Compositions and methods for detecting or quantifying hepatitis C virus
This disclosure provides oligomers, compositions, and kits for detecting and quantifying Hepatitis C virus (HCV), including different genotypes and variants thereof, and related methods and uses. In some embodiments, oligomers target the 5′ untranslated region of HCV and are configured to provide substantially equivalent quantification of different genotypes and variants of HCV.
1. A composition or kit comprising:
at least first and second amplification oligomers, wherein:
the first amplification oligomer comprises a target-hybridizing sequence comprising at least 10 contiguous nucleotides of SEQ ID NO: 2, including at least a portion of the nucleotide sequence containing positions 5, 7, 12, and 15 of SEQ ID NO: 2; and
the second amplification oligomer comprises a target-hybridizing sequence comprising at least 10 contiguous nucleotides of SEQ ID NO: 3 including at least a portion of the nucleotide sequence containing positions 5, 7, 12, and 15 of SEQ ID NO: 3;
the target-hybridizing sequences of the first and second amplification oligomers each comprise at least about 14 contiguous nucleotides of Hepatitis C virus sequence; and
the kit or composition comprises at least one of: (i) an amplification oligomer that is a promoter-primer; or (ii) a probe oligomer, wherein the probe oligomer comprises a non-nucleotide detectable label and/or wherein at least about half of the sugar moieties in the probe oligomer are 2′-O-methyl-ribose.
2. The composition or kit of claim 1 , wherein the first amplification oligomer comprises a target-hybridizing sequence comprising at least one, two, three, or four of SEQ ID NOs: 23-27; or wherein the first amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous nucleotides of SEQ ID NO: 2; or wherein the first amplification oligomer comprises the sequence of SEQ ID NO: 2.
3. The composition or kit of claim 1 , wherein the second amplification oligomer comprises a target-hybridizing sequence comprising at least one, two, three, or four of SEQ ID NOs: 28-32; or wherein the second amplification oligomer comprises at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous nucleotides of SEQ ID NO: 3; or wherein the second amplification oligomer comprises the sequence of SEQ ID NO: 3.
4. The composition or kit of claim 1 , further comprising a third amplification oligomer, wherein the third amplification oligomer comprises at least about 14 contiguous nucleotides of antisense Hepatitis C virus sequence and is configured to specifically hybridize downstream of position 78 of SEQ ID NO: 75, and the third amplification oligomer does not anneal downstream of an HCV genomic position selected from a position in at least one HCV type that corresponds to position 120, 125, 130, 135, 140, 145, or 150 of SEQ ID NO: 75.
5. The composition or kit of claim 4 , wherein the at least one HCV type includes one or more of HCV types 1a, 1b, 2b, 3b, 4b, 5a, and 6a.
6. The composition or kit of claim 1 , further comprising a third amplification oligomer, wherein the third amplification oligomer comprises at least about 14 contiguous nucleotides of antisense Hepatitis C virus sequence and is configured to specifically hybridize downstream of position 78 of SEQ ID NO: 75, and: wherein the third amplification oligomer is configured to specifically hybridize to a site comprising at least a portion of the nucleotide sequence containing positions 80-119 of SEQ ID NO: 75; or wherein the third amplification oligomer comprises a target-hybridizing sequence comprising at least 10 contiguous nucleotides of SEQ ID NO: 6 or 7; or wherein the third amplification oligomer comprises a target-hybridizing sequence comprising at least one, two, three, or four of SEQ ID NOs: 33-37; or wherein the third amplification oligomer comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 contiguous nucleotides of SEQ ID NO: 7; or wherein the third amplification oligomer comprises the sequence of SEQ ID NO: 7; or wherein the third amplification oligomer comprises the sequence of at least one, two, three, four, or five of SEQ ID NOs: 42-47; or wherein the third amplification oligomer comprises the sequence of SEQ ID NO: 5.
7. The composition or kit of claim 1 , wherein the first and second amplification oligomers are present in relative molar amounts (first:second) ranging from about 8.5:1.5 to about 1.5:8.5, about 7.5:2.5 to about 2.5:7.5, about 8:2 to about 7:3, about 7:3 to about 6:4, about 6:4 to about 5:5, about 5:5 to about 4:6, about 4:6 to about 3:7, or about 3:7 to about 2:8; or wherein the first and second amplification oligomers are present in relative molar amounts (first:second) ranging from about 6:4 to about 1.5:8.5, about 4:6 to about 6:4, or about 4.5:5.5 to about 5.5:4.5.
8. The composition or kit of claim 1 , wherein the composition or kit further comprises an initial amplification oligomer, and the initial amplification oligomer comprises a target-hybridizing sequence comprising at least one, two, three, four, five, six, or seven of SEQ ID NOs: 33-41; or wherein the initial amplification oligomer comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 contiguous nucleotides of SEQ ID NO: 6; or wherein the initial amplification oligomer comprises the sequence of SEQ ID NO: 6; or wherein the initial amplification oligomer comprises the sequence of at least one, two, three, four, or five of SEQ ID NOs: 42-47; or wherein the initial amplification oligomer comprises the sequence of SEQ ID NO: 4.
9. The composition or kit of claim 1 , wherein the probe oligomer comprises a target-hybridizing sequence comprising at least one or two of SEQ ID NOs: 50-52; or wherein the probe oligomer comprises the sequence of SEQ ID NO: 48 or 49; or wherein the probe oligomer comprises at least 11, 12, 13, 14, or 15 contiguous nucleotides of SEQ ID NO: 12; or wherein the probe oligomer comprises a target-hybridizing sequence comprising at least 11, 12, 13, 14, or 15 contiguous nucleotides of SEQ ID NO: 13; or wherein the probe oligomer comprises a first self-complementary region at its 5′ end and a second self-complementary region at its 3′ end; or wherein the probe oligomer comprises a first self-complementary region at its 5′ end and a second self-complementary region at its 3′ end and wherein the self-complementary regions can hybridize to form about 4 to 7 Watson-Crick or wobble base pairs; or wherein the probe oligomer comprises a first self-complementary region at its 5′ end and a second self-complementary region at its 3′ end and wherein the self-complementary regions can hybridize to form about 5 Watson-Crick or wobble base pairs; or wherein the probe oligomer comprises the sequence of SEQ ID NO: 12; or wherein the probe oligomer comprises a target-hybridizing sequence comprising the sequence of SEQ ID NO: 13.
10. The composition or kit of claim 9 , wherein the probe oligomer comprises a non-nucleotide detectable label.
11. The composition or kit of claim 10 , wherein the non-nucleotide detectable label is a fluorescent label.
12. The composition or kit of claim 10 , wherein the probe oligomer comprises a quencher.
13. The composition or kit of claim 12 , wherein the non-nucleotide detectable label is a fluorescent label and the quencher absorbs fluorescence to a greater extent when the probe is free than when the probe is annealed to a target nucleic acid.
14. The composition or kit of claim 11 , wherein the fluorescent label is fluorescein, hexachlorofluorescein, carboxyrhodamine, or acridine.
15. The composition or kit of claim 12 , wherein the quencher is DABCYL.
16. The composition or kit of claim 13 , wherein the fluorescent label is attached to the 5′-terminus of the probe oligomer and the quencher is attached to the 3′-terminus of the probe oligomer, or the fluorescent label is attached to the 3′-terminus of the probe oligomer and the quencher is attached to the 5′-terminus of the probe oligomer.
17. The composition or kit of claim 9 , wherein at least about half, at least about 90%, or all of the sugar moieties in the probe oligomer are 2′-O-methyl-ribose.
18. The composition or kit of claim 1 , wherein the kit or composition comprises at least one amplification oligomer that is a promoter-primer; or wherein the composition or kit comprises one or more promoter-primers comprising the sequence of SEQ ID NO: 8, 9, 10, or 11.
19. The composition or kit of claim 18 , wherein the composition or kit comprises a third amplification oligomer that comprises at least about 14 contiguous nucleotides of antisense Hepatitis C virus sequence, is configured to specifically hybridize downstream of position 78 of SEQ ID NO: 75, and is a promoter-primer.
20. The composition or kit of claim 18 , wherein one or more of the promoter-primers comprises a T7 promoter located 5′ of the target-hybridizing sequence.
21. A method of:
detecting Hepatitis C virus nucleic acid in a sample, comprising:
providing a composition according to claim 1 that further comprises the sample and a third amplification oligomer,
performing a nucleic acid amplification reaction in the composition which produces one or more amplicons in the presence of a Hepatitis C virus nucleic acid,
and detecting the amplicon, wherein:
the third amplification oligomer comprises at least about 14 contiguous nucleotides of antisense Hepatitis C virus sequence and is configured to specifically hybridize to downstream of position 78 of SEQ ID NO: 75; and
the one or more amplicons are produced through extension of the first and third amplification oligomers or second and third amplification oligomers in the presence of the Hepatitis C virus nucleic acid.