US2014199684A1PendingUtilityA1
Methods for amplifying hepatitis c virus nucleic acids
Est. expiryJan 21, 2029(~2.5 yrs left)· nominal 20-yr term from priority
Inventors:Ann Dak-Yee KwongJames D. FrantzDouglas John BartelsChao LinBenjamin ShamesSheila SeepersaudJudith A. LippkeTara Lynn KiefferYi ZhouEileen Z. ZhangJames C. Sullivan
C12Q 1/6883C12Q 2531/113C12Q 1/707
54
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Claims
Abstract
A method of amplifying an HCV nucleic acid in an HCV infected sample comprises amplifying a segment of a DNA template that is complementary to a genome of HCV RNA from the sample by a two-stage PCR, wherein a first stage PCR employs a first outer primer and a second outer primer, and a second stage PCR employs a first inner primer and a second inner primer. The nucleotide sequence of the first outer primer comprises a nucleotide sequence as set forth in SEQ ID NO: 2; or SEQ ID NO:9, wherein optionally 1, 2 or 3 nucleotides are other nucleotides than those of SEQ ID NO: 9. The nucleotide sequence of the second outer primer comprises a nucleotide sequence set forth in SEQ ID NO: 3 or 4; or a nucleotide sequence as set forth in SEQ ID NO: 10 or 11, wherein optionally 1, 2 or 3 nucleotides are other nucleotides than those of SEQ ID NO: 10 and 11. The nucleotide sequence of the first inner primer comprises a nucleotide sequence as set forth in SEQ ID NO: 5; or SEQ ID NO:12, wherein optionally 1, 2 or 3 nucleotides are other nucleotides than those of SEQ ID NO: 12. The nucleotide sequence of the second inner primer comprises a nucleotide sequence as set forth in SEQ ID NO: 6 or 7; or a nucleotide sequence as set forth in SEQ ID NO: 13 or 14, wherein optionally 1, 2 or 3 nucleotides are other nucleotides than those of SEQ ID NO: 13 and 14.
Claims
exact text as granted — not AI-modified1 - 53 . (canceled)
54 . A kit for assaying a hepatitis C virus (HCV) nucleic acid from an HCV infected sample, comprising a pair of outer PCR primers comprising a first outer primer and a second outer primer, wherein
i) the nucleotide sequence of the first outer primer comprises SEQ ID NO: 2:
(SEQ ID NO: 2)
5′-GAGTAGTGTTGGGTCG-3′,
and
ii) the nucleotide sequence of the second outer primer comprises SEQ ID NO: 3 or SEQ ID NO: 4:
(SEQ ID NO: 3)
5′-CACGCTGTGATAAATG-3′
or
(SEQ ID NO: 4)
5′-CAVGCTGTGATATATG-3′,
wherein V is A, C or G; and
iii) the nucleotide sequence of the first inner primer comprises SEQ ID NO: 5:
(SEQ ID NO: 5)
5′-GGTGCTTGCGAGTGCC-3′,
and
iv) the nucleotide sequence of the second inner primer comprises SEQ ID NO: 6 or SEQ ID NO: 7:
(SEQ ID NO: 6)
5′-TAGCCAGCCGTGAACC-3′
or
(SEQ ID NO: 7)
5′-TARCCAGCRACGAACC-3′,
wherein R is A or G.
55 . The kit of claim 54 , wherein:
i) the nucleotide sequence of the first outer primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 9 wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 9:
(SEQ ID NO: 9)
5′-CAAGACTGCTAGCCGAGTAGTGTTGGGTCG-3′,
and
ii) the nucleotide sequence of the second outer primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 10 or SEQ ID NO: 11, wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 10 and SEQ ID NO: 11:
(SEQ ID NO: 10)
5′-CCGGGCAYGAGACACGCTGTGATAAATG-3′,
or
(SEQ ID NO: 11)
5′-TCGGGCACGAGACAVGCTGTGATATATG-3′,
wherein Y is C, T or U, and V is A, C or G.
56 . (canceled)
57 . The kit of claim 54 , wherein:
i) the nucleotide sequence of the first inner primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 12 wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 12:
(SEQ ID NO: 12)
5′-GTACTGCCTGATAGGGTGCTTGCGAGTGCC-3′;
and
ii) the nucleotide sequence of the second inner primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 13 or SEQ ID NO: 14, wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 13 and SEQ ID NO: 14:
(SEQ ID NO: 13)
5′-TCTCCCCCGCTGTAGCCAGCCGTGAACC-3′
or
(SEQ ID NO: 14)
5′-TCTCCCCCGCTGTARCCAGCRACGAACC-3′,
wherein R is A or G.
58 . The kit of claim 57 , wherein:
i) the nucleotide sequence of the first inner primer comprises SEQ ID NO: 15:
(SEQ ID NO: 15)
5′-AAGTACTGCCTGATAGGGTGCTTGCGAGTGCC-3′;
and
ii) the nucleotide sequence of the second inner primer comprises SEQ ID NO: 16 or SEQ ID NO: 17:
(SEQ ID NO: 16)
5′-ATCTCCCCCGCTGTAGCCAGCCGTGAACC-3′
or
(SEQ ID NO: 17)
5′-ATCTCCCCCGCTGTARCCAGCRACGAACC-3′,
wherein R is A or G.
59 . The kit of claim 54 , further comprising an RT primer, wherein the nucleotide sequence of the RT primer comprises SEQ ID NO: 1:
5′-(A) n -AAAA-3′ (SEQ ID NO: 1), wherein n is an integer of 1-26.
60 - 63 . (canceled)
64 . A kit for assaying a hepatitis C virus (HCV) nucleic acid from an HCV infected sample, comprising:
a) an RT primer, wherein the nucleotide sequence of the RT primer comprises SEQ ID NO: 1 at its 3′ end:
(SEQ ID NO: 1)
5′-(A) n -AAAA-3′,
wherein n is an integer of 1-26;
b) a pair of outer PCR primers comprising a first outer primer and a second outer primer, wherein
i) the nucleotide sequence of the first outer primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 9 wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 9:
(SEQ ID NO: 9)
5′-CAAGACTGCTAGCCGAGTAGTGTTGGGTCG-3′,
and
ii) the nucleotide sequence of the second outer primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 10 or SEQ ID NO: 11, wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 10 and SEQ ID NO: 11:
(SEQ ID NO: 10)
5′-CCGGGCAYGAGACACGCTGTGATAAATG-3′,
or
(SEQ ID NO: 11)
5′-TCGGGCACGAGACAVGCTGTGATATATG-3′,
wherein Y is C, T or U, and V is A, C or G; and
c) a pair of inner PCR primers comprising a first inner primer and a second inner primer, wherein
i) the nucleotide sequence of the first inner primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 12 wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 12:
(SEQ ID NO: 12)
5′ -GTACTGCCTGATAGGGTGCTTGCGAGTGCC-3′;
and
ii) the nucleotide sequence of the second inner primer comprises at its 3′ end a nucleotide sequence as set forth SEQ ID NO: 13 or SEQ ID NO: 14, wherein optionally one, two or three nucleotides thereof independently are other nucleotides than those of SEQ ID NO: 13 and SEQ ID NO: 14:
(SEQ ID NO: 13)
5′-TCTCCCCCGCTGTAGCCAGCCGTGAACC-3′
or
(SEQ ID NO: 14)
5′-TCTCCCCCGCTGTARCCAGCRACGAACC-3′,
wherein R is A or G.
65 . The kit of claim 64 , wherein
the nucleotide sequence of the RT primer comprises SEQ ID NO: 8:
(SEQ ID NO: 8)
5′-AAAAAAAAAAAAAAAAAAAA-3′
66 . A kit for assaying a hepatitis C virus (HCV) nucleic acid from an HCV infected sample, comprising:
a) an RT primer, wherein the nucleotide sequence of the RT primer comprises SEQ ID NO: 8:
(SEQ ID NO: 8)
5′-AAAAAAAAAAAAAAAAAAAA-3′;
b) a pair of outer PCR primers comprising a first outer primer and a second outer primer, wherein
i) the nucleotide sequence of the first outer primer comprises SEQ ID NO: 9:
(SEQ ID NO: 9)
5′-CAAGACTGCTAGCCGAGTAGTGTTGGGTCG-3′,
and
ii) the nucleotide sequence of the second outer primer comprises SEQ ID NO: 10 or SEQ ID NO: 11:
(SEQ ID NO: 10)
5′-CCGGGCAYGAGACACGCTGTGATAAATG-3′,
or
(SEQ ID NO: 11)
5′-TCGGGCACGAGACAVGCTGTGATATATG-3′,
wherein Y is C, T or U, and V is A, C or G; and
c) a pair of inner PCR primers comprising a first inner primer and a second inner primer, wherein
i) the nucleotide sequence of the first inner primer comprises SEQ ID NO: 15:
(SEQ ID NO: 15)
5′-AAGTACTGCCTGATAGGGTGCTTGCGAGTGCC-3′;
and
ii) the nucleotide sequence of the second inner primer comprises SEQ ID NO: 16 or SEQ ID NO: 17:
(SEQ ID NO: 16)
5′-ATCTCCCCCGCTGTAGCCAGCCGTGAACC-3′
or
(SEQ ID NO: 17)
5′-ATCTCCCCCGCTGTARCCAGCRACGAACC-3′,
wherein R is A or G.Join the waitlist — get patent alerts
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