Compositions and Methods for Detecting Toxigenic Clostridium Difficile
AssigneeGen-Probe Incorporated
InventorPatrick Lynn Peterson
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Abstract
Provided herein are compositions, kits, and methods for detecting at least one of a C. diffcile tcdA, tcdB, tcdC, cdtA, or cdtB nucleic acid in a sample. In son embodiments, one or more alleles of tcdC such as 117del tcdC or 184T tcdC are detected.
Claims211
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.
A composition comprising forward and reverse tcdC amplification oligomers, one or both of a first tcdC detection oligomer and a second tcdC detection oligomer, forward and reverse tcdA amplification oligomers, and at least one tcdA detection oligomer, wherein:
- the first tcdC detection oligomer, if present, is configured to specifically hybridize to a first tcdC sequence, which is the sequence of SEQ ID NO:
- 3, at a site comprising position 117 thereof;
- the second tcdC detection oligomer, if present, is configured to specifically hybridize to a third tcdC sequence, which is the sequence of SEQ ID NO:
- 5, at a site comprising position 184 thereof;
- 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 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;
- 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.
A composition or kit comprising either or both of:
- (i) a first tcdC detection oligomer and a first tcdC primary probe oligomer;
- and
- (ii) a second tcdC detection oligomer and a second tcdC primary probe oligomer, wherein:
- the at least one first tcdC detection oligomer, if present, is an invader oligomer 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 first tcdC primary probe oligomer, if present, is configured to form an invasive cleavage substrate with the first tcdC detection oligomer in the presence of the first tcdC sequence;
- the second tcdC detection oligomer, if present, is an invader oligomer 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 second tcdC primary probe oligomer, if present, is configured to form an invasive cleavage substrate with the second tcdC detection oligomer in the presence of the third tcdC sequence.
The composition, kit, or method of any one of the preceding claims, wherein the first tcdC detection oligomer is present.
The composition, kit, or method of any one of the preceding claims, 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.
The composition, kit, or method of any one of the preceding claims, wherein the second tcdC detection oligomer is present and 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.
The composition, kit, or method of any one of claims 2-93, wherein the tcdA detection oligomer comprises the sequence of any one of SEQ ID NOs:
- 57, 61, 66, or 67, with up to two mismatches.
The composition, kit, or method of any one of the preceding claims, 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.
A composition or kit comprising a forward tcdB amplification oligomer, an additional forward tcdB amplification oligomer, and a reverse amplification oligomer, wherein the forward tcdB amplification oligomer competes for hybridization to a tcdB nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:
- 260-279 or 339 and/or the forward tcdB amplification oligomer comprises the sequence of any one of SEQ ID NOs:
- 260-279 or 339 with up to two mismatches;
- wherein the additional forward tcdB amplification oligomer competes for hybridization to a tcdB nucleic acid under stringent conditions with an oligomer having a sequence consisting of SEQ ID NO:
- 260-279 or 339 and/or the additional forward tcdB amplification oligomer comprises the sequence of any one of SEQ ID NOs:
- 260-279 or 339 with up to two mismatches;
- wherein the additional forward tcdB amplification oligomer is different from the forward tcdB amplification oligomer;
- and wherein the forward tcdB amplification oligomer, additional forward tcdB amplification oligomer, and reverse amplification oligomer are collectively configured to produce a tcdB amplicon having a size of from 80 to 400 nucleotides.
The composition, kit, or method of any one of the preceding claims, 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.
The composition, kit, or method of anyone of the preceding claims, 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.
A detection oligomer comprising the sequence set forth in positions 7-18 of any one of SEQ ID NOs:
- 18-20, 37-39, 46-48, 53, 54, 58, 62, 68, 69, 138-182, 188-191, 201-204, 213-219, 238-242, or 299-316, wherein the detection oligomer further comprises sufficient additional sequence to specifically hybridize to a C. difficile tcdA, tcdB, tcdC, cdtA, or cdtB nucleic acid.
The method of claim 217 or 218, wherein the invasive oligomer has a sequence comprising the sequence of any one of SEQ ID NOs:
- 14, 15, 40-42, 63-65, 74-76, 86-92, 183, 192-196, 205, 220, 221, 223-225, 260-280, or 317 with up to two mismatches.
The composition, kit, detection oligomer, or method of any one of the preceding claims, wherein at least one oligomer comprises at least one methylated cytosine.
The composition, kit, detection oligomer, or method of any one of the preceding claims, wherein the sequences of SEQ ID NOs include adenine methylation as indicated in the Table of Sequences.
The composition, kit, detection oligomer, or method of any one of the preceding claims, wherein the sequences of SEQ ID NOs include cytosine methylation as indicated in the Table of Sequences.
Dependencies are inferred from the claim text.