Aptamers for clostridium difficile diagnostics
The present disclosure relates generally to the field of nucleic acids and, more particularly, to aptamers capable of binding to toxins produced by Clostridium difficile ; diagnostic kits and methods comprising such aptamers; and methods of making and using such aptamers.
1. A method of detecting the presence of a C. difficile toxin in a test sample comprising contacting said test sample with an aptamer that binds to C. difficile toxin A wherein said aptamer comprises SEQ ID NO: 9.
2. The method of claim 1 , wherein said aptamer further comprises at least one additional chemical modification, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position.
3. The method of claim 2 , wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′-amino (2′-NH 2 ), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.
4. The method of claim 1 , wherein said aptamer has a K d for C. difficile toxin A of 30 nM or less.
5. The method of claim 1 , wherein said aptamer is nuclease resistant.
6. The method of claim 1 , wherein said aptamer has a slow off rate.
7. The method of claim 1 , wherein the method of detecting is selected from a pull-down assay, dot blot assay, PCR assay or sandwich assay.
8. The method of claim 7 , wherein the method of detecting is a sandwich assay.
9. The method of claim 8 , wherein the sandwich assay is an aptamer-target-antibody assay wherein the aptamer is immobilized on a substrate and the antibody is used to detect target bound to the immobilized aptamer.
10. The method of claim 8 , wherein the sandwich assay is an antibody-target-aptamer assay wherein the antibody is immobilized on a substrate and the aptamer is used to detect target bound to the immobilized antibody.
11. The method of claim 8 , wherein the sandwich assay is an aptamer-target-aptamer assay wherein a first aptamer is immobilized on a substrate and a second aptamer is used to detect target bound to the first, immobilized aptamer.
12. The method of claim 7 , wherein the method of detecting provides a quantitative measure of C. difficile toxin A.