METHODS AND REAGENTS FOR GENOTYPING HCV

1 . 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′.