Compositions and Methods for Detecting Toxigenic Clostridium Difficile
Provided herein are compositions, kits, and methods for detecting at least one of a C. difficile tcdA, tcdB, tcdC, cdtA, or cdtB nucleic acid in a sample. In some embodiments, one or more alleles of tcdC such as 117del tcdC or 184T tcdC are detected.
1 . A composition or kit comprising forward and reverse tcdC amplification oligomers and further comprising one or both of a first and a second tcdC detection oligomer, wherein:
the first tcdC detection oligomer, if present, is configured to specifically hybridize to a first tcdC sequence at a site comprising position 117 thereof, but exhibits distinguishably different hybridization to a second tcdC sequence, wherein the first tcdC sequence is the sequence of SEQ ID NO: 3 and the second tcdC sequence is the sequence of SEQ ID NO: 4;
the second tcdC detection oligomer, if present, is configured to specifically hybridize to the sequence of a third tcdC sequence at a site comprising position 184 thereof, but exhibits distinguishably different hybridization to the sequence of a fourth tcdC sequence, wherein the third tcdC sequence is the sequence of SEQ ID NO: 5 and the fourth tcdC sequence is the sequence of SEQ ID NO: 2; and
the forward and reverse tcdC amplification oligomers are configured to produce a tcdC amplicon, wherein the tcdC amplicon comprises position 117 of SEQ ID NO: 3 or SEQ ID NO: 4 if the first tcdC detection oligomer is present and/or position 184 of SEQ ID NO: 5 if the second tcdC detection oligomer is present.
2 . (canceled)
3 . A method of detecting a C. difficile tcdC allele, the C. difficile tcdC allele comprising a 117del mutation or a 184T mutation, the method comprising:
preparing a composition according to claim 1 which further comprises a sample comprising or suspected of comprising C. difficile nucleic acid;
subjecting the composition to amplification conditions; and
detecting the presence of the C. difficile 117del tcdC allele or 184T tcdC allele in the sample by determining whether at least one of the first tcdC detection oligomer or the second tcdC detection oligomer hybridized to a tcdC amplicon.
4 - 10 . (canceled)
11 . The composition or kit of claim 1 , wherein the first tcdC detection oligomer is present and competes for hybridization to a tcdC nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO: 86-92, 183, 192, or 193.
12 - 16 . (canceled)
17 . The composition or kit of claim 11 , wherein the first tcdC detection oligomer is present and has no mismatches to the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 3′-terminal nucleotides of SEQ ID NO: 86-92, 183, 192, or 193.
18 . The composition or kit of claim 17 , wherein the first tcdC detection oligomer is present and comprises the sequence of of SEQ ID NO: 86 or 183.
19 . (canceled)
20 . The composition or kit of claim 1 , wherein:
(i) the first tcdC detection oligomer is present and comprises the sequence any one of SEQ ID NOs: 89-92; and
(ii) the composition or kit further comprises an additional first tcdC detection oligomer that comprises the sequence any one of SEQ ID NOs: 89-92, which is different from the first tcdC detection oligomer.
21 . The composition or kit of claim 1 , wherein the composition or kit further comprises at least one first tcdC primary probe oligomer that is configured to form an invasive cleavage structure in the presence of the first tcdC detection oligomer and a polynucleotide comprising the sequence of SEQ ID NO: 3.
22 - 27 . (canceled)
28 . The composition or kit of claim 21 , wherein the first tcdC primary probe oligomer comprises the sequence of any one of SEQ ID NOs: 93-137 or 184-187.
29 - 31 . (canceled)
32 . The composition or kit of claim 1 , wherein the second tcdC detection oligomer is present.
33 . The composition or kit of claim 32 , wherein the second tcdC detection oligomer comprises the sequence of any one of SEQ ID NOs: 194-196 or 205, with up to two mismatches.
34 - 35 . (canceled)
36 . The composition or kit of claim 33 , wherein the second tcdC detection oligomer has no mismatches to the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 3′-terminal nucleotides of SEQ ID NO: 194-196 or 205.
37 . The composition or kit of claim 36 , wherein the second tcdC detection oligomer comprises the sequence of any one of SEQ ID NOs: 194-196 or 205.
38 . The composition or kit of claim 1 , wherein the composition or kit further comprises at least one second tcdC primary probe oligomer that is configured to form an invasive cleavage structure in the presence of the second tcdC detection oligomer and a polynucleotide comprising the sequence of SEQ ID NO: 5.
39 - 56 . (canceled)
57 . The composition or kit of claim 1 , wherein the forward tcdC amplification oligomer comprises the sequence of any one of SEQ ID NOs: 243-248.
58 - 62 . (canceled)
63 . The composition or kit of claim 1 , wherein the reverse tcdC amplification oligomer comprises the sequence of any one of SEQ ID NOs: 249-259.
64 . The composition or kit of claim 1 , wherein the composition or kit further comprises at least forward and reverse tcdA amplification oligomers, and at least one tcdA detection oligomer, wherein:
the forward and reverse tcdA amplification oligomers are configured to produce a tcdA amplicon having a size of from 80 to 400 nucleotides; and
the tcdA detection oligomer is configured to specifically hybridize to the tcdA amplicon.
65 - 72 . (canceled)
73 . The composition or kit of claim 64 , wherein the forward tcdA amplification oligomer comprises the sequence of any one of SEQ ID NOs: 59-60, 64, or 65.
74 - 88 . (canceled)
89 . The composition or kit of claim 1 , wherein the composition or kit further comprises forward and reverse tcdB amplification oligomers, wherein:
the forward and reverse tcdB amplification oligomers are configured to produce a tcdB amplicon having a size of from 80 to 400 nucleotides; and
the tcdB detection oligomer is configured to specifically hybridize to the tcdB amplicon.
90 - 123 . (canceled)
124 . The composition or kit of claim 1 , wherein the composition or kit further comprises forward and reverse cdtB amplification oligomers, and at least one cdtB detection oligomer, wherein:
the forward and reverse cdtB amplification oligomers are configured to produce a cdtB amplicon having a size of from 80 to 400 nucleotides; and
the cdtB detection oligomer is configured to specifically hybridize to the cdtB amplicon.
125 - 150 . (canceled)
151 . The composition or kit of claim 1 , wherein the composition or kit further comprises forward and reverse cdtA amplification oligomers, and at least one cdtA detection oligomer, wherein:
the forward and reverse cdtA amplification oligomers are configured to produce a cdtA amplicon having a size of from 80 to 400 nucleotides; and
the cdtA detection oligomer is configured to specifically hybridize to the cdtA amplicon.
152 - 211 . (canceled)