Compositions and methods for amplifying and characterizing HCV nucleic acid
View Patent ↗Disclosed are nucleic acid oligomers for amplifying one or more selected regions of HCV nucleic acid. Also disclosed are methods for specific amplification and characterization of HCV nucleic acid using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1. A method for determining at least partial genotype information for hepatitis C virus type la (HCV-la) in a sample, the method comprising:
(1) contacting a sample, said sample suspected of containing HCV-la, with
at least two amplification oligomers for amplifying at least one target region of an HCV-la target nucleic acid, wherein said at least one HCV-la target region is selected from the group consisting of
(a) a first target region comprising nucleotide positions 8522 to 9372 of SEQ ID NO:155 or the complement thereof, wherein if the first target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs:65, 43, and 34; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs:69, 38, and 41;
(b) a second target region comprising nucleotide positions 7788 to 8838 of SEQ ID NO:155 or the complement thereof, wherein if the second target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 57, 63, and 28; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 40, 35, and 42;
(c) a third target region comprising nucleotide positions 6966 to 7970 of SEQ ID NO:155 or the complement thereof, wherein if the third target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 60, 58, and 36; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 31, 73, and 62;
(d) a fourth target region comprising nucleotide positions 6076 to 7117 of SEQ ID NO:155 or the complement thereof, wherein if the fourth target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 55, 70, and 29; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs:46, 45, and 61;
(e) a fifth target region comprising nucleotide positions 5094 to 6304 of SEQ ID NO:155 or the complement thereof, wherein if the fifth target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 39, 44, and 68; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 47, 51, and 59;
(f) a sixth target region comprising nucleotide positions 4258 to 5297 of SEQ ID NO:155 or the complement thereof, wherein if the sixth target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 52, 49, and 72; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 37, 75, and 53; and
(g) a seventh target region comprising nucleotide positions 3434 to 4482 of SEQ ID NO:155 or the complement thereof, wherein if the seventh target region is amplified, then the at least two amplification oligomers comprise (i) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 77, 79, and 81; and (ii) at least one oligomer comprising a target-hybridizing sequence comprising a nucleotide sequence selected from SEQ ID NOs: 78, 80, and 71;
(2) performing at least one in vitro nucleic acid amplification reaction, wherein any HCV-la target nucleic acid present in said sample is used as a template for generating at least one amplification product corresponding to at least one of the first through seventh target regions; and
(3) detecting the nucleobase at one or more nucleotide positions within the at least one amplification product, thereby determining at least partial genotype information for the HCV-la in said sample.
2. The method of claim 1 , wherein
if the first target region is amplified, the target hybridizing region of the at least one oligomer of (a)(i) is selected from the group consisting of
SEQ ID NOs: 65, 43, and 34 and the target hybridizing region of
the at least one oligomer of (a)(ii) is selected from the group consisting of
SEQ ID NOs: 69, 38, and 41;
if the second region is amplified, the target hybridizing region of the at least one oligomer of (b)(i) is selected from the group consisting of
SEQ ID NOs: 57, 63, and 28;
and the target hybridizing region of the at least one oligomer of (b)(ii) is selected from the group consisting of
SEQ ID NOs: 40, 35, and 42;
if the third target region is selected, the target hybridizing region of the at least one oligomer of (c)(i) is selected from the group consisting of
SEQ ID NOs: 60, 58, and 36
and the target hybridizing region of the at least one oligomer of (c)(ii) is selected from the group consisting of
SEQ ID NOs: 31, 73, and 62;
if the fourth target region is amplified, the target hybridizing region of the at least one oligomer of (d)(i) is selected from the group consisting of
SEQ ID NOs: 55, 70, and 29
and the target hybridizing region of the at least one oligomer of (d)(ii) is selected from the group consisting of
SEQ ID NOs: 46, 45, and 61;
if the fifth target region is amplified, the target hybridizing region of the at least one oligomer of (e)(i) is selected from the group consisting of
SEQ ID NOs: 39, 44, and 68 and
the target hybridizing region of the at least one oligomer of (e)(ii) is selected from the group consisting of
SEQ ID NOs: 47, 51, and 59;
if the sixth target region is amplified, the target hybridizing region of the at least one oligomer of (f)(i) is selected from the group consisting of
SEQ ID NOs: 52, 49, and 72 and
the target hybridizing region of the at least one oligomer of (f)(ii) is selected from the group consisting of
SEQ ID NOs: 37, 75, and 53;
if the seventh target region is amplified, the target hybridizing region of the at least one oligomer of (g)(i) is selected from the group consisting of
SEQ ID NOs: 77, 79, and 81 and
the target hybridizing region of the at least one oligomer of (g)(ii) is selected from the group consisting of
SEQ ID NOs: 78, 80, and 71.
3. The method of claim 1 , where the detecting step comprises sequencing the at least one amplification product.
4. The method of claim 3 , wherein the detecting step comprises a sequencing method selected from the group consisting of: single molecule real time (SMRT) sequencing, chain terminator sequencing (Sanger sequencing), nanopore sequencing, massively parallel sequencing, pyrosequencing, polony sequencing, sequencing by ligation, ion semiconductor sequencing, and DNA nanoball sequencing.
5. The method of claim 1 , wherein the detecting step comprises detecting, in a hybridization assay, an ability of the at least one amplification product to hybridize to a SNP-specific probe oligomer and wherein the SNP-specific detection probe oligomer comprises a detectable label.
6. The method of claim 1 , wherein the detecting step comprises detecting, in an amplification-based assay, an ability of a SNP-specific amplification oligomer to amplify a region of the at least one amplification product.
7. The method of claim 1 , wherein the method further comprises contacting the sample with at least one capture probe oligomer comprising a nucleotide sequence that hybridizes to the HCV-la target nucleic acid, wherein the at least one capture probe further comprises a nucleotide sequence or moiety that binds to an immobilized probe.
8. The method of claim 1 , wherein each of said first through seventh target regions are amplified to produce at least one amplification product corresponding to each of said target regions, and wherein the detecting step comprises detecting the nucleobase at one or more positions within each of said amplification products.