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. An amplification primer pair for amplifying a portion of a HCV NS3 1b genomic sequence consisting of a forward primer and a reverse primer, wherein:
the forward primer is selected from the group consisting of:
(i) the nucleic acid sequence of SEQ ID NO: 14 or an active fragment thereof, and
(ii) the nucleic acid sequence of SEQ ID NO: 15 or an active fragment thereof; and
the reverse primer is selected from the group consisting of:
(i) the nucleic acid sequence of SEQ ID NO: 16 or an active fragment thereof,
(ii) the nucleic acid sequence of SEQ ID NO: 19 or an active fragment thereof, and
(iii) the nucleic acid sequence of SEQ ID NO: 20 or an active fragment thereof;
wherein the forward primer and the reverse primer 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 a HCV NS3 1b genomic sequence consisting of:
(i) a forward primer comprising the nucleic acid sequence of SEQ ID NO: 11 or an active fragment thereof,
(ii) a forward primer comprising the nucleic acid sequence of SEQ ID NO: 12 or an active fragment thereof,
(iii) a forward primer comprising the nucleic acid sequence of SEQ ID NO: 13 or an active fragment thereof,
(iv) a forward primer comprising the nucleic acid sequence of SEQ ID NO: 14 or an active fragment thereof,
(v) a forward primer comprising the nucleic acid sequence of SEQ ID NO: 15 or an active fragment thereof,
(vi) a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 16 or an active fragment thereof,
(vii) a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 17 or an active fragment thereof,
(viii) a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 18 or an active fragment thereof,
(ix) a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 19 or an active fragment thereof, and
(x) a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 20 or an active fragment thereof,
wherein the forward primers and the reverse primers are deoxyribonucleic acid primers, and wherein an active fragment is 15 or more nucleotides in length.
3. A kit for amplifying a portion of a HCV NS3 1b genomic sequence by nested PCR comprising:
(a) a container comprising a set of inner primers, and
(b) a container comprising a set of outer primers, wherein the set of outer primers consists of the collection of primers of claim 2 .
4. A kit for amplifying a portion of a HCV NS3 1b genomic sequence by nested PCR comprising:
(a) a container comprising a set of inner primers, and
(b) a container comprising a set of outer primers, wherein the set of outer primers consists of the amplification primer pair of claim 1 .
5. The kit of claim 4 , wherein at least some of the inner primers are labeled with a detectable moiety.
6. The kit of claim 4 , 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 consists of the amplification primer pair 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 9 , wherein at least some of the inner primers are labeled with a detectable moiety.
11. The method of claim 9 , wherein at least some of the inner primers are tagged with tag sequences.
12. The method of claim 11 , wherein the tag sequences are M13 tag sequences.
13. The method of claim 9 , further comprising sequencing a product from the second nucleic acid amplification reaction to detect an HCV NS3 polymorphism.
14. The method of claim 8 , wherein the set of outer primers comprises the collection of primers of claim 2 .