Reagents and methods for detecting HCV
The present disclosure relates to oligonucleotide sequences for amplification primers and their use in performing nucleic acid amplifications of HCV, in particular regions that encode the NS3 polypeptide. In some embodiments the primers are used in nested PCR methods for the detection or sequencing of HCV NS3. The oligonucleotide sequences are also provided assembled as kits that can be used to amplify and detect or sequence HCV NS3.
1. A collection of primers for amplifying a portion of an HCV NS3 1a genomic sequence comprising:
one or more forward primers each comprising a nucleic acid sequence selected from the group consisting of:
(i) SEQ ID NO: 1,
(ii) SEQ ID NO: 2 or an active fragment thereof,
(iii) SEQ ID NO: 3 or an active fragment thereof, and
(iv) SEQ ID NO: 4; and
one or more reverse primers each comprising a nucleic acid sequence selected from the group consisting of:
(i) SEQ ID NO: 6 or an active fragment thereof,
(ii) SEQ ID NO: 7 or an active fragment thereof,
(iii) SEQ ID NO: 8 or an active fragment thereof, and
(iv) SEQ ID NO: 9;
wherein the one or more forward primers and the one or more reverse primers are deoxyribonucleic acid primers, and wherein an active fragment is 15 or more nucleotides in length.
2. A collection of primers for amplifying a portion of an HCV NS3 1a genomic sequence, wherein the collection of primers comprises forward primers and reverse primers, the forward primers comprising: a primer comprising SEQ ID NO: 1 or an active fragment thereof, a primer comprising SEQ ID NO: 2 or an active fragment thereof, a primer comprising SEQ ID NO: 3 or an active fragment thereof, a primer comprising SEQ ID NO: 4 or an active fragment thereof, and a primer comprising SEQ ID NO: 5 or an active fragment thereof, and the reverse primers comprising a primer comprising SEQ ID NO: 6 or an active fragment thereof, a primer comprising SEQ ID NO: 7 or an active fragment thereof, a primer comprising SEQ ID NO: 8 or an active fragment thereof, a primer comprising SEQ ID NO: 9 or an active fragment thereof, and a primer comprising SEQ ID NO: 10 or an active fragment thereof.
3. A kit for amplifying HCV NS3 by nested PCR comprising a set of inner primers and a set of outer primers, wherein the set of outer primers comprises a collection of primers as defined in claim 1 .
4. A kit for amplifying HCV NS3 by nested PCR comprising a set of inner primers and a set of outer primers, wherein the set of outer primers comprises a collection of primers as defined in claim 2 .
5. The kit of claim 3 , wherein at least some of the inner primers are labeled with a detectable moiety.
6. The kit of claim 3 , wherein at least some of the inner primers are tagged with tag sequences.
7. The kit of claim 6 , wherein the tag sequences are M13 tag sequences.
8. A method of amplifying HCV NS3 in a sample comprising contacting a sample with a set of outer primers, wherein the set of outer primers comprises the collection of primers of claim 1 and submitting the resulting mixture to a first nucleic acid amplification reaction.
9. The method of claim 8 , further comprising contacting a product from the first nucleic acid amplification reaction with a set of inner primers and submitting the resulting mixture to a second nucleic acid amplification reaction.
10. The method of claim 8 , wherein the set of outer primers comprises a collection of primers as defined in claim 2 .
11. The method of claim 9 , wherein at least some of the inner primers are labeled.
12. The method of claim 9 , wherein at least some of the inner primers are tagged with tag sequences.
13. The method of claim 12 , wherein the tag sequences are M13 tag sequences.
14. The method of claim 9 , further comprising sequencing a product from the second nucleic acid amplification reaction to detect an HCV NS3 polymorphism.