Compositions and methods for detecting toxigenic
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.
1. A composition or kit comprising:
a detection oligomer comprising the sequence set forth in positions 7-18 of SEQ ID NO: 305, wherein the detection oligomer further comprises sufficient additional sequence to specifically hybridize to a C. difficile tcdB nucleic acid, and wherein the detection oligomer is non-extendable; and
at least one secondary detection oligomer, comprising a secondary detection oligomer that is configured to interact with a fragment of the detection oligomer, comprises at least two labels, and comprises the sequence of SEQ ID NO: 12.
2. The composition or kit of claim 1 , wherein the detection oligomer comprises the sequence set forth in SEQ ID NO: 305.
3. The composition or kit of claim 1 , wherein the detection oligomer comprises a label.
4. The composition or kit of claim 1 , wherein the detection oligomer has a length of 15 to 55 nucleotides.
5. The composition or kit of claim 1 , wherein the at least two labels include a FRET pair.
6. The composition or kit of claim 1 , wherein the at least two labels include a quencher.
7. The composition or kit of claim 1 , wherein the fragment of the detection oligomer is a 5′-terminal flap of at least six nucleotides.
8. The composition or kit of claim 1 , wherein the composition or kit comprises a plurality of secondary detection oligomers, wherein the secondary detection oligomers comprise FRET cassettes.
9. The composition or kit of claim 1 , wherein the kit or composition further comprises a tedA primary detection oligomer configured to specifically hybridize to a tedA amplicon having a size from 80 to 400 nucleotides; and at least one additional secondary detection oligomer, wherein the secondary detection oligomers include a secondary detection oligomer configured to generate a positive signal in the presence of a tcdB nucleic acid and in the presence of a tcdA nucleic acid.
10. The composition or kit of claim 1 , wherein the composition or kit comprises a nuclease with structure-specific activity toward a three-strand structure formed by 3′-end invasion.
11. The composition or kit of claim 1 , wherein the composition or kit comprises a flap endonuclease or 5′-nuclease.
12. The composition or kit of claim 1 , wherein the composition or kit comprises a FEN1 nuclease.