.33 (3 / 3) tcdA / tcdB, cdtB GP1793 33.33 (3 / 3) - tcdA / tcdB GP1886 333.3 (3 / 3) (33.33 for 1 / 3) 33.33 (3 / 3) tcdA / tcdB, cdtB GP1887 33.33 (3 / 3) (3.33 for 2 / 3) - tcdA / tcdB GP1888 333.3 (3 / 3) (33.33 for 1 / 3) 333.3 (3 / 3) (33.33 for 2 / 3) tcdA / tcdB, cdtBGP2178 33.33 (3 / 3) (3.33 for 1 / 3) - tcdA / tcdB

[0191] Conclusion: Strains tested with only tcdA / tcdB showed 33.33 CFU / mL for analytical sensitivity with 100% detection of target for all replicates (3 / 3). Strain GP1886 tested with multiple targets, cdtB and tcdA / tcdB, showed 333.33 CFU / mL for tcdA / tcdB analytical sensitivity with 100% of detection of target for all replicates (3 / 3) and 33.33 CFU / mL for cdtB with 100% detection (3 / 3). GP1888 had similar sensitivity, but had 66% cdtB detection (2 / 3 replicates) at 33.33 CFU / mL. Positive control detection was 33.33 CFU / mL for tcdA / tcdB and cdtB targets.Example 6: Multiplex Assay for Detection of Hypervirulent C. difficile

[0192] Primers and probes targeting the C. difficile tcdB, cdtB, and tcdC genes were evaluated in a multiplex PCR assay for detection of hypervirulent C. difficile. For multiplexing, tcdB probe was labeled with FAM / BHQ1, cdtB probe was labeled with Cal Orange 560 / BHQ1, tcdC 117delA probe was labeled with Cal Fluor Red / BHQ2, and tcdC 183 / 184 TTPRO4-670 probe was labeled with dQuasar / BHQ2. The primers and probes used are shown in Table 18 below.Table 18. tcdB, cdtB, and fc C Primer and Probe SequencesTarget Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C difftcdB FP01 Primer 26C difftcdB FP02 Primer 31C difftcdB RP01 Primer 38tcdBC difftcdB PR01 Probe 1 5mC at residues 4, 13, 15, 5’ FAM and 16 3’ BHQ1 C difftcdB PR02 Probe 3 5mC at residues 13, 15, and 5’ FAM16 3’ BHQ1Target Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*C diff cdtB FP01 Primer 35C diff cdtB RP02 Primer 21cdtBC diff cdtB PR01 Probe 25 5mC at residues 2, 8, and 5 ’ Cal Fluor 16 Orange 5603’ BHQ1tcdC FP01 Primer 6tcdC RP01 Primer 13tcdC tcdC 183 / 184TT Probe 11 5’ dQuasar PRO4-670 3’ BHQ2 117delA PR05 Probe 24 Propyne C at residue 1 5’ ROX pdU at residues 3, 4, 5, 7, 8, 3’ BHQ29, 10, 13, 15, 17, 18, and 19* Listed SEQ ID NOs indicated unmodified sequences. Modifications, if any, are indicated in the “Modifications” column.

[0193] The primer and probe combination shown in Table 18 was tested in multiplex PCR with two different targets: (1) tcdC plasmid tcdC 117T-120T 183 & 184T (clone ID 100735) and (2) genomic DNA of C. difficile ribotype 27 (strain 4118). Targets were tested at 1, 10, 100, 1,000, 10,000, and 100,000 copies / rxn. Results are summarized in Tables 19-22 below (“RB27” = genomic DNA of C. difficile ribotype 27; “735” = plasmid tcdC 117T-120T 183 & 184T).Table 19. Results for tcdB Probe (FAN Channel)Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnRB27 1 1 37.03 - 761,621.25 1,180,854.61 RB27 10 3 34.82 0.25 3,083,344.98 81,332.14 RB27 100 3 32.13 0.32 3,455,641.00 166,460.57 RB27 1000 3 29.14 0.37 3,606,190.13 73,363.62 RB27 10000 3 25.60 0.03 3,894,574.10 68,612.50 RB27 100000 3 22.53 0.45 3,768,838.48 192,163.60 735 1 0 - - 35,632.87 59,015.96 735 10 1 35.18 891,631.46 1,440,160.22 735 100 0 - 37,998.18 25,333.23 735 1000 0 - 17,586.26 36,537.75 735 10000 0 - 26,906.87 47,723.43 735 100000 0 - 42,491.89 13,190.71Mix 1 0 - 77,419.48 3,842.96Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnMix 10 3 35.32 0.93 2,794,930.36 359,486.08 Mix 100 3 33.11 0.72 3,189,140.76 215,452.77 Mix 1000 3 29.76 0.12 3,388,038.36 55,591.06 Mix 10000 3 26.46 0.33 3,373,520.89 256,196.08Mix 100000 3 23.05 0.09 3,501,445.74 104,710.31 Table 20. Results for cdtB Probe (Cal Fluor Orange Channel)Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnRB27 1 3 41.90 2.31 45,226.62 27,343.38 RB27 10 3 39.51 2.73 90,588.56 84,352.27 RB27 100 3 33.49 0.08 303,593.56 32,437.47 RB27 1000 3 29.92 0.24 398,060.47 28,374.39 RB27 10000 3 26.41 0.08 492,530.11 20,281.52 RB27 100000 3 23.12 0.17 535,376.86 49,774.65 735 1 0 - 1,859.74 1,268.39 735 10 1 37.09 60,453.02 96,032.53 735 100 0 - 9,869.47 1,651.05 735 1000 0 - 12,811.32 1,653.30 735 10000 0 - 13,453.79 1,542.46 735 100000 0 - 17,384.08 2,043.93 Mix 1 2 41.90 0.93 24,249.37 18,811.98 Mix 10 3 38.60 1.66 99,868.31 56,524.87 Mix 100 3 33.93 0.19 264,162.16 3,960.62 Mix 1000 3 30.19 0.08 370,599.79 6,639.31 Mix 10000 3 26.79 0.09 427,048.42 20,345.16 Mix 100000 3 23.36 0.08 484,503.04 16,512.19 Table 21. Results for tcdC Probe tcdC 183 / 184TT PRO4-670 (dQuasar Channel) Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnRB27 1 3 38.16 0.93 300,408.09 69,727.57 RB27 10 2 36.32 0.59 380,035.85 311,125.79 RB27 100 3 32.86 0.20 892,536.91 112,594.73 RB27 1000 3 29.72 0.47 1,098,844.29 99,931.91 RB27 10000 3 26.40 0.02 1,388,330.05 66,836.10 RB27 100000 3 23.02 0.21 1,471,894.24 116,147.80 735 1 0 - - 26,357.01 11,396.96 735 10 3 41.67 3.90 162,399.53 177,969.19 735 100 3 38.80 0.68 110,287.06 11,980.69 735 1000 3 36.45 0.58 133,797.32 14,566.73 735 10000 3 32.80 0.55 144,138.59 12,420.76735 100000 3 29.14 0.45 179,939.25 15,204.95Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnMix 1 2 37.65 0.37 307,211.77 240,513.79 Mix 10 3 37.64 2.12 288,380.61 207,219.24 Mix 100 3 33.85 0.53 424,004.40 68,921.36 Mix 1000 3 30.17 0.11 530,800.00 27,861.10 Mix 10000 3 26.75 0.34 583,647.06 35,977.39 Mix 100000 3 23.70 0.07 611,222.84 30,399.15 Table 22. Results for tcdC Probe 117delA PR05 (ROX Channel)Target copies / N Ct Mean Ct SD RFU Mean RFU SD rxnRB27 1 0 - - 23,128.01 6,544.74 RB27 10 0 - - 26,438.85 15,463.16 RB27 100 1 40.75 - 53,205.39 13,573.35 RB27 1000 3 38.53 1.46 71,991.93 10,651.39 RB27 10000 3 33.92 0.15 95,601.00 5,784.29 RB27 100000 3 30.67 0.75 103,173.89 5,106.32 735 1 1 39.72 - 52,480.33 110,737.93 735 10 3 40.07 3.08 261,919.64 190,936.72 735 100 3 33.59 0.45 663,246.70 80,981.74 735 1000 3 31.13 0.28 766,794.49 80,509.36 735 10000 3 27.08 0.31 808,578.53 84,535.14 735 100000 3 23.46 0.14 940,872.19 82,529.14 Mix 1 2 41.32 1.43 89,267.57 67,610.84 Mix 10 3 36.78 1.40 346,733.77 173,845.55 Mix 100 3 33.56 0.32 463,426.22 37,183.58 Mix 1000 3 29.87 0.28 556,216.99 71,101.14 Mix 10000 3 26.38 0.35 627,214.66 87,629.32Mix 100000 2 23.04 0.24 332,678.07 571,402.01

[0194] Conclusion: All primers and probes amplify and detect their respective targets with no cross-reactivity.Example 7: Evaluation of Alternative tcdC Primer Design

[0195] Primer and probe combinations targeting the C. difficile tcdC gene were evaluated for the ability to detect different tcdC gBlocks in singleplex PCR reactions. This experiment evaluated four alternative primer designs, each used in combination with probe tcdC probe 117delA PR05 (SEQ ID NO:24 with the following modifications: pdU at residues 3, 4, 5, 7, 8, 9, 10, 13, 15, 17, 18, and 19; labeled with 5’ HEX / 3’ BHQ1). The different primer combinations are shown in Table 23 below.Table 23. tcdC Primer CombinationsPrimer Set Forward Primer Reverse Primer(SEQ ID NO) (SEQ ID NO)tcdC set5 92A 29 40tcdC set 92G 30 40tcdC set8 92A 8 19tcdC set8 92G 9 19

[0196] Oligo combinations were tested with four different tcdC gBlocks (tcdC WT, tcdC 92A 117 del, tcdC 92G 117 del, and tcdC 87 del) (SEQ ID NOs:72-75, respectively; see Table 37, infra). Target gBlocks were diluted to concentrations of 1, 10, 100, and 1,000 copies / pL. Target gBlocks were tested at 5, 50, 500, and 5,000 copies / rxn (5 pL sample / rxn; three replicates for each sample condition). Results are summarized in Table 24 below.Table 24. Results for tcdC Alternative Designsted C set5 S »2A ted C set5 ‘ 2G ted C set8 ‘ 2A tcd< 2 set89 2G Target c / pL No. Ct Ct No. Ct Ct No. Ct Ct No. Ct Ct Pos Avg SD Pos Avg SD Pos Avg SD Pos Avg SD 10e3 0 / 3 - - 0 / 3 - - 0 / 3 - - 0 / 3 - - tcdC WT 10e2 0 / 3 - - 0 / 3 - - 0 / 3 - - 0 / 3 - - lOel 0 / 3 - - 0 / 3 - - 0 / 3 - - 0 / 3 - - WeO 0 / 2 - - 0 / 2 - - 0 / 2 - - 0 / 2 - - 10e3 3 / 3 31.1 0.1 3 / 3 32.4 0.3 3 / 3 29.7 0.2 3 / 3 30.5 0.1 tcdC 92A 10e2 3 / 3 34.6 0.4 3 / 3 36.0 0.4 3 / 3 33.4 0.2 3 / 3 34.2 0.4 117 del lOel 3 / 3 38.0 0.6 3 / 3 39.9 0.2 3 / 3 36.5 0.5 3 / 3 38.0 0.2WeO 2 / 2 40.3 1.0 2 / 2 43.7 0.4 2 / 2 41.0 0.7 2 / 2 42.1 1.5 10e3 3 / 3 31.4 0.2 3 / 3 32.7 0.2 3 / 3 29.9 0.3 3 / 3 31.0 0.2 tcdC 92G 10e2 3 / 3 34.7 0.1 3 / 3 36.2 0.1 3 / 3 33.5 0.4 3 / 3 34.6 0.2 117 del lOel 3 / 3 38.0 0.2 3 / 3 40.1 0.4 3 / 3 36.7 0.4 3 / 3 38.2 0.6WeO 2 / 2 41.2 0.3 1 / 2 43.0 - 2 / 2 40.5 1.7 2 / 2 42.0 0.8 10e3 3 / 3 30.4 0.6 3 / 3 32.3 0.1 3 / 3 29.4 0.1 3 / 3 31.4 0.2 tcdC 87 del 10e2 3 / 3 33.9 0.3 3 / 3 35.4 0.4 3 / 3 32.6 0.0 3 / 3 34.7 0.1 lOel 3 / 3 37.5 0.1 3 / 3 39.3 0.6 3 / 3 36.3 0.2 3 / 3 38.0 0.5 WeO 2 / 2 41.8 1.2 1 / 2 42.4 - 2 / 2 38.9 0.0 2 / 2 40.8 0.3Neg - 0 / 4 - - 0 / 4 - - 0 / 4 - - 0 / 4 - -

[0197] Conclusion: The alternative tcdC oligomer designs are specific to the 117 del. No amplification of WT sequence was observed. Each primer and probe combination amplifies all three 117 del variants. There is no need to use degenerate base at position 92, and 87 del is amplified.

[0198] There is better fluorescence level with primers containing A at position 92. There is better PCR performance with primer set892A.Example 8: Analytical Sensitivity (LoD) Study

[0199] The purpose of this study was to establish the Limit of Detection (LoD) of the Panther Fusion C. difficile assay using dilutions of the selected strains spiked in negative matrix (processed negative stool in STM). LoD is defined as the lowest concentration of the C. difficile strain (expressed in CFU / mL) that can be consistently detected in a sample with 95% confidence. The primers and probes used are shown in Table 25 below.Table 25. tcdA, tcdB, cdtB, and tcdC Primer and Probe SequencesTarget Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*tcdA FPOl Primer 12tcdA tcdA FP02 Primer 32tcdA PR02B Q670 Probe 10 5mC at residues 9, 14,23, 24, and 25 5’ Q670 pdU at residues 6, 7, 3’ BHQ2 10, and 20tcdB FP05 Primer 18tcdB FP06 Primer 33tcdB RP01 Primer 38tcdBtcdB RP03 Primer 36tcdB PR03B FAM Probe 2 5mC at residues 4, 13,15, and 16 5’ FAM pdU at residues 9, 20, 3’ BHQ1 21, and 22tcdB PR04B FAM Probe 4 5mC at residues 13,15, and 16 5’ FAM pdU at residues 9, 20, 3’ BHQ1 and 21cdtB FP03 Primer 14cdtB RP07 Primer 15cdtB cdtB PR03 HEX Probe 16 5mC at residues 3, 9, 5’ HEX 10, 15, 18, 21, 24, 25, 3’ BHQ1 and 26tcdC_CC_Fw_set8_92A Primer 18Target Oligo Name Oligo SEQ Modifications Detectable Type ID Labels NO*tcdC_CC_Re_set8 Primer 19tcdC tcdC A117del PR05A Probe 23 pdU at residues 3, 4, 5, 5’ Cal Red 6107, 8, 9, 10, 13, 15, 17, 3’ BHQ218, 19, and 20Materials and Methods

[0200] The LoD was conducted with serial dilutions of two toxigenic C. difficile strains and two hypervirulent ribotype 027 C. difficile strains (see Table 26), spiked into negative matrix. The tested concentrations were based on the results of a preliminary LoD study (not shown). The negative matrix was prepared from freshly collected negative stools, swabbed into STM (with a ratio of 1 swab per 2.9 mL of STM) and pooled. Each concentration was tested in 13 replicates with two cartridge lots (#550 / DEVl and #575 / DEV2; each containing the same amplification reaction formulation), on three instruments, for a total of 39 replicates by concentration and by reagent lot. The LoD was determined by selecting the probit analysis giving the highest p value for the Pearson Goodness of Fit per cartridge batch.

[0201] The LoD was confirmed by testing the predicted LoD95% value on one Panther Fusion instrument, with one cartridge lot and in 20 replicates. The LoD is confirmed when a minimum of 95% of positive calls are observed at the LoD95% predicted concentration.Table 26. C. difficile strains used for LoD determinationStrain type Hologic ID ReferenceC. difficile toxigenic GP0844 9689 (ATCC)C. difficile toxigenic GP1887 43598 (ATCC)C. difficile toxigenic,GP1886 BAA- 1805 (ATCC)ribotype 027C. difficile toxigenic, GP2687 AR- 1071 (CDC)ribotype 027Results

[0202] The positive rates by concentration and by cartridge lot are summarized in Error! Reference source not found.7 for each tested strain. The GP1886 at 250 CFU / mL count 34 total replicates instead of 39, due to instrument error during the test. The missing replicates were not reprocessed. A probit analysis was performed with JMP software (version 18) to predict LoD 95% by strain and predicted values are reported in Table 28. LoD for toxigenic C. difficile ranges from 10 CFU / mL (GP0844) to 212 CFU / mL (GP1886), and from 19 CFU / mL (GP2687) to 157 CFU / mL (GP1887) for ribotype 027 C. difficile.

[0203] The high variability observed amongst the strains is probably due to the expression of the LoD in CFU / mL. The quantification in CFU / mL only considers living cells from the stock solution that might contain a proportion of dead cells (these dead cells still contain DNA that is extracted and amplified by PCR) and is variable depending on the strain or the batch of the same strain. In parallel to the serial dilutions of the strains, a quantification curve was run with serial dilutions of NATtrol C. difficile NAP1 strain (quantified in copies / mL) to convert CFU / mL into c / mL. For toxigenic C. difficile, the LoD is 143 and 193 c / mL (for GP1886 and GP0844, respectively) and is 320 and 339 c / mL for ribotype 027 C. difficile (GP2687 and GP1887, respectively). This suggests that the LoD is consistent for the two types of tested strains when expressed in copies / mL.

[0204] The predicted LoD was then confirmed by testing each strain at lx LoD in 20 replicates with #550 / DEVl cartridge lot. Results are presented in Table 29 and demonstrate that >95% positivity was observed for GP1886, GP1887 and GP2687 strains at lx LoD concentration. 18 / 20 positive calls were observed for GP0844 and were repeated with 19 / 20 reactivity, confirming the LoD.

[0205] The LoD confirmation was repeated with #663 / DEV3 cartridge lot (containing the same amplification reaction formulation as lots #550 / DEVl and #575 / DEV2 above), and 100% positivity was observed for all tested strains (Table 29).

[0206] Table 30 shows the LoD95% expressed in CFU / swab allowing a comparison of the sensitivity with the Cepheid C. difficile / Epi assay (Cepheid; Sunnyvale, CA) which has a LoD95% of 460 CFU / Swab for toxigenic C. difficile strains and 75 CFU / Swab for ribotype 027 C. difficile strains. The results are within specifications, bothC. difficile toxigenic and C. difficile ribotype 027 LoD95% are within 0.5 Log of Cepheid’s LoD95%.Table 27. Positivity by concentration and cartridge lotStrain ID Cone. % Toxigenic Pos by cartridge lot(CFU / mL)550 / DEV1 575 / DEV260 100 10030 100 10015 97 100GP08448 92 824 64 692 36 44500 100 100250 100 97125 77 82GP188683 67 7956 49 4937 31 21800 100 100400 100 100200 90 100GP1887100 72 8250 54 6225 46 3860 100 10030 97 10015 79 90GP268710 92 797 69 564 54 49Table 28. Predicted LoD by strainStrain Strain type Predicted Lower 95% Upper 95% Pearson Deviance LoD 95% ID LoD 95% (CFU / mL) (CFU / mL) converted (CFU / mL) in c / mL GP0844 toxigenic 10 8 17 0.958 0.951 193 GP1886 toxigenic 212 161 340 0.628 0.449 143 GP1887 ribotype 027 157 115 265 0.612 0.432 339GP2687 ribotype 027 19 15 31 0.753 0.653 320Table 29. LoD confirmation by cartridge lotTested con centration Positivi ty by cartridge lot (%) Strain Strain type lx LoD lx LoD #550 / DEV1 #550 / DEV1 #663 / DEV3 ID (CFU / mL) (C / mL) retestGP0844 toxigenic 10 193 90 95 100 GP1886 toxigenic 212 143 95 - 100 GP1887 ribotype 027 157 339 95 - 100GP2687 ribotype 027 19 320 100 - 100Table 30. LoD95% expressed in CFU / swabStrain ID Strain type LoD95% LoD95%(CFU / mL) (CFU / Swab)GP0844 toxigenic 10 29GP1886 toxigenic 212 615GP1887 ribotype 027 157 455GP2687ribotype 027 19 55Example 9: Cross-reactivity and Microbial interference

[0207] The purpose of this study was to determine whether the presence of commonly found microorganisms in clinical samples will impact assay performance. The primers and probes used are shown in Table 25 (see Example 8, supra).Materials and Methods

[0208] The analytical specificity was assessed with a total of 59 microorganisms, consisting of viruses, bacteria, commonly found in stool specimen. Among these, C. sordellii is identified as the most likely strain to cause a potential cross-reaction with the assay due to the high sequence similarity between its toxin genes and the C.difficile toxin genes located in the Pathogenicity locus. In addition, four non-toxigenic C. difficile strains and human genomic DNA were assessed (see Table 31). The bacteria were tested in pools and spiked into processed negative stools in STM at 1E+06 CFU / mL. The viruses, quantified in TCID50 / mL units, were tested individually at concentrations ranging from 1.0E+03 to 1.0E+05 TCID50 / mL in processed negative stools in STM (see Table 31). Additionally, a human genomic DNA concentration of 1E+06 Copies / mL was also tested in processed negative stools in STM.

[0209] Furthermore, four plasmids representing different tcdC gene variants (tcdC Al 17T-A120T, tcdC Al 17G, tcdC Al 17T, and tcdC wild type) were included in thestudy to challenge the assay specificity of the tcdC A117del target. These plasmids represent the wild type tcdC sequence and three mutations at the position 117 and 120 of the tcdC sequence that are not associated with the hypervirulent ribotype 027 C. difficile.

[0210] For the microbial interference study, all potential cross-reacting organisms were evaluated in the presence of toxigenic C. difficile strain GP1887 and hypervirulent ribotype 027 C. difficile strain GP1886. These strains were spiked at concentrations of 471 CFU / mL and 636 CFU / mL, respectively (3x LoD95%).Table 31. Micoorganisms tested for specificity and microbial interferencePool Strains Provider Reference Tested Cone.Citrobacter freundii ATCC 8090 1E+06 CFU / mLA Enterobacter aerogenes ATCC 13048 1E+06 CFU / mL Klebsiella oxytoca ATCC 33497 1E+06 CFU / mL Serratia marcescens ATCC 13880 1E+06 CFU / mL Abiotophia defectiva ATCC 49176 1E+06 CFU / mLB Acinetobacter baumannii ATCC 19606 1E+06 CFU / mL Aeromonas hydrophila ATCC 7966 1E+06 CFU / mL Alcaligenes faecalis subsp. ATCC 15554 1E+06 CFU / mL faecalisCampylobacter coli ATCC 33559 1E+06 CFU / mLC Campylobacter jejuni subsp. ATCC 33560 1E+06 CFU / mL jejuniBacillus cereus ATCC 12826 1E+06 CFU / mL Bacteroides fragilis ATCC 25285 1E+06 CFU / mL Clostridium bifermentans ATCC 17836 1E+06 CFU / mL (Clostridium orbiscindens) ATCC 49531 1E+06 CFU / mLD Flavonifractor plautiiClostridium perfringens ATCC 13124 1E+06 CFU / mL Clostridium scindens ATCC 35704 1E+06 CFU / mL Clostridium septicum ATCC 12464 1E+06 CFU / mLE Clostridium sordellii ATCC 9714 1E+06 CFU / mL Clostridium sporogenes ATCC 13663 1E+06 CFU / mL Edwardsiella tarda ATCC 15947 1E+06 CFU / mL Enterobacter cloacae ATCC 13047 1E+06 CFU / mLF Enterococcus faecalis vanB ATCC 51299 1E+06 CFU / mL Escherichia coli ATCC 11775 1E+06 CFU / mL Escherichia coli O157: H7 ATCC 700927 1E+06 CFU / mL Lactobacillus acidophilus ATCC 4356 1E+06 CFU / mL Listeria monocytogenes ATCC 13513 1E+06 CFU / mLG Peptostreptococcus anaerobius ATCC 27337 1E+06 CFU / mL Plesiomonas shigelloides ATCC 14029 1E+06 CFU / mL Porphyromonas ATCC 25260 1E+06 CFU / mL asaccharolyticaPrevotella melaninogenica ATCC 25845 1E+06 CFU / mLProteus mirabilis ATCC 29906 1E+06 CFU / mLPool Strains Provider Reference Tested Cone.H Providencia alcalifaciens ATCC 9886 1E+06 CFU / mL Pseudomonas aeruginosa ATCC 10145 1E+06 CFU / mL Vibrio parahaemolyticus ATCC 17802 1E+06 CFU / mL Salmonella enterica ssp. ATCC 14028 1E+06 CFU / mL entericaI Salmonella enterica subsp. ATCC 13314 1E+06 CFU / mL arizonaeSalmonella enterica subsp. ATCC 43973 1E+06 CFU / mL diarizonaeSerratia liquefaciens ATCC 27592 1E+06 CFU / mL Helicobacter pylori ATCC 43504 1E+06 CFU / mL Shigella boydii ATCC 9207 1E+06 CFU / mL Shigella dysenteriae ATCC 11835 1E+06 CFU / mLJ Shigella sonnei ATCC 29930 1E+06 CFU / mL Staphylococcus aureus ATCC 43300 1E+06 CFU / mL Staphylococcus epidermidis ATCC 14990 1E+06 CFU / mLK Streptococcus agalactiae ATCC 13813 1E+06 CFU / mL Candida albicans ATCC 10231 1E+06 CFU / mL Clostridium butyricum ATCC 19398 1E+06 CFU / mLL Clostridium novyi ATCC 19402 1E+06 CFU / mL Clostridium haemolyticum ATCC 9650 1E+06 CFU / mLM Clostridium difficile (non- ATCC 43593 1E+06 CFU / mL toxigenic)Clostridium difficile (non- ATCC 43601 1E+06 CFU / mL toxigenic)N Clostridium difficile (non- ATCC BAA- 1801 1E+06 CFU / mL toxigenic)Clostridium difficile (non- ATCC 700057 1E+06 CFU / mL toxigenic)AdenovirusType 40 Zeptometrix 0810084CF 1.0E+04TCDI50 / mL AdenovirusType 41 Zeptometrix 0810085CF 1.0E+03TCDI50 / mL Rotavirus Zeptometrix 0810041CF 1.0E+04TCDI50 / mL Norovirus group I Zeptometrix 0810086CF 1.0E+04TCDI50 / mL No Norovirus group II Zeptometrix 0810087CF 1.0E+04TCDI50 / mL pool Enterovirus type 68 (Isolate 1 Zeptometrix 0810300CF 1.0E+04TCDI50 / mL 2014)Echovirus Type 11 Culture Zeptometrix 0810501CF 1.0E+05TCDI50 / mL FluidCoxsackie virus culture Fluid Zeptometrix 0810075CF 1.0E+05TCDI50 / mL (type B4)Cytomegalovirus Zeptometrix 0810003CF 1.0E+03TCDI50 / mLHuman Genomic DNA Promega G304A 1.0E+06 Copies / mLResults

[0211] As shown in Table 32, no cross-reactivity was observed among all microorganisms and human genomic DNA tested. These results support that the C. difficile assay does not cross-react with non-target organisms commonly found in stool specimens.In addition, no amplification was observed for wild type and other Al 17 and A120 plasmid variants, suggesting that tcdC A117del PCR target is specific to the Al 17del deletion and A120T mutation, which are associated with the hypervirulent ribotype 027.Table 32. Specificity results - positivity and Ct mean by targetSample ID tcdA tcdB tcdC cdtB A117delCdiffCPos-000630 1 / 1 1 / 1 1 / 1 1 / 1 Negative Control 0 / 1 0 / 1 0 / 1 0 / 1 Pool A 0 / 3 0 / 3 0 / 3 0 / 3 Pool B 0 / 3 0 / 3 0 / 3 0 / 3 Pool C 0 / 3 0 / 3 0 / 3 0 / 3 Pool D 0 / 3 0 / 3 0 / 3 0 / 3 Pool E 0 / 3 0 / 3 0 / 3 0 / 3 Pool F 0 / 3 0 / 3 0 / 3 0 / 3 Pool G 0 / 3 0 / 3 0 / 3 0 / 3 Pool H 0 / 3 0 / 3 0 / 3 0 / 3 Pool I 0 / 3 0 / 3 0 / 3 0 / 3 Pool J 0 / 3 0 / 3 0 / 3 0 / 3 Pool K 0 / 3 0 / 3 0 / 3 0 / 3 Pool L 0 / 3 0 / 3 0 / 3 0 / 3 Pool M 0 / 3 0 / 3 0 / 3 0 / 3 Pool N 0 / 3 0 / 3 0 / 3 0 / 3 Adenovirus Type 40 0 / 3 0 / 3 0 / 3 0 / 3 Adenovirus Type 41 0 / 3 0 / 3 0 / 3 0 / 3 Rotavirus 0 / 3 0 / 3 0 / 3 0 / 3 Norovirus group I 0 / 3 0 / 3 0 / 3 0 / 3 Norovirus group II 0 / 3 0 / 3 0 / 3 0 / 3 Enterovirus type 68(Isolate 1 0 / 3 0 / 3 0 / 3 0 / 32014)Echovirus Type 11 Culture Fluid 0 / 3 0 / 3 0 / 3 0 / 3 Coxsackie virus culture Fluid 0 / 3 0 / 3 0 / 3 0 / 3(type B4)Cytomegalovirus 0 / 3 0 / 3 0 / 3 0 / 3 tcdC 4T 0 / 3 0 / 3 0 / 3 0 / 3 tcdC 117G 0 / 3 0 / 3 0 / 3 0 / 3 tcdC 117T 0 / 3 0 / 3 0 / 3 0 / 3 tcdC WT 0 / 3 0 / 3 0 / 3 0 / 3gDNA 0 / 3 0 / 3 0 / 3 0 / 3

[0212] Results summarized in Table 33 and Table 34 show that 100% positivity is observed for both C. difficile strains in presence of all tested microorganisms, bacteria, and virus. The presence of human genomic DNA at 1E+06 copies / mL impacts the detection of tcdA gene, for the two tested C. difficile strains. However, this misseddetection of tcdA does not impact the final reported result regarding the toxigenic or hypervirulent ribotype 027 C. difficile detection, as tcdB is 100% detected. As shown in Table 33 and Table 34, 100% detection of tcdA target was observed in the presence of 1.0E+05 copies / mL of human gDNA. In addition, no impact on tcdA was observed with samples containing clinical matrix (processed stool in STM), suggesting that 1E+06 c / mL of human gDNA is much higher than the clinically relevant concentration present in true clinical specimens.

[0213] Altogether, these results support that the detection of C. difficile toxigenic and ribotype 027 strains by Panther Fusion C. difficile assay, is not impacted by the presence of commonly found microorganisms in stool specimens.Table 33. Microbial interference results - positivity and Ct mean by target for the toxigenic C. difficileToxigenic Ct meanMicrobial interferent C. tcdA tcdB tcdCA117del cdtB difficiledetection N MeanSD N MeanSD N Mean SD N MeanSD CdiffCPos-000630 N / A 1 / 1 31.7. 1 / 1 31.2. 1 / 1 32.0. 1 / 1 31.4.Negative Control N / A 0 / 1 0 / 1 0 / 1 0 / 1.no interferent 100% 3 / 3 35.1 0.1 3 / 3 35.70.4 0 / 3 0 / 3. Pool A 100% 3 / 3 35.20.1 3 / 3 36.1 0.2 0 / 3 0 / 3. Pool B 100% 3 / 3 35.80.6 3 / 3 36.1 1.1 0 / 3 0 / 3. Pool C 100% 3 / 3 35.80.9 3 / 3 36.00.3 0 / 3 0 / 3. Pool D 100% 3 / 3 35.80.2 3 / 3 36.40.6 0 / 3 0 / 3. Pool E 100% 3 / 3 35.80.5 3 / 3 35.70.2 0 / 3 0 / 3. Pool F 100% 3 / 3 35.80.3 3 / 3 35.80.3 0 / 3 0 / 3. Pool G 100% 3 / 3 35.80.3 3 / 3 36.20.4 0 / 3 0 / 3. Pool H 100% 3 / 3 35.80.0 3 / 3 35.90.4 0 / 3 0 / 3. Pool I 100% 3 / 3 35.80.3 3 / 3 35.90.4 0 / 3 0 / 3.Pool J 100% 3 / 3 35.80.5 3 / 3 36.50.7 0 / 3 0 / 3.Pool K 100% 3 / 3 35.80.8 3 / 3 36.60.9 0 / 3 0 / 3. Pool L 100% 3 / 3 35.80.5 3 / 3 36.00.4 0 / 3 0 / 3. Pool M 100% 3 / 3 35.80.2 3 / 3 36.20.9 0 / 3 0 / 3. Pool N 100% 3 / 3 35.80.3 3 / 3 36.1 0.6 0 / 3 0 / 3. Adenovirus Type 40 100% 3 / 3 35.8 1.0 3 / 3 35.80.1 0 / 3 0 / 3.Adenovirus Type 41 100% 3 / 3 35.80.3 3 / 3 35.40.1 0 / 3 0 / 3.Rotavirus 100% 3 / 3 35.80.1 3 / 3 36.00.4 0 / 3 0 / 3. Norovirus group I 100% 3 / 3 35.80.5 3 / 3 36.1 0.5 0 / 3 0 / 3.Norovirus group II 100% 3 / 3 35.80.3 3 / 3 35.80.6 0 / 3 0 / 3.Enterovirus type 68 (Isolate 1 2014) 100% 3 / 3 35.80.8 3 / 3 35.70.2 0 / 3 0 / 3.Echovirus Type 11 Cumture Fluid 100% 3 / 3 35.80.6 3 / 3 36.1 0.2 0 / 3 0 / 3.Coxsackie virus culture Fluid (type B4) 100% 3 / 3 35.80.4 3 / 3 35.90.3 0 / 3 0 / 3.Cytomealovirus 100% 3 / 3 35.80.2 3 / 3 36.40.2 0 / 3 0 / 3. Human gDNA 1E+06 c / mL 100% 1 / 3 35.8. 3 / 3 35.70.4 0 / 3 0 / 3.Human gDNA 1E+05 c / mL 100% 3 / 3 35.80.6 3 / 3 36.00.3 0 / 3 0 / 3.Neg Matrix (no spiked strain) 0% 3 / 3. 0 / 3. 0 / 3 3 / 3.Table 34. Microbial interference results - positivity and Ct mean by target for the hypervirulent ribotype 027 C. difficileRibotype Ct meanMicrobial interferent 027 tcdA tcdB tcdCA117del cdtB detection N MeanSD N MeanSD N Mean SD N MeanSD CdiffCPos-000630 N / A 1 / 1 31.7. 1 / 1 31.2. 1 / 1 32.0. 1 / 1 31.4.Negative Control N / A 0 / 1. 0 / 1. 0 / 1 0 / 1no interferent 100% 3 / 3 34.4 0.1 3 / 3 35.1 0.5 3 / 3 34.9 0.1 3 / 3 34.3 0.7 Pool A 100% 3 / 3 34.3 0.2 3 / 3 34.8 0.3 3 / 3 34.8 0.2 3 / 3 34.3 0.3 Pool B 100% 3 / 3 34.0 0.2 3 / 3 34.7 0.3 3 / 3 34.4 0.0 3 / 3 33.7 0.4 Pool C 100% 3 / 3 34.0 0.3 3 / 3 34.6 0.2 3 / 3 34.7 0.3 3 / 3 34.2 0.2 Pool D 100% 3 / 3 33.7 0.4 3 / 3 34.3 0.3 3 / 3 34.2 0.2 3 / 3 33.8 0.3 Pool E 100% 3 / 3 34.4 1.1 3 / 3 34.9 0.4 3 / 3 34.8 0.6 3 / 3 34.6 0.7 Pool F 100% 3 / 3 33.6 0.6 3 / 3 34.1 0.3 3 / 3 34.2 0.2 3 / 3 34.0 0.1 Pool G 100% 3 / 3 34.0 0.2 3 / 3 34.5 0.1 3 / 3 34.5 0.1 3 / 3 34.2 0.2 Pool H 100% 3 / 3 33.9 0.2 3 / 3 34.3 0.2 3 / 3 34.3 0.2 3 / 3 33.9 0.4 Pool I 100% 3 / 3 34.0 0.2 3 / 3 34.4 0.4 3 / 3 34.3 0.4 3 / 3 34.2 0.2 Pool J 100% 3 / 3 34.1 0.0 3 / 3 34.4 0.3 3 / 3 34.5 0.2 3 / 3 33.8 0.2 Pool K 100% 3 / 3 34.1 0.1 3 / 3 34.5 0.2 3 / 3 34.4 0.2 3 / 3 33.9 0.2 Pool L 100% 3 / 3 33.9 0.4 3 / 3 34.2 0.6 3 / 3 34.4 0.5 3 / 3 34.2 0.1 Pool M 100% 3 / 3 34.4 0.2 3 / 3 34.7 0.3 3 / 3 34.5 0.0 3 / 3 34.1 0.3 Pool N 100% 3 / 3 34.0 0.1 3 / 3 34.5 0.2 3 / 3 34.7 0.2 3 / 3 34.2 0.3 Adenovirus Type 40 100% 3 / 3 34.5 0.4 3 / 3 34.6 0.4 3 / 3 35.1 0.1 3 / 3 34.4 0.0 Adenovirus Type 41 100% 3 / 3 34.7 0.4 3 / 3 35.5 0.5 3 / 3 35.2 0.2 3 / 3 34.8 0.5 Rotavirus 100% 3 / 3 34.2 0.1 3 / 3 34.9 0.3 3 / 3 35.0 0.2 3 / 3 35.0 0.3 Norovirus group I 100% 3 / 3 34.0 0.4 3 / 3 34.2 0.2 3 / 3 34.4 0.2 3 / 3 34.4 0.4 Norovirus group II 100% 3 / 3 34.4 0.1 3 / 3 34.7 0.4 3 / 3 34.6 0.1 3 / 3 34.3 0.1 Enterovirus type 68 (Isolate 1 2014) 100% 3 / 3 33.9 0.4 3 / 3 34.2 0.0 3 / 3 34.4 0.1 3 / 3 34.0 0.4 Echovirus Type 11 Cumture Fluid 100% 3 / 3 34.6 1.1 3 / 3 34.9 0.4 3 / 3 35.0 0.9 3 / 3 35.1 0.5 Coxsackie virus culture Fluid (type B4) 100% 3 / 3 33.9 0.4 3 / 3 34.6 0.2 3 / 3 34.2 0.2 3 / 3 34.4 0.3Cytomealovirus 100% 3 / 3 34.0 0.0 3 / 3 34.4 0.2 3 / 3 34.4 0.3 3 / 3 33.8 0.2 Human gDNA 1E+06 c / mL 100% 1 / 3 36.3. 3 / 3 34.8 0.3 3 / 3 35.0 0.3 3 / 3 34.9 0.4 Human gDNA 1E+05 c / mL 100% 3 / 3 34.4 0.3 3 / 3 34.9 0.3 3 / 3 34.9 0.2 3 / 3 34.6 0.4Neg Matrix (no spiked strain) 0% 0 / 3. 3 / 3. 0 / 3 0 / 3.SEQUENCESTable 35. Exemplary Oligonucleotide SequencesSEPTargetID Sequence (5’ 3’) Function geneNO1 AAACAGGATGGACACCAGGT tcdB detection probe2 AAACAGGATGGACACCAGGTTT tcdB detection probe3 AAAGAGGATGGACGCCAGG tcdB detection probe4 AAAGAGGATGGACGCCAGGTT tcdB detection probe5 AATGACCTCCTCATGGTCTTC tcdC amplification oligomer 6 AATTAAATTCTTTAAGAGCACAAAGG tcdC amplification oligomer 7 AGAAATGACCTCCTCATGGTC tcdC amplification oligomer 8 AGCACAAAGGATATTGCTCT tcdC amplification oligomerSEOTargetID Sequence (5’ 3’) Function geneNO9 AGCACAAAGGGTATTGCTCT tcdC amplification oligomer 10 AG GAAT T AC T AGAC GAAC AT GAC C C tcdA detection probe 11 AGGTCATTTCTAATTAAAC tcdC detection probe 12 ATATTAGACGGAATAAACTTAGGTGC tcdA amplification oligomer 13 AT C AGAC AAT T T G C T AT T T AAAGT T T C tcdC amplification oligomer 14 AT C CAT T T AT C C C AAAT AAC AAT T T C cdtB amplification oligomer 15 ATCCATTTCGTTCATATGAATCTG cdtB amplification oligomer 16 ATCTGTATCCAAATCTTCGTCTTCCCA cdtB detection probe 17 AT GC C C CAAAT AGAGT GT T T G tcdB amplification oligomer 18 ATGCCCCAGATAGAATCTTTG tcdB amplification oligomer 19 CAGAACAAGCT GGT GAGGAT tcdC amplification oligomer 20 CAGAACAAGCT GGT GAGGATA tcdC amplification oligomer 21 CATATTTCTTATAGCCTTGTTCTGC cdtB amplification oligomer 22 CAT GGT CTT CAGAACAAGCT G tcdC amplification oligomer 23 CATTTATTTTGGTGTGTTTT tcdC detection probe A117del24 CATTTATTTTGGTGTGTTT tcdC detection probe A117del25 C C AGAT T CAT AT GAAC GAAAT G GAT cdtB detection probe 26 CTAATGCTCCAAATAGAGTATTTGC tcdB amplification oligomer 27 GAAATGACCTCCTCATGGTCT tcdC amplification oligomer 28 GACCTCCTCATGGTCTTCAG tcdC amplification oligomer 29 GAGCACAAAGGATATT GCT CT tcdC amplification oligomer 30 GAGCACAAAGGGTATTGCTCT tcdC amplification oligomer 31 GCCCCAGATAGAATCTTTGG tcdB amplification oligomer 32 GCTAAAACACCCACCTTAGCTTC tcdA amplification oligomer 33 GGTATTACCTAATGCTCCAAATAGA tcdB amplification oligomer 34 GT CTT CAGAACAAGCT GGT GA tcdC amplification oligomer 35 GTT GAT GT CT GATT GGGAAGAC cdtB amplification oligomer 36 GTTTTGTACCATCATTTTCTAA tcdB amplification oligomer 37 GTTTTGTACCCTCATTTTCTAA tcdB amplification oligomer 38 GTTTTGTGCCATCATTTTCTAAG tcdB amplification oligomer 39 T CTT CAGAACAAGCT GGT GAG tcdC amplification oligomerSEOTargetID Sequence (5’ 3’) Function geneNO40 TTCAGAACAAGCTGGTGAGG tcdC amplification oligomer 59 GCRCCAYATAGA tcdB target-hybridizing core sequence60 GGT AT T AC CT AAT GC RC CAYAT AGAYTMT T T G S tcdB target-hybridizing region 61 ATGCRCCAYATAGA tcdB target-hybridizing core sequence62 GGT AT T AC CT AAT GC RC CAYAT AGAYTMT T T G tcdB target-hybridizing region 63 GTTTTGTRCCMTCATTTTCTAAN tcdB amplification oligomer (N is G or is absent)64 AGCACAAAGG tcdC target-hybridizing core sequence65 AATTAAATTCTTTAAGAGCACAAAGGRTATTGC tcdC target-hybridizing region TCT66 NAGCACAAAGGRTATTGCTCT tcdC amplification oligomer (N is G or is absent)67 CAGAACAAGCT G tcdC target-hybridizing core sequence68 CAT GGT CTT CAGAACAAGCT GGT GAGGATA tcdC target-hybridizing region 69 GACCTCCTCATGGTC tcdC target-hybridizing core sequence70 AGAAATGACCTCCTCATGGTCTTCAG tcdC target-hybridizing region 71 AAASAGGATGGACACCAGGNNN tcdB amplification oligomer (N is T or is absent)Table 36. Exemplary Nucleotide Modifications and LabelsSEODetectable 112 Unmodified Sequence (5’ 3’1 Nucleotide Modification(s)Labels NO:5mC at residues 4, 13, 15, 5’ FAM 41 AAACAGGATGGACACCAGGTand 16 3’ BHQ1 AAAGAGGATGGACGCCAGG 5mC at residues 13, 15, and 5’ FAM 4216 3’ BHQ15’ Cal Orange 43 C C AGAT T CAT AT GAAC GAAAT G GAT 5mC at residues 2, 8, and 16 5603’ BHQ1 AAACAGGATGGACACCAGGTTT 5mC at residues 4, 13, 15, 5’ FAM 44and 16 3’ BHQ1SEODetectable 112 Unmodified Sequence (5’ 3’) Nucleotide Modification(s)Labels NO:5mC at residues 13, 15, and 5’ FAM 45 AAAGAGGATGGACGCCAGGTT16 3’ BHQ1 AG GAAT T AC T AGAC GAAC AT GAC C C 5mC at residues 9, 14, 23, 24, 5’ FAM 46and 25 3’ BHQ1 47 ATCTGTATCCAAATCTTCGTCTTCCCA 5mC at residues 3, 9, 10, 15, 5’ BHQ118, 21, 24, 25, and 26 3’ ROX 5mC at residues 4, 13, 15, 5’ FAM and 16 3’ BHQ1 48 AAACAGGATGGACACCAGGTTTpdU at residues 9, 20, 21, and225mC at resides 13, 15, and 16 5’ FAM 49 AAAGAGGATGGACGCCAGGTTpdU at residues 9, 20, and 21 3’ BHQ1 50 AG GAAT TAG T AGAC GAAC AT GAC C C 5mC at residues 9, 14, 18, 23, 5’ FAM 24, and 25 3’ BHQ1 pdU at residues 6, 7, 10, and2051 CATTTATTTTGGTGTGTTT pdU at residues 3, 4, 5, 7, 8, 5’ CalFluor 9, 10, 13, 15, 17, 18, and 19 Orange 5603’ BHQ1 or5’ HEX 3’ BHQ1 52 CATTTATTTTGGTGTGTTT Propyne C at residue 1 5’ FAM pdU at residues 3, 4, 5, 7, 8, 3’ BHQ1 9, 10, 13, 15, 17, 18, and 19 or5’ ROX 3’ BHQ2 53 CATTTATTTTGGTGTGTTT 5mC at residue 1 5’ Cal Red 610 pdU at residues 3, 4, 5, 7, 8, 3’ BHQ2 9, 10, 13, 15, 17, 18, and 1954 AG GAAT TAG T AGAC GAAC AT GAC C C 5mC at residues 9, 14, 23, 24, 5’ Quasar 670 and 25 3’ BHQ2 pdU at residues 6, 7, 10, and2055 AAACAGGATGGACACCAGGTTT 5mC at residues 4, 13, 15, 5’ FAM and 16 3’ BHQ1 pdU at residues 9, 20, 21, and2256 AAAGAGGATGGACGCCAGGTT 5mC at residues 13, 15, and 5’ FAM 16 3’ BHQ1SEODetectable 112 Unmodified Sequence (5’ 3’) Nucleotide Modification(s)Labels NO:pdU at residues 9, 20, and 2157 CATTTATTTTGGTGTGTTTT pdU at residues 3, 4, 5, 7, 8, 5’ ROX 9, 10, 13, 15, 17, 18, 19, and 3’ BHQ2 2058 ATCTGTATCCAAATCTTCGTCTTCCCA 5mC at residues 3, 9, 10, 15, 5’ HEX 18, 21, 24, 25, and 26 3’ BHQ1Table 37. C. difficile tcdC gBlocksSEO gBlock name Sequence112NO72 tcdC WT TAAGAAAATAATTAAATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGG CATTTATTTTAGGCGTGTTTTTTGGCAATATATCCTCACCAGCTTGTTCT GAAGAC CAT GAG GAG GT CAT T T C T AAC C AAAC AT C AGT T AT AGAT T C T C A AAAAACAGAAATAGAAACTTTAAATAGCAAATT GT CT GAT GCT GAACCAT GGTTCAAAATG73 tcdC 92A 117del TAAGAAAATAATTAAATTCTTTAAGAGCACAAAGGaTATTGCTCTACTGG 120T 183T CATTTATTTTGGTGTGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTG AAGAC CAT GAG GAG GT CAT T T C T AA t C AAAC AT C AGT TAT AGAT T C T C AA AAAACAGAAATAGAAACTTTAAATAGCAAATT GT CT GAT GCT GAACCAT G GTTCAAAATG74 tcdC 92G 117del TAAGAAAATAATTAAATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGG 120T 183T CATTTATTTTGGTGTGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTG AAGAC CAT GAG GAG GT CAT T T C T AA t C AAAC AT C AGT TAT AGAT T C T C AA AAAACAGAAATAGAAACTTTAAATAGCAAATT GT CT GAT GCT GAACCAT G GTTCAAAATG75 tcdC 87del 92A TAAGAAAATAATTAAATTCTTTAAGAGCACAAGGaTATTGCTCTACTGGC 117del 120T ATTTATTTTGGTGTGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGA 183T AGAC CAT GAG GAG GT CAT T T C T AA t C AAAC AT C AGT TAT AGAT T C T C AAA AAACAGAAATAGAAACTTTAAATAGCAAATT GT CT GAT GCT GAACCAT GG TTCAAAATGTable 38. C. difficile Plasmid Insert SequencesSEP Plasmid name Insert Sequence112NO76 tcdC WT GGTAACGAATTTAGTAATGAAGGAAAAGGAAGCTCTAAGAAAATAATTAA ATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGGCATTTATTTTAGGCG TGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAG GT CAT T T C T AAC C AAAC AT C AGT TAT AGAT T C T C AAAAAAC AGAAAT AGA AACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAATGAAAG ACGACGAAAAGAAAGCTATTGAAGCTGAAAATCAACGTAAAGCTGAAGAAGCTAAAAAAGCTGAAGAAGCTAAAAAGGCTGAAGAACAACGCAAAAAAGASEO Plasmid name Insert Sequence112NO AGAAGAGGAGAAGAAAGGATATGATACTGGTATTACTTATGACCAATTAG CTAGAACACCTGATGATTATAAGTACAAAAAGGTAAAATTTGAAGGTAAG GTTATTCAAGTTATTGAAGATGGTGATGAGGTGCAAATAAGATTAGCTGT GT CT GGAAATTAT GATAAGGTT GTACTAT GTAGTT77 tcdC sc-1 A117A GGTAACGAATTTAGTAATGAAAGAAAAGGAAGCTCTAAGAAAATAATTAA ATTCTTTAAGAGCACAAAGGATATTGCTCTACTGGCATTTATTTTGGTGT GTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAGG T CAT T T C T AAT C AAAC AT C AGT T AT AGAT T C T C AAAAAAC AGAAAT AGAA ACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAATGAAAGA CGACGAAAAGAAAGCTATTGAAGCTGAAAATCAACGTAAAGCTGAAGAAG CTAAAAAGGCTGAAGAACAACGTAAAAAAGAAGAAGAAGAGAAGAAAGGA TATGATACTGGTATTACTTATGACCAATTAGCTAGAACACCTGATGATTA T AAGT ACAAAAAGGT AAAAT T T GAAGGT AAGGT TAT T CAAGT TAT T GAAG AT GGT GAT GAGGT GCAAATAAGATTAGCT GT GT CT GGAAATTAT GATAAG GT C GTACTAT GT AGT T78 tcdC 117T GGTAACGAATTTAGTAATGAAGGAAAAGGAAGCTCTAAGAAAATAATTAA ATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGGCATTTATTTTTGGCG TGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAG GT CAT T T C T AAC C AAAC AT C AGT TAT AGAT T C T C AAAAAAC AGAAAT AGA AACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAATGAAAG ACGACGAAAAGAAAGCTATTGAAGCTGAAAATCAACGTAAAGCTGAAGAA GCTAAAAAAGCTGAAGAAGCTAAAAAGGCTGAAGAACAACGCAAAAAAGA AGAAGAGGAGAAGAAAGGATATGATACTGGTATTACTTATGACCAATTAG CTAGAACACCTGATGATTATAAGTACAAAAAGGTAAAATTTGAAGGTAAG GTTATTCAAGTTATTGAAGATGGTGATGAGGTGCAAATAAGATTAGCTGT GT CT GGAAATTAT GATAAGGTT GTACTAT GTAGTT79 tcdC 117G GGTAACGAATTTAGTAATGAAGGAAAAGGAAGCTCTAAGAAAATAATTAA ATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGGCATTTATTTTGGGCG TGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAG GT CAT T T C T AAC C AAAC AT C AGT TAT AGAT T C T C AAAAAAC AGAAAT AGA AACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAATGAAAG ACGACGAAAAGAAAGCTATTGAAGCTGAAAATCAACGTAAAGCTGAAGAA GCTAAAAAAGCTGAAGAAGCTAAAAAGGCTGAAGAACAACGCAAAAAAGA AGAAGAGGAGAAGAAAGGATATGATACTGGTATTACTTATGACCAATTAG CTAGAACACCTGATGATTATAAGTACAAAAAGGTAAAATTTGAAGGTAAG GTTATTCAAGTTATTGAAGATGGTGATGAGGTGCAAATAAGATTAGCTGT GT CT GGAAATTAT GATAAGGTT GTACTAT GTAGTT80 tcdCl 17T-120T GGTAACGAATTTAGTAATGAAGGAAAAGGAAGCTCTAAGAAAATAATTAA 183 & 184T ATTCTTTAAGAGCACAAAGGGTATTGCTCTACTGGCATTTATTTTTGGTG TGTTTTTTGGCAATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAG GT CAT T T C T AAT T AAAC AT C AGT TAT AGAT T C T C AAAAAAC AGAAAT AGA AACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAATGAAAG ACGACGAAAAGAAAGCTATTGAAGCTGAAAATCAACGTAAAGCTGAAGAA GCTAAAAAAGCTGAAGAAGCTAAAAAGGCTGAAGAACAACGCAAAAAAGA AGAAGAGGAGAAGAAAGGATATGATACTGGTATTACTTATGACCAATTAG CTAGAACACCTGATGATTATAAGTACAAAAAGGTAAAATTTGAAGGTAAG GTTATTCAAGTTATTGAAGATGGTGATGAGGTGCAAATAAGATTAGCTGT GT CT GGAAATTAT GATAAGGTT GTACTAT GTAGTT81 tcdB 3905-5038 AAAT CT GGAGAAT GGAAGGT GGTT CAGGT CATACT GTAACT GAT GATATA GATCACTTCTTTTCAGCACCATCAATAACATATAGAGAGCCACACTTATC TAT AT AT GAC GT AT T GGAAGT ACAAAAAGAAGAACT T GAT T T GT CAAAAGATTTAATGGTATTACCTAATGCTCCAAATAGAGTATTTGCTTGGGAAACASEO Plasmid name Insert Sequence112NO GGATGGACACCAGGTTTAAGAAGCTTAGAAAATGATGGCACAAAACTGTT AGAC C GT AT AAGAGAT AAC TAT GAAG GT GAGTTTTATTG GAGAT AT T T T G CTTTTATAGCTGATGCTTTAATAACAACATTAAAACCAAGATATGAAGAT ACT AAT AT AAGAAT AAAT T T AGAT AGT AAT ACT AGAAGT T T T AT AGT T C C AAT AAT AAC T AC AGAAT AT AT AAGAGAAAAAT TAT CAT AT TCTTTCTATG GTTCAGGAGGAACTTATGCATTGTCTCTTTCTCAATATAATATGGGTATA AAT AT AGAAT T AAGT GAAAGT GAT GT T T GGAT TAT AGAT GT T GAT AAT GT T GT GAGAGAT GTAACTATAGAAT CT GATAAAATTAAAAAAGGT GATTTAA T AGAAG GT AT TTTATCTACACTAAGTATT GAAGAGAAT AAAAT T AT C T T A AATAGCCATGAGATTAATTTTTCTGGTGAGGTAAATGGAAGTAATGGATT TGTTTCTT T AAC AT T T T C AAT T T T AGAAG GAAT AAAT G C AAT TAT AGAAG TTGATTTATTATCTAAATCATATAAATTACTTATTTCTGGCGAATTAAAA AT AT T GAT GT T AAAT T C AAAT CAT AT T C AAC AGAAAAT AGAT TAT AT AG G AT T C AAT AG C GAAT T AC AGAAAAAT AT AC CAT AT AG C T T T GT AGAT AGT G AAG GAAAAGAGAAT G GT T T T AT T AAT G GT T C AAC AAAAGAAG GT T T AT T T GTATCTGAATTACCTGATGTAGTTCTTATAAGTAAGGTTTATATGGATGA TAGTAAGCCTT CAT T T G GAT AT TAT AGT AAT AAT T T GAAAGAT GT C AAAG T TAT AAC T AAAGAT AAT GT T AAT AT AT TAACAGGTTATTATCTTAA82 tcdB 3215-3730 GGT ACT AT AT T T GAT ACT GT AAAT GGT AAGT T AGT AAAAAAAGT AAAT T T AGATACTACACACGAAGTAAATACTTTAAATGCTGCATTTTTTATACAAT CAT T AAT AGAAT AT AAT AGT T CT AAAGAAT CT CT T AGT AAT T T AAGT GT A GCAATGAAAGTCCAAGTTTACGCTCAATTATTTAGTACTGGTTTAAATAC TAT TAG AGAT G C AG C C AAAGT T GT T GAAT T AGT AT C AAC T G CAT T AGAT G AAACTATAGACTTACTTCCTACATTATCTGAAGGATTACCTATAATTGCA ACTATTAT AGAT GGT GTAAGTTTAGGT GCAGCAAT CAAAGAGCTAAGT GA AAC GAGT GAC C CAT TAT TAAGACAAGAAAT AGAAG C T AAGAT AG GT AT AA TGGCAGTAAATTTAACAACAGCTACAACTGCAATCATTACTTCATCTTTG GGGATAGCTAGTGGATTTAGTATACTTTTAGTTCCTTTAGCAGGAATTTC AGCAGGTATACCAAG83 tcdB 2503-3099 GGATGTAATATGTTTAGCTACTCTATCAACGTAGAGGAGACTTATCCTGG AAAAT TAT TAG T T AAAGT T AAAGAT AAAAT AT C AGAAT T AAT G C CAT C T A TAAGT CAAGACT CTATTATAGTAAGT GCAAAT GAAT AT GAAGTTAGAATA AAT AGT GAAGGAAGAAGAGAATTATT GGAT GATT CT GGT GAAT GGAT AAA T AAAGAAGAAAGT AT TAT AAAG GAT AT T T CAT C AAAAGAAT AT AT AT CAT T T AAT C C TAAAGAAAAT AAAAT TAG AGT AAAAT C T AAAAAT TTACCTGAG C T AT C TAG AT TAT T AC AAGAAAT T AGAAAT AAT T C T AAT T C AAGT GAT AT T GAACT AGAAGAAAAAGT AAT GT T AACAGAAT GT GAGAT AAAT GT TAT T T CAAATATAGATACGCAAATTGTTGAGGAAAGGATTGAAGAAGCTAAGAAT TTAACTTCTGACTCTATTAATTATATAAAAGATGAATTTAAACTAATAGA AT CTATTT CT GAT GCACTAT GT GAC TT AAAAGAAG AGAAT GAATTAGAAG ATTCTCATTTTATATCTTTTGAGGACATATCAGAGACTGATGAGGG84 tcdB 7236-7517 GGT TAT AT AAAT AT AGAAGAT AAGAT GT T CT AT T T T GGT GAAGAT GGT GT CATGCAGATTGGAGTATTTAATACACCAGATGGATTTAAATACTTTGCAC ATCAAAATACTTTGGATGAGAATTTTGAGGGAGAATCAATAAACTATACT G GT T G GT T AGAT T T AGAT GAAAAGAGAT AT T AT T T TAG AGAT GAAT AT AT TGCAGCAACTGGTTCAGTTATTATTGATGGTGAGGAGTATTATTTTGATC CT GATACAGCT CAATTAGT GATTAGT GAAT AG85 cdtB AM478 ACTTAAAGTTAATATGAAAAAGGGTAAAGAATATAAATTTAGAATAGAGC TAG AAGAT AAAAAT T T AG GT T GAAT AGAT AAT T T GT CAT C AC C T AAT C T T T AT T G G GAAT T AGAT G GT AT T AAGAAAAT TAT AC GAG C AGAAAAT T T AT T C T T AAGAGAT T AT T C T AAT AT AGAAAAAAAT GAT C CAT T T AT C C C AAAT A ACAATTTCTTTGACCCAAGGTTGATGTCTGATTGGGAAGACGAAGATTTGGAT AC AGAT AAT GAT AAT AT AC GAGAT T CAT AT GAAC GAAAT G GAT AT ACSEO Plasmid name Insert Sequence112NO TATTAAGGACTTAATTGCAGTTAAGTGGGAAGATAGCTTTGCAGAACAAG GCTATAAGAAATAT GTAT CAAATTATTTAGAGT CAAATACT GCT GGAGAT CCATATACAGATTATGAAAAAGCTTCAGGTTCTTTTGACAAGGCTATAAA86 cdtB A765 CACTTAAAGTTAATATGAAAAAGGGTAAAGAATATAAAGTTAGAATAGAG C T AC AAGAT AAAAAT T T AG GT T C AAT AGAT AAT T T AT CAT C AC C T AAT C T T T AT T G G GAAT T AGAT G GT AT GAAGAAAAT TAT AC C AGAAGAAAAT T T AT T C T T AAGAGAT T AT T C T AAT AT AGAAAAAGAT GAT C CAT T T AT C C C AAAT AAC AAT T T C T T T GAC C C AAAGT T GAT GT C T GAT T G G GAAGAC GAAGAT T T G GAT AC AGAT AAT GAT AAT AT AC C AGAT T CAT AT GAAC GAAAT G GAT AT A CTATTAAGGACTTAATTGCAGTTAAGTGGGAAGATAGTTTTGCAGAACAA GGCTATAAGAAATAT GTAT CAAATTATTTAGAGT CAAATACT GCT GGAGA TCCATATACAGATTATGAAAAAGCTTCAGGTTCTTTTGACAAGGCTATAAA87 tcdA 4086 T C T C AAGT T AAT AC AT T AAAC G C AG CAT T C T T T AT T C AAT CAT T AAT AGA TTATAGTAG C AAT AAAGAT GT AC T GAAT GAT TTAAGTACCTCAGTTAAGG TTCAACTTTATGCTCAACTATTTAGTACAGGTTTAAATACTATATATGAC T C T AT C C AAT T AGT AAAT T T AAT AT C AAAT G C AGT AAAT GAT AC TAT AAA TGTACTACCTACAATAACAGAGGGGATACCTATTGTATCTACTATATTAG ACGGAATAAACTTAGGTGCAGCAATTAAGGAATTACTAGACGAACATGAC CCATTACTAAAAAAAGAATTAGAAGCTAAGGTGGGTGTTTTAGCAATAAA TATGTCATTATCTATAGCTGCAACTGTAGCTTCAATTGTTGGAATAGGTG CTGAAGTTACTATTTTCTTATTACCTATAGCTGGTATATCTGCTable 39. Exemplary C. difficile tcdC Reference SequencesSEO112 Sequence Comments NO:AT GT T T T C T AAAAAAAAT GAG G GT AAC GAAT T T AGT AAT GAAG G AAAAGGAAGCTCTAAGAAAATAATTAAATTCTTTAAGAGCACAA AGGGTATTGCTCTACTGGCATTTATTTTAGGCGTGTTTTTTGGC AATATATCCTCACCATCTTGTTCTGAAGACCATGAGGAGGTCAT T T C T AAC C AAAC AT C AGT TAT AGAT T C T C AAAAAAC AGAAAT AG AAACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAA AT GAAAGAC GAC GAAAAGAAAG C T AT T GAAG C T GAAAAT C AAC G Clostridioides difficile TAAAGCT GAAGAAGCTAAAAAAGCT GAAGAAGCTAAAAAGGCT G strain PC020 tcdC gene, 88 AAGAACAACGCAAAAAAGAAGAAGAGGAGAAGAAAGGATATGAT complete cds ACTGGTATTACTTATGACCAATTAGCTAGAACACCTGATGATTA T AAGT ACAAAAAGGT AAAAT T T GAAGGT AAGGT TAT T CAAGT TA GenBank: MG569922.1 TT GAAGAT GGT GAT GAGGT GCAAATAAGATTAGCT GT GT CT GGA AAT TAT GAT AAGGT T GT ACT AT GT AGT T AT AAAAAAT CAAT AAC TCCTTCAAGAGTATTAGAGGATGATTACATAACTATAAGAGGTA TAAGT GCT GGAACTATAACTTAT GAAT CAACTAT GGGT GGAAAT ATAACTATACCAGGGATAGCTGTAGAGAAAATTAATTAAA AT GT T T T C T AAAAAAAAT GAG G GT AAC GAAT T T AGT AAT GAAG G Clostridioides difficile AAAAGGAAGCTCTAAGAAAATAATTAAATTCTTTAAGAGCACAA tcdC gene,89 AGGGTATTGCTCTACTGGCATTTATTTTGGTGTGTTTTTTGGCA ATATATCCTCACCAGCTTGTTCTGAAGACCATGAGGAGGTCATT single nucleotide deletion T CT AAt t AAACAT CAGT TAT AGAT T CT CAAAAAACAGAAAT AGA at position A117SEO112 Sequence Comments NO:AACTTTAAATAGCAAATTGTCTGATGCTGAACCATGGTTCAAAA T GAAAGAC GAG GAAAAGAAAG C T AT T GAAG C T GAAAAT C AAC GT AAAGCT GAAGAAGCTAAAAAAGCT GAAGAAGCTAAAAAGGCT GA AGAACAACGCAAAAAAGAAGAAGAGGAGAAGAAAGGATATGATA CTGGTATTACTTATGACCAATTAGCTAGAACACCTGATGATTAT AAGT ACAAAAAGGT AAAAT T T GAAGGT AAGGT TAT T CAAGT TAT T GAAGAT GGT GAT GAGGT GCAAATAAGATTAGCT GT GT CT GGAA AT TAT GAT AAGGT T GT ACT AT GT AGT T AT AAAAAAT CAAT AACT CCTTCAAGAGTATTAGAGGATGATTACATAACTATAAGAGGTAT AAGT GCT GGAACTATAACTTAT GAAT CAACTAT GGGT GGAAATA TAACTATACCAGGGATAGCTGTAGAGAAAATTAATTAA

[0214] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition or kit for determining the presence or absence of a toxigenic C. difficile strain in a sample, said composition or kit comprising a set of oligonucleotides comprising each of (a)-(d):(a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid, wherein the first amplification oligomer set comprises first and second / ct / d -specific amplification oligomers respectively comprising first and second fct / 4-specific target-hybridizing sequences, wherein(i) the first tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:12, including from 0 to 19 nucleotide analogs; and(ii) the second / ct / d -specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs;(b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid, wherein the second amplification oligomer set comprises first and second aB-specific amplification oligomers respectively comprising first and second / ct / TL specific target-hybridizing sequences, wherein(i) the first tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 17, SEQ IDN0:18, SEQ ID NO:26, SEQ ID N0:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO:60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the second tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs;(c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid, wherein the third amplification oligomer set comprises first and second tcdC-specific amplification oligomers respectively comprising first and second tcdC-specific target-hybridizing sequences, wherein(i) the first tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO:65 and that includes at least the sequence of SEQ ID NO: 64, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO:66, including from 0 to 19 nucleotide analogs; and(ii) the second tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:68 and that includes at least the sequence of SEQ ID NO: 67, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:70 and that includes at least the sequence of SEQ ID NO: 69, including from 0 to 19 nucleotide analogs; and(d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid, wherein the third amplification oligomer set comprises first and second ct / ZS-specific amplification oligomers respectively comprising first and second ct / ZS-specific target-hybridizing sequences, wherein(i) the first cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, including from 0 to 19 nucleotide analogs; and(ii) the second ct / ZS-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequenceshown in SEQ ID NO: 15, including from 0 to 19 nucleotide analogs.

2. The composition or kit of claim 1, wherein the first / ct / 4 -specific targethybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 12, including from 0 to 19 nucleotide analogs.

3. The composition or kit of claim 1 or 2, wherein the second tcdA-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs.

4. The composition or kit of any one of claims 1 to 3, wherein the first tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

5. The composition or kit of any one of claims 1 to 4, wherein the second aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs.

6. The composition or kit of any one of claims 1 to 3, wherein the second amplification oligomer set further comprises third and fourth aB-specific amplification oligomers respectively comprising third and fourth aB-specific target-hybridizing sequences, wherein(i) the third aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO:60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the fourth aB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs.

7. The composition or kit of claim 6, wherein the third aB-specific targethybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:26, SEQ ID NO:31, or SEQ ID NO:33, including from 0 to 19 nucleotide analogs.

8. The composition or kit of claim 6 or 7, wherein the fourth aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38, including from 0 to 19 nucleotide analogs.

9. The composition or kit of claim 6, whereinthe first aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 18, including from 0 to 19 nucleotide analogs,the second aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:36, including from 0 to 19 nucleotide analogs,the third tcdB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:33, including from 0 to 19 nucleotide analogs, and the fourth aB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:38, including from 0 to 19 nucleotide analogs.

10. The composition or kit of any one of claims 1 to 9, wherein the first tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO:30, including from 0 to 19 nucleotide analogs.

11. The composition or kit of claim 10, wherein the first fct / C-specific targethybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:8, including from 0 to 19 nucleotide analogs.

12. The composition or kit of any one of claims 1 to 11, wherein the second tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs.

13. The composition or kit of claim 12, wherein the second tcdC-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 19, including from 0 to 19 nucleotide analogs.

14. The composition or kit of any one of claims 1 to 13, wherein the first cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 14, including from 0 to 19 nucleotide analogs.

15. The composition or kit of any one of claims 1 to 14, wherein the second cdtB-specific target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs.

16. The composition or kit of any one of claims 1 to 15, further comprising at least one / ct / 4- specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a tcdA target sequence amplifiable by the first and second fct / 4-specific amplification oligomers.

17. The composition or kit of claim 16, wherein the Zct / d -specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 10, including from 0 to 19 nucleotide analogs.

18. The composition or kit of claim 17, wherein the Zct / d -specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 10, including from 0 to 19 nucleotide analogs.

19. The composition or kit of any one of claims 1 to 18, further comprising at least one tcdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a tcdB target sequence amplifiable by the first and second aB-specific amplification oligomers.

20. The composition or kit of claim 19, wherein the tcdB-specific detection probe target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

21. The composition or kit of claim 19, wherein the composition or kit comprises at least two tcdB-specific detection probes, wherein a first tcdB-specific detection probe comprises a first tcdB-specific detection probe target-hybridizing sequence and a second tcdB-specific detection comprises a second tcdB-specific detection probe target-hybridizing sequence, andwherein each of the first and second tcdB-specific detection probe target hybridizing sequences is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO:71, including from 0 to 19 nucleotide analogs.

22. The composition or kit of claim 21, whereinthe first tcdB-specific detection probe target hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:2, including from 0 to 19 nucleotide analogs, andthe second tcdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:4, including from 0 to 19 nucleotide analogs.

23. The composition or kit of any one of claims 1 to 18, further comprising at least one tcdC-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a tcdC target sequence amplifiable by the first and second tcdC-specific amplification oligomers, wherein the tcdC-specific detection probe target-hybridizing sequence is capable of detecting a single base pair deletion at nucleotide 117 of the tcdC gene.

24. The composition or kit of claim 23, wherein the tcdC-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO:23 or SEQ ID NO:24, including from 0 to 19 nucleotide analogs.

25. The composition or kit of any one of claims 1 to 24, further comprising at least one ctdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to a ctdB target sequence amplifiable by the first and second ctdB-specific amplification oligomers.

26. The composition or kit of claim 25, wherein the ctdB-specific detection probe target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

27. The composition or kit of claim 26, wherein the ctdB-specific detection probe target-hybridizing sequence consists of the nucleotide sequence shown in SEQ ID NO: 16, including from 0 to 19 nucleotide analogs.

28. The composition or kit of any one of claims 16 to 27, wherein one or more of the detection probes comprises a detectable label.

29. The composition or kit of claim 28, wherein the detectable label is a fluorescent or chemiluminescent label.

30. The composition or kit of claim 28, wherein the detectable label is a fluorescent label and each of the one or more detection probes further comprises a non-fluorescent quencher.

31. An oligonucleotide for determining the presence or absence of a toxigenic C. difficile strain in a sample, wherein said oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0-20 nucleotide analogs.

32. The oligonucleotide of claim 31, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NOs:41-58.

33. The oligonucleotide of claim 31 or 32, wherein the 3’ end of said oligonucleotide is attached to a solid support.

34. A reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample, said reaction mixture comprising a set of oligonucleotides as specified in any one of claims 1 to 30.

35. A reaction mixture for determining the presence or absence of a toxigenic C. difficile strain in a sample, said reaction mixture comprising the oligonucleotide of claim 31 or 32.

36. A method for determining the presence or absence of a toxigenic C. difficile strain in a sample, the method comprising:(1) contacting a sample, said sample suspected of containing the toxigenic C. difficile strain, with an oligomer combination comprising(a) a first amplification oligomer set capable of amplifying a target region of a C. difficile toxin A gene (tcdA) target nucleic acid, wherein the first amplification oligomer set comprises first and second tcdA-specific amplification oligomers respectively comprising first and second tcdA-specific target-hybridizing sequences, wherein(i) the first tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 12, including from 0 to 19 nucleotide analogs; and(ii) the second tcdA-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:32, including from 0 to 19 nucleotide analogs;(b) a second amplification oligomer set capable of amplifying a target region of a C. difficile toxin B gene (tcdB) target nucleic acid, wherein the second amplification oligomer set comprises first and second tcdB-specific amplification oligomers respectively comprising first and second tcdB-specific target-hybridizing sequences, wherein(i) the first tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, orSEQ ID NO:33, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 25 contiguous nucleotides contained in the sequence of SEQ ID NO: 60 and that includes at least the sequence of SEQ ID NO:59, including from 0 to 19 nucleotide analogs; and(ii) the second tcdB-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, including from 0 to 19 nucleotide analogs; and a nucleotide sequence as shown in SEQ ID NO: 63, including from 0 to 19 nucleotide analogs;(c) a third amplification oligomer set capable of amplifying a target region of a C. difficile toxin C gene (tcdC) target nucleic acid, wherein the third amplification oligomer set comprises first and second tcdC-specific amplification oligomers respectively comprising first and second tcdC-specific target-hybridizing sequences, wherein(i) the first tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29, or SEQ ID NO: 30, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 26 contiguous nucleotides contained in the sequence of SEQ ID NO:65 andthat includes at least the sequence of SEQ ID NO: 64, including from 0 to 19 nucleotide analogs; anda nucleotide sequence as shown in SEQ ID NO:66, including from 0 to 19 nucleotide analogs; and (ii) the second tcdC-specific target-hybridizing sequence is selected from the group consisting ofa sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:39, or SEQ ID NO:40, including from 0 to 19 nucleotide analogs;a sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO:68 and that includes at least the sequence of SEQ ID NO: 67, including from 0 to 19 nucleotide analogs; anda sequence that is from 20 to 22 contiguous nucleotides contained in the sequence of SEQ ID NO: 70 and that includes at least the sequence of SEQ ID NO: 69, including from 0 to 19 nucleotide analogs; and(d) a fourth amplification oligomer set capable of amplifying a target region of a C. difficile binary toxin subunit B gene (cdtB) target nucleic acid, wherein the third amplification oligomer set comprises first and second cdtB-specific amplification oligomers respectively comprising first and second cdtB-specific target-hybridizing sequences, wherein(i) the first cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 14, including from 0 to 19 nucleotide analogs; and(ii) the second cdtB-specific target-hybridizing sequence is a sequence having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:15, including from 0 to 19 nucleotide analogs;(2) performing an in vitro nucleic acid amplification reaction, wherein any C. difficile tcdA, tcdB, tcdC, and ctdB target nucleic acid present in the sample is used as a template for generating one or more amplification products corresponding to the C. difficile tcdA, tcdB, tcdC, and ctdB target regions;(3) detecting the presence or absence of one or more tcdA, tcdB, tcdC, and ctdB amplification products respectively corresponding to the C. difficile tcdA, tcdB, tcdC, and ctdB target regions, wherein the tcdC amplification product contains a single base pair deletion corresponding to nucleotide 117 of the tcdC gene, and wherein detecting the tcdC amplification product comprises detection of said single base pair deletion,whereby the combined presence of the tcdA, tcdB, ctdB, and tcdC amplification products indicates the presence of the toxigenic C. difficile strain in the sample.

37. The method of claim 36, wherein the detecting step (3) comprises contacting the in vitro nucleic acid amplification reaction withat least one tcdA-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdA amplification product, and / or at least one tcdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdB amplification product, and / or at least one tcdC-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the tcdC amplification product, whereinthe tcdC-specific detection probe target-hybridizing sequence is capable of detecting the single base pair deletion at nucleotide 117 of the tcdC gene, and / orat least one ctdB-specific detection probe comprising a target-hybridizing sequence configured to specifically hybridize to the ctdB amplification product.

38. The method of claim 36 or 37, wherein the method is a multiplex method in which the first, second, third, and fourth amplification oligomer sets are used for amplification of any C. difficile tcdA, IcdB. tcdC, and ctdB target nucleic acid in the same in vitro nucleic acid amplification reaction.

39. A method for synthesizing an oligonucleotide, comprising the steps of: (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 19 additional times, thereby generating at least 20 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 20 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide,wherein the oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-10, 12, 14-20, 22-24, 26-34, and 36-40, including from 0 to 19 nucleotide analogs.

40. A method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide, wherein each of the synthesizing the first oligonucleotide and the synthesizing the second oligonucleotide comprises the steps of:(a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3’ position to the solid support;(b) coupling a 5’ position of the nucleobase residue furthest from the solid support to a 3’ position of another nucleobase residue;(c) repeating step (b) at least 19 additional times, thereby generating at least 20 contiguous nucleobase residues coupled to the solid support; and(d) cleaving the at least 20 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, andwherein the first oligonucleotide and the second oligonucleotide respectively comprise the nucleotide sequences of any one ofSEQ ID NO: 12 and SEQ ID NO:32, including from 0 to 19 nucleotide analogs;SEQ ID NO: 18 and SEQ ID NO:36, including from 0 to 19 nucleotide analogs;SEQ ID NO: 18 and SEQ ID NO:38, including from 0 to 19 nucleotide analogs;SEQ ID NO:33 and SEQ ID NO: 36, including from 0 to 19 nucleotide analogs;SEQ ID NO:33 and SEQ ID NO: 38, including from 0 to 19 nucleotide analogs;SEQ ID NO:14 and SEQ ID NO:15, including from 0 to 19 nucleotide analogs;SEQ ID NO:8 and SEQ ID NO: 19, including from 0 to 19 nucleotide analogs; orSEQ ID NO:29 and SEQ ID NO:40, including from 0 to 19 nucleotide analogs.

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