US2007259339A1PendingUtilityA1
Methods and reagents for genotyping hcv
Est. expiryDec 23, 2025(expired)· nominal 20-yr term from priority
C12Q 1/707C12N 2770/24211
48
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Claims
Abstract
The present invention is directed to methods and reagents for determining the genotype of a hepatitis C virus (HCV) species present in a test sample. The invention more particularly relates to mixtures of degenerate amplification and sequencing primers, and methods of using such primers, that are complementary to a plurality of HCV species, and are capable of generating nucleotide sequence information for a region of NS5B of HCV that is, for each species, indicative of the type and/or subtype, of the species present in the sample.
Claims
exact text as granted — not AI-modified1 . A method for determining the genotype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) determining the nucleotide sequence of at least a portion of the NS5b region of HCV indicative of the genotype of said HCV species present in the test sample, wherein the corresponding nucleotide sequence of a plurality of HCV species is indicative of a distinct genotype of that HCV species; (b) correlating the nucleotide sequence of said portion of the NS5b region determined in (a) with the genotype of one of said plurality of HCV species.
2 . The method of claim 1 , wherein the portion of the NS5b region of HCV consists essentially of the region from about nucleotide position 8344 to about 8547 of SEQ ID NO: 1.
3 . The method of claim 1 , wherein the portion of the NS5b region of HCV consists essentially of the region from nucleotide position 8344 to 8547 of SEQ ID NO: 1.
4 . A method for determining the genotype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) determining the nucleotide sequence of at least a portion of the NS5b region of HCV indicative of the genotype of said HCV species present in the test sample, wherein the corresponding nucleotide sequence of each of a plurality of HCV species having HCV genotypes 1, 2, 3, 4, 5, and 6 is indicative of a distinct genotype of that HCV species; (b) correlating the nucleotide sequence of said portion of the NS5b region determined in (a) with one of said HCV genotypes 1, 2, 3, 4, 5 and 6.
5 . The method of claim 4 , wherein the portion of the NS5b region of HCV consists essentially of the region from about nucleotide position 8344 to about 8547 of SEQ ID NO: 1.
6 . The method of claim 4 , wherein the portion of the NS5b region of HCV consists essentially of the region from nucleotide position 8344 to 8547 of SEQ ID NO: 1.
7 . A method for determining the genotype and subtype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) determining the nucleotide sequence of at least a portion of the NS5b region of HCV indicative of the genotype and subtype of said HCV species present in the test sample, wherein the corresponding nucleotide sequence of each of a plurality of HCV species having HCV genotypes 1, 2, 3, 4, 5, and 6, and each of the HCV subtypes set forth in Table 1, is indicative of a distinct genotype and subtype of that HCV species; (b) correlating the nucleotide sequence of said portion of the NS5b region determined in (a) with one of said HCV genotypes 1, 2, 3, 4, 5 and 6 and one of said subtypes set forth in Table 1.
8 . The method of claim 7 , wherein the portion of the NS5b region of HCV, consists essentially of the region from about nucleotide position 8344 to about 8547 of SEQ ID NO: 1.
9 . The method of claim 7 , wherein the portion of the NS5b region of HCV consists essentially of the region from nucleotide position 8344 to 8547 of SEQ ID NO: 1.
10 . A method for determining the genotype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) providing a mixture of degenerate oligonucleotide sequencing primers capable of generating nucleotide sequence of at least a portion of the NS5b region of a plurality of HCV species, wherein the corresponding nucleotide sequence of each of said plurality of HCV species is indicative of a distinct genotype of that HCV species; (b) determining the nucleotide sequence of said portion of the NS5b region indicative of the genotype of said HCV species present in the test sample; and (c) correlating the nucleotide sequence of said portion of the NS5b region of said HCV species determined in (b) with a genotype of one of said plurality of HCV species.
11 . The method of claim 10 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8256 to about 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8611 to about 8633 of SEQ ID NO: 1, or its complement.
12 . The method of claim 10 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8256 to 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8611 to 8633 of SEQ ID NO: 1, or its complement.
13 . The method of claim 10 , wherein the mixture of degenerate oligonucleotide sequencing primers comprise degenerate oligonucleotide sequences defined by the following formulas, or complements thereof:
SEQ ID NO:1:
5′-TAT GAY ACC CGC TGY TTY GAY TC-3′;
and
SEQ ID NO:2:
5′-VGT CAT RGC ITC YGT RAA GGC TC-3′.
14 . A method for determining the genotype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) providing a mixture of degenerate oligonucleotide sequencing primers capable of generating nucleotide sequence of at least a portion of the NS5b region of a plurality of HCV species, wherein the corresponding nucleotide sequence of each of said plurality of HCV species is indicative of one of HCV genotypes 1, 2, 3, 4, 5 and 6; (b) determining the nucleotide sequence of said portion of the NS5b region indicative of the genotype of said HCV species present in the test sample; and (c) correlating the nucleotide sequence of said portion of the NS5b region of said HCV species determined in (b) with one of HCV genotypes 1, 2, 3, 4, 5 and 6.
15 . The method of claim 14 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8256 to about 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8611 to about 8633 of SEQ ID NO: 1, or its complement.
16 . The method of claim 14 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8256 to 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8611 to 8633 of SEQ ID NO: 1, or its complement.
17 . The method of claim 14 , wherein the mixture of degenerate oligonucleotide sequencing primers comprise degenerate oligonucleotide sequences defined by the following formulas, or complements thereof:
SEQ ID NO:1:
5′-TAT GAY ACC CGC TGY TTY GAY TC-3′;
and
SEQ ID NO:2:
5′-VGT CAT RGC ITC YGT RAA GGC TC-3′.
18 . A method for determining the genotype of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) providing a mixture of degenerate oligonucleotide sequencing primers capable of generating nucleotide sequence of at least a portion of the NS5b region of a plurality of HCV species, wherein the corresponding nucleotide sequence of each of said plurality of HCV species is indicative of one of HCV genotypes 1, 2, 3, 4, 5 and 6 and one of the subtypes; (b) determining the nucleotide sequence of said portion of the NS5b region indicative of the genotype and subtype of said HCV species present in the test sample; and (c) correlating the nucleotide sequence of said portion of the NS5b region of said HCV species determined in (b) with an HCV genotype and subtype.
19 . The method of claim 18 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8256 to about 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8611 to about 8633 of SEQ ID NO: 1, or its complement.
20 . The method of claim 18 , wherein the mixture of degenerate oligonucleotide sequencing primers comprises degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8256 to 8278, or its complement, and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8611 to 8633 of SEQ ID NO: 1, or its complement.
21 . The method of claim 18 , wherein the mixture of degenerate oligonucleotide sequencing primers comprise degenerate oligonucleotide sequences defined by the following formulas, or complements thereof:
SEQ ID NO:1:
5′-TAT GAY ACC CGC TGY TTY GAY TC-3′;
and
SEQ ID NO:2:
5′-VGT CAT RGC ITC YGT RAA GGC TC-3′.
22 . A method for amplifying a portion of the NS5b region of a hepatitis C virus (HCV) species present in a test sample, comprising:
(a) providing a mixture of degenerate oligonucleotide PCR primers comprising: degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8245 to about 8269, or its complement; and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from about nucleotide 8616 to about 8641 of SEQ ID NO: 1, or its complement; and (b) amplifying the nucleotide sequence of said portion of the NS5b region.
23 . The method of claim 22 , wherein the mixture of degenerate oligonucleotide PCR primers comprises:
degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8256 to 8278, or its complement; and degenerate nucleotide sequences complementary to the NS5b region of a plurality of HCV species from nucleotide 8611 to 8633 of SEQ ID NO: 1, or its complement.
24 . The method of claim 22 , wherein the mixture of degenerate oligonucleotide PCR primers comprise degenerate oligonucleotide sequences defined by the following formulas, or complements thereof:
SEQ ID NO:6:
5′- TGG SBT TYK CNT AYG AYA CYM GNT G - 3′
SEQ ID NO:5:
5′- GAR TAY CTV GTC ATR GCI TCY GTR AA - 3′
25 . The method of claim 22 , wherein the mixture of degenerate oligonucleotide PCR primers comprise degenerate oligonucleotide sequences defined by the following formulas, or complements thereof:
SEQ ID NO:3:
5′- TGG GGT TCK CGT ATG AYA CCC GCT G - 3′
SEQ ID NO:4:
5′- TGG GGT TCK CIT ATG AYA CYM GIT G - 3′
SEQ ID NO:5:
5′- GAR TAY CTV GTC ATR GCI TCY GTR AA - 3′
26 . A mixture of degenerate oligonucleotide PCR primers, wherein the mixture comprises a plurality of oligonucleotide PCR primers defined by one or more of the following formulas:
SEQ ID NO:3:
5′- TGG GGT TCK CGT ATG AYA CCC GCT G - 3′
SEQ ID NO:4:
5′- TGG GGT TCK CIT ATG AYA CYM GIT G - 3′
SEQ ID NO:5:
5′- GAR TAY CTV GTC ATR GCI TCY GTR AA - 3′
27 . The mixture of degenerate oligonucleotide PCR primers of claim 26 , wherein the mixture comprises a plurality of oligonucleotide PCR primers defined by the following formula:
SEQ ID NO:3:
5′- TGG GGT TCK CGT ATG AYA CCC GCT G - 3′
28 . The mixture of degenerate oligonucleotide PCR primers of claim 26 , wherein the mixture comprises a plurality of oligonucleotide PCR primers defined by the following formula:
SEQ ID NO:4:
5′- TGG GGT TCK CIT ATG AYA CYM GIT G - 3′
29 . The mixture of degenerate oligonucleotide PCR primers of claim 26 , wherein the mixture comprises a plurality of oligonucleotide PCR primers defined by the following formula:
SEQ ID NO:5:
5′- TGG GGT TCK CIT ATG AYA CYM GIT G - 3″
30 . A mixture of degenerate oligonucleotide sequencing primers, wherein the mixture comprises a plurality of oligonucleotide sequencing primers defined by one or more of the following formulas:
SEQ ID NO:1:
5′- TAT GAY ACC CGC TGY TTY GAY TC - 3′;
and
SEQ ID NO:2:
5′- VGT CAT RGC ITC YGT RAA GGC TC - 3′.
31 . The mixture of degenerate oligonucleotide sequencing primers of claim 30 , wherein the mixture comprises a plurality of oligonucleotide sequencing primers defined by the following formula:
SEQ ID NO:1:
5′- TAT GAY ACC CGC TGY TTY GAY TC - 3′;
and
32 . The mixture of degenerate oligonucleotide sequencing primers of claim 30 , wherein the mixture comprises a plurality of oligonucleotide sequencing primers defined by the following formula:
SEQ ID NO:2:
5′- VGT CAT RGC ITC YGT RAA GGC TC -3′.Cited by (0)
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