U.S. patent application number 11/615789 was filed with the patent office on 2007-11-08 for methods and reagents for genotyping hcv.
Invention is credited to Marcellinus Beld, Remko Gouw, Toumy Guettouche, James Hnatyszyn, Carola van der Meer.
Application Number | 20070259339 11/615789 |
Document ID | / |
Family ID | 38218857 |
Filed Date | 2007-11-08 |
United States Patent
Application |
20070259339 |
Kind Code |
A1 |
Hnatyszyn; James ; et
al. |
November 8, 2007 |
METHODS AND REAGENTS FOR GENOTYPING HCV
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.
Inventors: |
Hnatyszyn; James; (Madison,
WI) ; Beld; Marcellinus; (Leiden, NL) ;
Guettouche; Toumy; (Stuttgart, DE) ; Gouw; Remko;
(Katwijk aan Zee, NL) ; van der Meer; Carola;
(Amstelhoek, NL) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE;UTAH OFFICE
405 South Main Street
Suite 800
SALT LAKE CITY
UT
84111-3400
US
|
Family ID: |
38218857 |
Appl. No.: |
11/615789 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60753761 |
Dec 23, 2005 |
|
|
|
Current U.S.
Class: |
435/5 ;
536/24.33; 536/25.3 |
Current CPC
Class: |
C12Q 1/707 20130101;
C12N 2770/24211 20130101 |
Class at
Publication: |
435/005 ;
536/024.33; 536/025.3 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C07H 21/04 20060101 C07H021/04 |
Claims
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: TABLE-US-00026 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: TABLE-US-00027 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: TABLE-US-00028 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: TABLE-US-00029 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: TABLE-US-00030 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: TABLE-US-00031
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: TABLE-US-00032 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: TABLE-US-00033 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: TABLE-US-00034 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: TABLE-US-00035 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: TABLE-US-00036 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: TABLE-US-00037 SEQ ID NO:2: 5'- VGT CAT RGC ITC YGT RAA
GGC TC -3'.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the filing date of
U.S. Provisional Patent Application No. 60/753,761, filed Dec. 23,
2005, the disclosure of which is incorporated, in its entirety, by
this reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention is generally directed to methods and
materials for genotyping a hepatitis C virus (HCV) species found in
a test sample.
BACKGROUND OF THE INVENTION
[0003] Hepatitis C virus (HCV) is estimated to infect at
approximately 170 million people worldwide, and is responsible for
chronic liver disease and increased risk of cirrhosis and
hepatocellular carcinoma. Treatment of HCV is principally limited
to antiviral regimens, the efficacies of which are largely
influenced by several biological parameters, such as the virus
genotype. HCV genotyping has therefore been widely used to predict
the response to antiviral therapy and to optimize the duration of
treatment. HCV genotyping has also become an essential tool for
epidemiological studies and for tracing sources of contamination by
HCV.
[0004] The plus-strand HCV RNA genome is approximately 9600
nucleotides in length and encodes at least one open-reading frame
with approximately 3010 amino acids. In infected cells, this
polyprotein is cleaved at multiple sites by cellular and viral
proteases to produce structural and non-structural (NS) proteins.
HCV isolates are characterized by a high degree of genetic
variability due to the lack of fidelity of the HCV RNA-dependent
RNA polymerase, which is encoded by the non-structural 5B (NS5B)
gene. In addition, as a result of endogenous mutation or infection
by a plurality of species, also gives rise to genetically variable
quasi-species of HCV within a single patient. Six main genotypes of
HCV, and over a hundred subtypes, have been described.
[0005] The genetic variability of HCV complicates the processes of
amplification, sequencing, and genotyping. These processes
typically rely upon use of oligonucleotide primers and probes
(e.g., PCR amplification primers, sequencing primers, and
site-specific probes) that are complementary to and are capable of
hybridizing to corresponding nucleic acid sequences of the HCV
genome. As a result of the high degree of variability of the HCV
genome, oligonucleotide primers and probes complementary to one
species of HCV may not be complementary to another species. Such
primers and probes must therefore be designed for specificity to
highly conserved regions. Alternatively, assays must use mixtures
of degenerate primers and probes that are complementary to all
species.
[0006] Some genotyping methods have focused on the 5' noncoding
(5'NC) region. The 5' non-coding (NC) region of HCV is highly
conserved, yet contains type-specific polymorphisms that can be
utilized to distinguish between genotypes. To date, most of the
commercially available 5'NC region genotyping assays have been
based on PCR amplification and fragment analysis by RFLP or
hybridization to oligonucleotide probes. These types of assay are
rapid but not as accurate as sequencing-based assays. For this
reason, alternative genomic regions have been proposed for use in
genotyping HCV, including the NS5B region.
[0007] The most accurate and direct method of genotyping HCV is to
sequence the virus genome in a region that is sufficiently
divergent among various species to distinguish between virus types
and subtypes. Equally importantly, databases for phylogenetic
analysis must be readily available to analyze the sequences
generated from these regions.
[0008] Commercially available sequencing-based HCV genotyping
assays include, for example, the TRUGENE HCV 5'NC Genotyping Kit
(Bayer HealthCare), which is a rapid sequencing-based assay
utilizing the 5'NC region of HCV. Sequence data generated by this
assay are directly analyzed utilizing a phylogenetic 5'NC region
database (TRUGENE HCV 5'NC software module v3.1.1). Previously,
5'NC databases have included sequences from various sources that
have never been fully validated and, in some cases, subtype
assignments for particular strains have been discordant when 5'NC
or NS5B sequences were analyzed.
[0009] There is a continuing need to improve sequencing-based HCV
assays, so as to improve identification of HCV types and subtypes
for purposes of clinical analaysis and therapeutic
intervention.
SUMMARY OF THE INVENTION
[0010] The present invention is generally directed to methods and
reagents for genotyping a hepatitis C virus (HCV) species found in
a test sample. More particularly, the present invention is directed
to an improved method of amplifying and sequencing a portion of the
NS5B region of an HCV species in a sample and determining its
genotype.
[0011] One aspect of the invention relates to a method for
determining the genotype of a hepatitis C virus (HCV) species
present in a test sample by sequencing at least a portion of the
NS5B region of HCV that, for each of a plurality of HCV species, is
indicative of the type and/or subtype of that species.
[0012] In one embodiment, the method comprises (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; and (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.
[0013] In another embodiment, the method comprises (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; and
(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.
[0014] In yet another embodiment, the method comprises (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 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; and (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 HCV subtypes set
forth in Table 1.
[0015] In one particular embodiment of the above methods, 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.
[0016] In another particular embodiment of the above methods, the
portion of the NS5b region of HCV consists essentially of the
region from nucleotide position 8344 to 8547 of SEQ ID NO: 1.
[0017] In another embodiment, the method of present invention
comprises (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
present in a test sample, 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.
[0018] In yet another embodiment, the method comprises (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.
[0019] In still another embodiment, the method comprises (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 set forth in Table 1; (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 (for example, one of HCV genotypes 1, 2,
3, 4, 5 and 6 and one of the subtypes set forth in Table 1).
[0020] In one particular embodiment, 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 [CLIP sequencing
primer M-NS5b-Cy5.5], 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 (for example, CLIP
sequencing primer M-NS5b-Cy5) of SEQ ID NO: 1, or its
complement.
[0021] In another particular embodiment, 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 (for example, CLIP sequencing
primer M-NS5b-Cy5.5), or its complement, and degenerate nucleotide
sequences complementary to the NS5b region of a plurality of HCV
species from nucleotide 8611 to 8633 (for example, CLIP sequencing
primer M-NS5b-Cy5) of SEQ ID NO: 1, or its complement.
[0022] In yet another particular embodiment, the mixture of
degenerate oligonucleotide sequencing primers comprise degenerate
oligonucleotide sequences defined by one or more of the following
formulas, or complements thereof: TABLE-US-00001 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'.
[0023] Another aspect of the invention relates to a method for
determining the genotype of a hepatitis C virus (HCV) species
present in a test sample by amplifying at least a portion of the
NS5B region of HCV that, for each of a plurality of HCV species, is
indicative of the type and/or subtype of that species.
[0024] In one embodiment, the method comprises (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
(for example, forward primers FF1 and/or FF2), 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 (for example, reverse primers RR1) of SEQ ID NO: 1, or
its complement; and (b) amplifying the nucleotide sequence of said
portion of the NS5b region.
[0025] In a particular embodiment, 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 (for example, CLIP sequencing
primers M-NS5b-Cy5.5), or its complement; and degenerate nucleotide
sequences complementary to the NS5b region of a plurality of HCV
species from nucleotide 8611 to 8633 (for example, CLIP sequencing
primers M-NS5b-Cy5) of SEQ ID NO: 1, or its complement.
[0026] In another particular embodiment, the mixture of degenerate
oligonucleotide PCR primers comprise degenerate oligonucleotide
sequences defined by one or more of the following formulas, or
complements thereof: TABLE-US-00002 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'
[0027] In yet another particular embodiment, the mixture of
degenerate oligonucleotide PCR primers comprise degenerate
oligonucleotide sequences defined by one or more of the following
formulas, or complements thereof: TABLE-US-00003 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'
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a schematic diagram showing the HCV NS5b domain
and relevant regions utilized in particular embodiments of the
invention.
[0029] FIG. 2 shows the nucleotide sequence of NS5b domain of HCV,
as set forth in GenBank Accession No. M67463. Primer regions are
highlighted and designated in the margins. FIG. 2 corresponds to
SEQ ID NO:12.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Definitions
[0031] While the terminology used in this application is standard
within the art, the following definitions of certain terms are
provided to assure clarity.
[0032] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation. Numeric ranges
recited herein are inclusive of the numbers defining the range and
include and are supportive of each integer within the defined
range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUBMB Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. Unless otherwise noted, the terms "a"
or "an" are to be construed as meaning "at least one of." The
section headings used herein are for organizational purposes only
and are not to be construed as limiting the subject matter
described. All documents, or portions of documents, cited in this
application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose. In the
case of any amino acid or nucleic sequence discrepancy within the
application, the figures control.
[0033] As used herein, the term "amplification" means the process
of increasing the relative abundance of one or more specific genes
or gene fragments in a reaction mixture with respect to other
genes.
[0034] The term "consisting essentially of" means, when used herein
in reference to specified nucleotide sequences, the specified
sequence and any additional sequence that does not materially
affect the complementarity of the sequence and ability of the
sequence to hybridize to a plurality of HCV types or subtypes.
[0035] As used herein, a "sample" refers to any substance
containing or suspected of containing a nucleic acid, such as RNA
or DNA, and includes samples of tissue or fluid isolated from an
individual or individuals, including but not limited to, for
example, skin, plasma, serum, spinal fluid, lymph fluid, synovial
fluid, urine, tears, blood cells, organs, tumors, and also to
samples of in vitro cell culture constituents (including but not
limited to conditioned medium resulting from the growth of cells in
cell culture medium, recombinant cells and cell components).
[0036] The term "nucleotide" means the nucleotides adenosine,
cytosine, guanine and thymine are represented by their one-letter
codes A, C, G, and T respectively. In representations of degenerate
primers, the symbol R refers to either G or A, the symbol Y refers
to either T/U or C, the symbol M refers to either A or C, the
symbol K refers to either G or T/U, the symbol S refers to G or C,
the symbol W refers to either A or T/U, the symbol B refers to "not
A", the symbol D refers to "not C", the symbol H refers to "not G",
the symbol V refers to "not T/U" and the symbol N refers to any
nucleotide. The symbol I represents inosine, which is a neutral
base that generally will pair with any C, T or A. In the
specification and claims of this application, a degenerate primer
refers to any or all of the combinations of base choices and to
either DNA or the corresponding RNA sequence (i.e., with T replaced
by U). Thus, a degenerate primer may represent a single species, or
a mixture of two species which fall within the choices, or a
mixture of three choices which fall with the choices, and so on up
to a mixture containing all the possible combinations.
[0037] The terms "nucleic acid", "polynucleotide" and
"oligonucleotide" refer to primers, probes, oligomer fragments to
be detected, oligomer controls and unlabeled blocking oligomers and
shall be generic to polydeoxyribonucleotides (containing
2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose),
and to any other type of polynucleotide which is an N-glycoside of
a purine or pyrimidine base, or modified purine or pyrimidine
bases. There is no intended distinction in length between the term
"nucleic acid", "polynucleotide" and "oligonucleotide", and these
terms will be used interchangeably. These terms refer only to the
primary structure of the molecule. Thus, these terms include
double- and single-stranded DNA, as well as double- and
single-stranded RNA. The oligonucleotide is comprised of a sequence
of approximately at least 6 nucleotides, preferably at least about
10-12 nucleotides, and more preferably at least about 15-25
nucleotides corresponding to a region of the designated nucleotide
sequence. The term "corresponding to," as used herein, as used
herein to define a nucleic acid sequence in terms of a reference
nucleotide sequence, means nucleotide sequences that match all or
part of the reference sequence, and nucleotide sequences that are
the complement of all or part of the reference sequence.
[0038] The oligonucleotide is not necessarily physically derived
from any existing or natural sequence but may be generated in any
manner, including chemical synthesis, DNA replication, reverse
transcription or a combination thereof. The terms "oligonucleotide"
or "nucleic acid" intend a polynucleotide of genomic DNA or RNA,
cDNA, semisynthetic, or synthetic origin which, by virtue of its
origin or manipulation: (1) is not associated with all or a portion
of the polynucleotide with which it is associated in nature; and/or
(2) is linked to a polynucleotide other than that to which it is
linked in nature; and (3) is not found in nature.
[0039] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends.
[0040] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points toward the
5' end of the other, the former may be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0041] The term "primer" may refer to more than one primer and
refers to an oligonucleotide, whether occurring naturally, as in a
purified restriction digest, or produced synthetically, which is
capable of acting as a point of initiation of synthesis along a
complementary strand when placed under conditions in which
synthesis of a primer extension product which is complementary to a
nucleic acid strand is catalyzed. Such conditions include the
presence of four different deoxyribonucleoside triphosphates and a
polymerization-inducing agent such as DNA polymerase or reverse
transcriptase, in a suitable buffer ("buffer" includes substituents
which are cofactors, or which affect pH, ionic strength, etc.), and
at a suitable temperature.
[0042] The primer is preferably single stranded for maximum
efficiency in amplification, but may alternatively be double
stranded. If double stranded, the primer is first treated to
separate its strands before being used to prepare extension
products. Preferably, the primer is an oligodeoxyribonucleotide.
The primer must be sufficiently long to prime the synthesis of
extension products in the presence of the agent for polymerization.
The exact lengths of the primers will depend on many factors,
including temperature and source of primer and use of the method.
For example, depending on the complexity of the target sequence,
the oligonucleotide primer typically contains 15-25 nucleotides,
although it may contain more or fewer nucleotides. Short primer
molecules generally require lower temperatures to form sufficiently
stable hybrid complexes with the template.
[0043] The term "extension primer" means a polynucleotide sequence
that is complementary to a template sequence, and which is capable
of hybridizing and extending a sequence under polymerase chain
reaction conditions.
[0044] The term "complement" and its related adjectival form
"complementary," when used in reference to two nucleic acid
sequences, means that when two nucleic acid sequences are aligned
in anti-parallel association (with the 5' end of one sequence
paired with the 3' end of the other sequence) the corresponding G
and C nucleotide bases of the sequences are paired, and the
corresponding A and T nucleotide bases are paired. Certain bases
not commonly found in natural nucleic acids may be included in the
nucleic acids of the present invention and include, for example,
inosine and 7-deazaguanine.
[0045] The term "location" or "position" of a nucleotide in a
genetic locus means the number assigned to the nucleotide in the
gene, generally taken from the CDNA sequence of the genomic
sequence of a gene.
[0046] The term "oligonucleotide primer" means a molecule comprised
of more than three deoxyribonucleotides or ribonucleotides. Its
exact length will depend on many factors relating to the ultimate
function and use of the oligonucleotide primer, including
temperature of the annealing reaction, and the source and
composition of the primer. Amplification primers must be
sufficiently long to prime the synthesis of extension products in
the presence of the agent for polymerization. The oligonucleotide
primer is capable of acting as an initiation point for synthesis
when placed under conditions which induce synthesis of a primer
extension product complementary to a nucleic acid strand. The
conditions can include the presence of nucleotides and an inducing
agent such as a DNA polymerase at a suitable temperature and pH. In
preferred embodiments, the primer is a single-stranded
oligodeoxyribonucleotide of sufficient length to prime the
synthesis of an extension product from a specific sequence in the
presence of an inducing agent. In one aspect of the present
invention, the oligonucleotide primers are from about 15 to about
30 nucleotides long, although a primer may contain more or fewer
nucleotides. The oligonucleotide primers are preferably at least
15, 16, 17, 18, 19 or 20 nucleotides long. More preferably, primers
will contain around 20-25 nucleotides. Sensitivity and specificity
of the oligonucleotide primers are determined by the primer length
and uniqueness of sequence within a given sample of template
nucleic acid. Primers which are too short, for example, may show
non-specific binding to a wide variety of sequences.
[0047] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA techniques, oligonucleotide synthesis
which are within the skill of the art. Such techniques are
explained fully in the literature. Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications or as commonly accomplished in the art or as
described herein. The foregoing techniques and procedures are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)); Oligonucleotide Synthesis (M. J.
Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S.
J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B.
Perbal, 1984); and a series, Methods in Enzymology (Academic Press,
Inc.), the contents of all of which are incorporated herein by
reference.
[0048] The term "reverse transcription" means the process of
generating a DNA complement to an RNA molecule, and is generally
accomplished with the use of a reverse transcriptase enzyme. A
primer may be used to initiate polymerization; this primer may be
one of a primer pair later used for PCR amplification. The RNA
molecule is then separated from the copied DNA ("cDNA") or degraded
by an RNAse H activity of an enzyme thus allowing the second strand
of cDNA to be generated by a template dependent DNA polymerase.
This method is disclosed in Units 3.7 and 15.4 of Current Protocols
in Molecular Biology, Eds. Ausubel, F. M. et al, (John Wiley &
Sons; 1995), the contents of which are incorporated herein by
reference.
[0049] The term "sequencing" means the determination of the order
of nucleotides in at least a part of a gene.
[0050] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA techniques, oligonucleotide synthesis
which are within the skill of the art. Such techniques are
explained fully in the literature. Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications or as commonly accomplished in the art or as
described herein. The foregoing techniques and procedures are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)); Oligonucleotide Synthesis (M. J.
Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S.
J. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B.
Perbal, 1984); and a series, Methods in Enzymology (Academic Press,
Inc.), the contents of all of which are incorporated herein by
reference.
[0051] Source of DNA
[0052] In one aspect of the invention, the method comprises first
obtaining from the patient sample a double-stranded polynucleotide
template encompassing the mutation of interest. The double-stranded
polynucleotide template may initially comprise genomic DNA, a
fragment of genomic DNA, or cDNA reverse transcribed from RNA. This
template will encompass not only the mutation of interest, but may
also encompass the region containing the length polymorphism giving
rise to multiple quasispecies.
[0053] A double-stranded polynucleotide template will typically be
prepared from a patient sample by treating a patient sample
containing DNA so as to make all or a portion of the DNA in the
sample accessible for hybridization with oligonucleotide primers,
for example by lysis, centrifugation to remove cellular debris and
proteolytic digestion to expose the DNA. The DNA template may
therefore contain only nuclear DNA, only mitochondrial DNA, or some
sub-fraction of nuclear or mitochondrial DNA obtained by isolation
from a tissue sample. The DNA template may also be prepared by
conversion, for example by reverse transcription, of a total mRNA
preparation or the genome of an RNA virus to cDNA; DNA isolated
from an individual bacterial colony growing on a plate or from an
enriched bacterial culture; and a viral DNA preparation where
substantially the entire viral genome is isolated.
[0054] DNA can be prepared from fluid samples, e.g., blood or urine
or tissue samples by any of a number of techniques, including
lysis, centrifugation to remove cellular debris and proteolytic
digestion to expose the DNA; salt precipitation or standard
SDS-proteinase K-phenol extraction. Samples can also be prepared
using kits, for example the Pure Gene DNA Isolation Kit
(Gentra).
[0055] Amplification of Nucleic Acids
[0056] The present invention includes methods and reagents for
amplification of DNA to provide an abundant source of DNA for
subsequent sequencing.
[0057] Typically, prior to sequencing, a sequencing template is
prepared by first amplifying a region of DNA that encompasses the
target region to be sequenced. It is not necessary that the
sequence to be amplified be present initially in a pure form; it
may be a minor fraction of a complex mixture or a portion of
nucleic acid sequence. The starting nucleic acid may contain more
than one desired specific nucleic acid sequence which may be the
same or different. Therefore, the present process is useful not
only for producing large amounts of one specific nucleic acid
sequence, but also for amplifying simultaneously more than one
different specific nucleic acid sequence located on the same or
different nucleic acid molecules if more than one of the base pair
variations in sequence is present.
[0058] The present invention is directed to methods and reagents,
including amplification and sequencing primers, used for genotyping
HCV. The present invention utilizes well-known methods for
amplifying specific nucleic acid sequences using the technique of
polymerase chain reaction (or PCR) or some other primer
extension-based methodology. Polymerase chain reaction (PCR)
methods are very widely known in the art. Such methods are
described, for example, in U.S. Pat. Nos. 4,683,195, 4,683,202, and
4,800,159; K. Mullis, Cold Spring Harbor Symp. Quant. Biol.,
51:263-273 (1986); and C. R. Newton & A. Graham, Introduction
to Biotechniques: PCR, 2.sup.nd Ed., Springer-Verlag (New York:
1997), the disclosures of which are incorporated herein by
reference. PCR involves the use of pairs of primers, one for each
complementary strand of the duplex DNA (wherein the coding strand
is referred to as the "sense strand" and its complementary strand
is referred to as the "anti-sense strand), that will hybridize at
sites located on either side of a region of interest in a gene.
Chain extension polymerization is then carried out in repetitive
cycles to increase the number of copies of the region of interest
exponentially. To briefly summarize, in the first step of the PCR
reaction, the nucleic acid molecules of the sample are transiently
heated, and then cooled, in order to denature double stranded
molecules. Forward and reverse primers are present in the
amplification reaction mixture at an excess concentration relative
to the sample target. When the sample is incubated under conditions
conducive to hybridization and polymerization, the primers
hybridize to the complementary strand of the nucleic acid molecule
at a position 3' to the sequence of the region desired to be
amplified that is the complement of the sequence whose
amplification is desired. Upon hybridization, the 3' ends of the
primers are extended by the polymerase. The extension of the primer
results in the synthesis of a DNA molecule having the exact
sequence of the complement of the desired nucleic acid sample
target. The PCR reaction is capable of exponentially amplifying the
desired nucleic acid sequences, with a near doubling of the number
of molecules having the desired sequence in each cycle. Thus, by
permitting cycles of hybridization, polymerization, and
denaturation, an exponential increase in the concentration of the
desired nucleic acid molecule can be achieved. The amplified
polynucleotide may be used as the template for a sequencing
reaction. Gelfand et al. have described a thermostable enzyme, "Taq
polymerase", derived from the organism Thermus aquaticus, which is
useful in this amplification process (see U.S. Pat. Nos. 4,889,818;
5,352,600; and 5,079,352 which are incorporated herein by
reference). Alternative amplification techniques such as NASBA,
3SR, Qb Replicase, and Branched Chain Amplification are known and
available to persons skilled in the art. The term "RT-PCR" refers
generally to amplification which includes a reverse transcription
step to permit amplification of RNA sequences.
[0059] Preparation of Polynucleotide Amplification Templates
[0060] The present invention relates to amplification and
sequencing of HCV and its variant forms. The method of the present
invention may employ, for example, DNA or RNA, including messenger
RNA, which DNA or RNA may be single stranded or double stranded. In
addition, a DNA-RNA hybrid which contains one strand of each may be
utilized. A mixture of any of these nucleic acids may also be
employed, or the nucleic acids produced from a previous
amplification reaction herein using the same or different primers
may be so utilized. The specific nucleic acid sequence to be
amplified may be only a fraction of a larger molecule or can be
present initially as a discrete molecule, so that the specific
sequence constitutes the entire nucleic acid.
[0061] It is not necessary that the sequence to be amplified be
present initially in a pure form; it may be a minor fraction of a
complex mixture or a portion of nucleic acid sequence. The starting
nucleic acid may contain more than one desired specific nucleic
acid sequence which may be the same or different. Therefore, the
present process is useful not only for producing large amounts of
one specific nucleic acid sequence, but also for amplifying
simultaneously more than one different specific nucleic acid
sequence located on the same or different nucleic acid molecules if
more than one of the base pair variations in sequence is
present.
[0062] The nucleic acid templates may be obtained from any source,
for example, from plasmids such as pBR322, from cloned DNA or RNA,
or from natural DNA or RNA from any source. DNA or RNA may be
extracted from blood, tissue material or amniotic cells by a
variety of techniques such as that described by Maniatis et al.,
Molecular Cloning (1982), 280-281.
[0063] The cells may be directly used without purification of the
nucleic acid if they are suspended in hypotonic buffer and heated
to about 90.degree.-100.degree. C., until cell lysis and dispersion
of intracellular components occur, generally about 1 to 15 minutes.
After the heating step the amplification reagents may be added
directly to the lysed cells. This direct cell detection method may
be used on peripheral blood lymphocytes and amniocytes.
[0064] The target nucleic acid contained in the sample will
initially be in the form of RNA, and is preferably reverse
transcribed into cDNA, and then denatured, using any suitable
denaturing method, including physical, chemical, or enzymatic
means, which are known to those of skill in the art. A preferred
physical means for strand separation involves heating the nucleic
acid until it is completely (>99%) denatured. Typical heat
denaturation involves temperatures ranging from about 80.degree. C.
to about 105.degree. C., for times ranging from a few seconds to
minutes. As an alternative to denaturation, the target nucleic acid
may exist in a single-stranded form in the sample, such as, for
example, single-stranded RNA or DNA viruses.
[0065] The denatured nucleic acid strands are then incubated with
preselected oligonucleotide primers, and, optionally, a labeled
oligonucleotide (referred to herein as a "probe") for purposes of
detecting the amplified sequence) under conditions that facilitate
the binding of the primers and probes to the single nucleic acid
strands. As known in the art, the primers are selected so that
their relative positions along a duplex sequence are such that an
extension product synthesized from one primer, when the extension
product is separated from its template (complement), serves as a
template for the extension of the other primer to yield a replicate
chain of defined length.
[0066] Amplification of HCV
[0067] One aspect of the present invention relates to a method for
determining the genotype of a hepatitis C virus (HCV) species
present in a test sample by amplifying at least a portion of the
NS5B region of HCV that, for each of a plurality of HCV species, is
indicative of the type and/or subtype of that species.
[0068] In one embodiment, the method comprises (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
(for example, forward primers FF1 and/or FF2), 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 (for example, reverse primers RR1) of SEQ ID NO: 1, or
its complement; and (b) amplifying the nucleotide sequence of said
portion of the NS5b region.
[0069] In a particular embodiment, 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 (for example, CLIP sequencing
primers M-NS5b-Cy5.5), or its complement; and degenerate nucleotide
sequences complementary to the NS5b region of a plurality of HCV
species from nucleotide 8611 to 8633 (for example, CLIP sequencing
primers M-NS5b-Cy5) of SEQ ID NO: 1, or its complement.
[0070] In another particular embodiment, the mixture of degenerate
oligonucleotide PCR primers comprise degenerate oligonucleotide
sequences defined by one or more of the following formulas, or
complements thereof: TABLE-US-00004 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'
[0071] In yet another particular embodiment, the mixture of
degenerate oligonucleotide PCR primers comprise degenerate
oligonucleotide sequences defined by one or more of the following
formulas, or complements thereof: TABLE-US-00005 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'
[0072] Sequencing of Nucleic Acids
[0073] Amplification of DNA as described above will result in an
abundant source of DNA for sequencing. The polynucleotide templates
prepared as described above are sequenced using any of the numerous
methods available and known to those in the art for sequencing
nucleotides.
[0074] Numerous methods are available and known to those in the art
for sequencing nucleotides, any of which may be used in the method
of the present invention. One well known method of sequencing is
the "chain termination" method first described by Sanger et al.,
PNAS (USA) 74(12): 5463-5467 (1977) and detailed in Sequenase.RTM.
2.0 product literature (Amersham Life Sciences, Cleveland) and more
recently elaborated in European Patent EP-B1-655506, the content of
which are all incorporated herein by reference. In this process,
DNA to be sequenced is isolated, rendered single stranded, and
placed into four vessels. In each vessel are the necessary
components to replicate the DNA strand, which include a
template-dependent DNA polymerase, a short primer molecule
complementary to the initiation site of sequencing of the DNA to be
sequenced and deoxyribonucleotide triphosphates for each of the
bases A, C, G and T, in a buffer conducive to hybridization between
the primer and the DNA to be sequenced and chain extension of the
hybridized primer. In addition, each vessel contains a small
quantity of one type of dideoxynucleotide triphosphate, e.g.
dideoxyadenosine triphosphate ("ddA"), dideoxyguanosine
triphosphate ("ddG"), dideoxycytosine triphosphate ("ddC"),
dideoxythymidine triphosphate ("ddT"). In each vessel, each piece
of the isolated DNA is hybridized with a primer. The primers are
then extended, one base at a time to form a new nucleic acid
polymer complementary to the template DNA. When a dideoxynucleotide
is incorporated into the extending polymer, the polymer is
prevented from further extension. Accordingly, in each vessel, a
set of extended polymers of specific lengths are formed which are
indicative of the positions of the nucleotide corresponding to the
dideoxynucleotide in that vessel. These sets of polymers are then
evaluated using gel electrophoresis to determine the sequence.
[0075] Sequencing of polynucleotides may be performed using either
single-stranded or double stranded DNA. Use of polymerase for
primer extension requires a single-stranded DNA template. In
preferred embodiments, the method of the present invention uses
double-stranded DNA in order to obtain confirmatory opposite strand
confirmation of sequencing results. Double stranded DNA templates
may be sequenced using either alkaline or heat denaturation to
separate the two complementary DNA templates into single strands.
During polymerization, each molecule of the DNA template is copied
once as the complementary primer-extended strand. Use of
thermostable DNA polymerases (e.g. Taq, Bst, Tth or Vent DNA
polymerase) enables repeated cycling of double-stranded DNA
templates in the sequencing reaction through alternate periods of
heat denaturation, primer annealing, extension and dideoxy
termination. This cycling process effectively amplifies small
amounts of input DNA template to generate sufficient template for
sequencing.
[0076] Sequencing may also be performed directly on PCR
amplification reaction products. Although the cloning of amplified
DNA is relatively straightforward, direct sequencing of PCR
products facilitates and speeds the acquisition of sequence
information. As long as the PCR reaction produces a discrete
amplified product, it will be amenable to direct sequencing. In
contrast to methods where the PCR product is cloned and a single
clone is sequenced, the approach in which the sequence of PCR
products is analysed directly is generally unaffected by the
comparatively high error rate of Taq DNA polymerase. Errors are
likely to be stochastically distributed throughout the molecule.
Thus, the overwhelming majority of the amplified product will
consist of the correct sequence. Direct sequencing of PCR products
has the advantage over sequencing cloned PCR products in that (1)
it is readily standardized because it is simple enzymatic process
that does not depend on the use of living cells, and (2) only a
single sequence needs to be determined for each sample.
[0077] The amplification methods used in the present invention may
also be simultaneously used in conjunction with sequencing. Methods
for simultaneous amplification and sequencing are widely known in
the art, and include coupled amplification and sequence (CAS)
(described by Ruano and Kidd, Proc. Nat'l. Acad. Sci. (USA) 88(7):
2815-2819 (1991), and in U.S. Pat. No. 5,427,911, which are
incorporated herein by reference), and CLIP amplification and
sequencing (described in U.S. Pat. No. 6,007,983, and in J. Clin.
Microbiology 41(4); 1586-1593 (April 2003) which are incorporated
herein by reference). CLIP sequencing subjects PCR amplification
fragments previously generated to simultaneous PCR amplification
and direct sequencing. In CAS sequencing, a sample is treated in a
first reaction stage with two primers and amplified for a number of
cycles to achieve 10,000 to 100,000-fold amplification. A ddNTP is
then added during the exponential phase of the amplification
reaction, and the reaction is processed for additional thermal
cycles to produce chain-terminated sequencing fragments. The CAS
process requires an intermediate addition of reagents (the ddNTP
reagents), which introduces opportunity for error or contamination
and increases the complexity of any apparatus which would be used
for automation. The CAS methodology is therefore preferably
combined with CLIP sequencing, which subjects PCR amplification
fragments previously generated to simultaneous PCR amplification
and direct sequencing. Simultaneous amplification and sequencing
using the CLIP.RTM. method may be accomplished, for example, using
the reagents described herein, under conditions similar to those
described in commercially available kits, such as the TRUGENE.RTM.
HCV genotyping kit (Bayer HealthCare LLC).
[0078] In particular aspects, the present invention relates to
sequencing of HCV. The double stranded DNA template used in the
method of the present invention may be derived from, for example,
DNA or RNA, including messenger RNA, which may be single stranded
or double stranded. In addition, the DNA template may be in the
form of a DNA-RNA hybrid which contains one strand of DNA and one
strand of RNA. A mixture of any of these nucleic acids may also be
employed, or the nucleic acids produced from a previous
amplification reaction herein using the same or different primers
may be so utilized. The specific nucleic acid sequence to be
amplified may be only a fraction of a larger molecule or can be
present initially as a discrete molecule, so that the specific
sequence constitutes the entire nucleic acid.
[0079] Genotyping HCV
[0080] The present invention includes a novel method and reagents
for genotyping HCV in a sample suspected of containing or known to
contain HCV.
[0081] The present invention addresses the above-mentioned problem,
by providing primers that encompass a region of HCV.
[0082] The sequencing primers of the present invention consist of
oligonucleotides specific to the NS5b region of HCV, which can be
used to amplify and sequence a portion of HCV. In accordance with
methods known to those in the art, a sample obtained from an
individual suspected of being infected with HCV is used to recover
viral RNA, either in the form of RNA or DNA. Viral HCV RNA obtained
from the sample is reverse transcribed to cDNA. The cDNA template
is then amplified, using Polymerase Chain Reaction or some other
primer extension based method. The resulting amplified fragment is
then initially sequenced with a set of primers the encompass the
desired NS5b region of HCV using cycle sequencing methods or
CLIP.TM. bi-directional sequencing.
[0083] One particular aspect of the present invention is a method
for amplifying and genotyping a portion of the NS5b region of HCV
in a sample suspected of containing HCV.
[0084] Sequencing of HCV
[0085] The present invention is generally directed to improved
methods and reagents for genotyping a hepatitis C virus (HCV)
species found in a test sample. In certain aspects of the
invention, the invention relates to a method of sequencing a
portion of the NS5B region of an HCV species in a sample and
determining its genotype.
[0086] Generally, the invention relates to a method for determining
the genotype of a hepatitis C virus (HCV) species present in a test
sample by sequencing at least a portion of the NS5B region of HCV
that, for each of a plurality of HCV species, is indicative of the
type and/or subtype of that species.
[0087] The present invention also includes a method and primers for
determining the sequence of the NS5b region of HCV, or a portion
thereof. The sequencing primers of the present invention include
both forward primers and reverse primers, which may be labeled with
a detectable label. For most common sequencing instruments, a
fluorescent label is desirable, although other labels types
including colored, chromogenic, fluorogenic (including
chemiluminescent) and radiolabels could also be employed. The
primer combination may include other reagents appropriate for
reverse transcription, amplification or sequencing, and may, of
course, include HCV genetic material for analysis.
[0088] Although the sequencing primers of the present invention
disclosed below are preferably selected from among primers having
the same sequence as disclosed below, it is contemplated that the
present invention includes degenerate sequences having the
equivalent specificity and function, which may be designed and
constructed in accordance with the skill in the art. Specifically,
the sequencing primers include fragments of the above primers of 15
or more nucleotides. The sequencing primers may also include
fragments of the above primers having 16, 17, 18, 19, or 20 or more
nucleotides. The design and construction of such degenerate
sequences is well know to those in the art.
[0089] Because sequencing primers, as opposed to amplification
primers, may not be mixed together if they do not have the same
location for the 3' base, only specific degenerate base positions
are illustrated below, although it is to be understood that the 3'
location may also be modified. For example, 5' length may be
changed to include regions of greater sequence conservation or to
modify melting temperature and stringency of binding. A
nondegenerate primer set is preferred if the success rate is found
to be sufficient with the primary primers. Reaction conditions may
also significantly affect performance. The potential modifications
of sequencing primers are illustrated in the following sections and
examples.
[0090] In one embodiment, the method of the invention includes
first determining the nucleotide sequence of at least a portion of
the NS5b region of HCV indicative of the genotype of said HCV
species. The portion of the NS5b region of HCV that is indicative
of its genotype will be the same region that corresponds to the
nucleotide sequence of a plurality of HCV species that is
indicative of the genotype of each of the plurality of HCV species.
Thus, one aspect of the invention is the identification of a region
that is common among HCV species that is indicative of the type
and/or subtype of the particular HCV species. Sequencing of this
region of any HCV species thus enables determination of the type
and subtype of that species, by correlating the nucleotide sequence
of said portion of the NS5b region determined in the above step
with the genotype of one of a plurality of HCV species of known
sequence/genotype.
[0091] In another embodiment, the method comprises (a) determining
the nucleotide sequence of at least a portion of the NS5b region of
HCV indicative of the genotype of said HCV species, wherein the
corresponding nucleotide sequence of each HCV species having
genotypes 1, 2, 3, 4, 5, and 6 is indicative of the genotype of
said species; and (b) correlating the nucleotide sequence of said
portion of the NS5b region determined in (a) with one of HCV
genotypes 1, 2, 3, 4, 5 and 6.
[0092] In yet another embodiment, the method comprises (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, wherein the corresponding nucleotide sequence of each
HCV species having genotypes 1, 2, 3, 4, 5, and 6, and each of the
subtypes set forth in Table 1, is indicative of the genotype and
subtype of said species; and (b) correlating the nucleotide
sequence of said portion of the NS5b region determined in (a) with
one of HCV genotypes 1, 2, 3, 4, 5 and 6 and one of the subtypes
set forth in Table 1.
[0093] In one particular embodiment of the above methods, 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.
[0094] In another particular embodiment of the above methods, the
portion of the NS5b region of HCV consists essentially of the
region from nucleotide position 8344 to 8547 of SEQ ID NO: 1.
[0095] In another embodiment, the method of present invention
comprises (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,
and wherein the corresponding nucleotide sequence of each of said
plurality of HCV species is indicative of the genotype of said
species; (b) determining the nucleotide sequence of said portion of
the NS5b region indicative of the genotype of said species; 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.
[0096] In yet another embodiment, the method comprises (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 species; 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.
[0097] In still another embodiment, the method comprises (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 set forth in Table 1; (b) determining the
nucleotide sequence of said portion of the NS5b region indicative
of the genotype of said species; 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 (for example,
one of HCV genotypes 1, 2, 3, 4, 5 and 6 and one of the subtypes
set forth in Table 1).
[0098] In one particular embodiment, 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 (for example, CLIP
sequencing primer M-NS5b-Cy5.5), 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
(for example, CLIP sequencing primer M-NS5b-Cy5) of SEQ ID NO: 1,
or its complement.
[0099] In another particular embodiment, 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 (for example, CLIP sequencing
primer M-NS5b-Cy5.5), or its complement, and degenerate nucleotide
sequences complementary to the NS5b region of a plurality of HCV
species from nucleotide 8611 to 8633 (for example, CLIP sequencing
primer M-NS5b-Cy5) of SEQ ID NO: 1, or its complement.
[0100] In yet another particular embodiment, the mixture of
degenerate oligonucleotide sequencing primers comprise degenerate
oligonucleotide sequences defined by one or more of the following
formulas, or complements thereof: TABLE-US-00006 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'.
[0101] Other variations of the above sequences may be utilized to
permit detection of other HCV variants.
EXAMPLE 1
HCV NS5b Genotyping by Sequencing
[0102] This following example describes a laboratory protocol to
produce bi-directional sequence of a 204 base pair fragment in the
NS5b region of Hepatitis C virus for the purpose of determining
genotype and subtype.
[0103] Generally, viral HCV RNA is extracted from the plasma sample
using a Qiagen QIAamp Viral RNA Mini Kit as described by the
manufacturer. Briefly, the extracted RNA specimens are reverse
transcribed using random hexamers.
[0104] Following cDNA synthesis, 10 .mu.L of the cDNA template is
amplified using specific primers to generate a 398 base pair
amplicon.
[0105] Following PCR amplification, a dye-primer CLIP sequencing
reaction is performed. The CLIP reaction sequences both strands of
the DNA simultaneously by using forward (sense) and reverse
(antisense) primers each labeled with different fluorescent dyes
(Cy 5.5, Cy 5; chain-extension reagents, and one of four
chain-terminating dideoxynucleotide triphosphates (ddNTPs):
dideoxyadenosine (ddATP), dideoxycytidine (ddCTP), dideoxyguanosine
(ddGTP), or dideoxythymidine (ddTTP) triphosphates. The reaction is
initiated with the addition of the sample and a thermostable DNA
polymerase with a high affinity for ddNTPs. As the reaction mixture
is thermally cycled, primers hybridize to template DNA and are
extended, then usually terminated somewhere along the target DNA
sequence. Four CLIP reactions yield both the forward and reverse
sequence of the target between the two CLIP primers. The reaction
proceeds through 40 cycles generating high levels of chain
terminated reaction products from each primer. Upon completion of
the cycling program, Stop Loading Dye solution is added to each
reaction tube and the reactions are heated to separate the
double-stranded DNA fragments. A fraction of each reaction is then
loaded onto the top of a MicroCel.TM. 500 cassette containing an
ultra thin vertical polymerized polyacrylamide gel that has a
formed matrix of specific pore size. The polyacrylamide gel
contains a high concentration of urea to maintain the DNA fragments
in a single-stranded denatured state. A buffered solution maintains
contact with both the top and bottom of the ultra thin gel. A high
voltage electric field is applied, forcing the negatively charged
fragments of DNA to migrate through the gel towards the anode. The
speed of migration of the DNA fragments is related to the size of
pores formed by the polyacrylamide matrix and DNA fragment size,
with the smaller fragments migrating faster. Near the bottom of the
polyacrylamide gel, a laser beam excites the fluorescent dye linked
to the DNA fragments moving past the laser, and detectors measure
the amount of light and wavelength produced by the fluorescent dye.
This light measurement is then collected by the sequencer and
transmitted to a workstation that stores the data. Each sequencing
reaction requires four lanes, one for each of the four chain
terminating dideoxynucleotides. (ddATP, ddCTP, ddGTP, ddTTP). The
forward and reverse CLIP sequences are combined and compared to the
sequence of a "best matched" reference sequence (W). Operators
review the alignment at flagged positions and edit the bases as
necessary. The software prepares an HCV NS5b report for each
sample.
[0106] Reverse Transcription (cDNA Synthesis)
[0107] cDNA is synthesized from genomic RNA derived from the sample
in accordance with the following protocol.
[0108] RT reagents described below (except SuperScript and RNase
inhibitor) are vortexed and microcentrifuged to recover volume. The
HCV NS5b RT Master Mix is prepared, using volumes calculated
according to the following formula: TABLE-US-00007 HCV NS5b RT
Master Mix RT Reagent Final Conc. Volume/sample Nuclease free water
3.90 .mu.L 10X PCR Buffer II 1X 2.50 .mu.L MgCl2 Solution (25 mM) 5
mM 5.00 .mu.L dNTP's (100 mM) 8 mM 2.00 .mu.L Random Hexamers 60
pg/.mu.L 1.00 .mu.L RNase Inhibitor (20 U/.mu.L) 0.4 U/.mu.L 0.50
.mu.L SuperScript III Reverse 0.8 U/.mu.L 0.10 .mu.L Transcriptase
(200 U/.mu.L) TOTAL RT reagent 15.00 .mu.L
[0109] RT reagents (15 .mu.L) are aliquoted into each labeled PCR
tube. Tubes are transferred to post amplification area dead air
hood. Sample and control RNA extracts are removed from freezer to
thaw at room temperature. Thawed RNA extract (10 .mu.L) is added to
the appropriate tube containing the RT reagent. The reagent and RNA
are mixed by gently tapping tray.
[0110] The tubes are placed in a thermocycler programmed to perform
the following steps: TABLE-US-00008 HCV NS5b RT Program # Cycles
Min Temp .degree. C. Process 1 10 25 Random Hexamer annealing 1 30
50 Reverse Transcription 1 15 75 SuperScript de-activation 1
Forever 4 Hold
[0111] RT samples are removed from the thermocycler and stored at
4.degree. C.
[0112] PCR Amplification
[0113] Amplification by polymerase chain reaction (PCR) is
performed as follows. PCR tubes are prepared and labeled for
samples. PCR reagents described below (except AmpliTaq Gold enzyme)
are vortexed and microcentrifuged to recover volume. Mixtures of
degenerate primers, which are designed to amplify multiple
clinically relevant HCV species, are prepared, having the following
nucleotide sequences:
[0114] Forward PCR Primer (FF-1): 5'-TGG GGT TCK CGT ATG AYA CCC
GCT G-3' (SEQ ID NO:7)
[0115] Forward PCR Primer (FF-2): 5'-TGG GGT TCK CIT ATG AYA CYM
GIT G-3' (SEQ ID NO:8)
[0116] Reverse PCR Primer (RR1): 5'-GAR TAY CTV GTC ATR GCI TCY GTR
AA-3' (SEQ ID NO:9)
[0117] A PCR Master Mix is prepared using the worksheet for reagent
volumes. Volumes are calculated according to the following formula:
TABLE-US-00009 HCV NS5b PCR Mastermix PCR reagent Final
Concentration Volume per sample Nuclease free water 23.25 .mu.L 10X
PCR Buffer II 0.8 X 4.00 .mu.L 25 mM MgCL2 Solution 1 mM 2.00 .mu.L
M-NS5B-FF1 (10 .mu.M) 0.3 .mu.M 1.50 .mu.L M-NS5B-FF2 (10 .mu.M)
0.6 uM 3.00 uL M-NS5B-RR1 (10 .mu.M) 1.2 .mu.M 6.00 .mu.L AmpliTaq
Gold (5 U/.mu.L) 0.025 U/.mu.L 0.25 .mu.L TOTAL PCR reagent 40.00
.mu.L
[0118] The PCR Master Mix (40 .mu.L) is aliquoted into each labeled
PCR tube. RT cDNA (10 .mu.L) is added to appropriately labeled
tubes containing PCR reagent, and tubes are placed in a
thermocycler programmed to perform the following steps:
TABLE-US-00010 HCV NS5b PCR Program # cycles Time Temp .degree. C.
Process 1 10 min 95 AmpliTaq activation 45 30 sec 94 Denaturation
30 sec 48 Annealing 1 min 68 Extension 1 10 min 68 Final Extension
1 Forever 4 Hold
[0119] PCR samples are removed from the thermocycler, and may be
stored at 4.degree. C. for up to two weeks or subjected to CLIP
sequencing, as described below.
[0120] CLIP Sequencing
[0121] Mixtures of degenerate sequencing primers, which are
designed to sequence multiple clinically relevant HCV species, are
prepared, having the following nucleotide sequences: TABLE-US-00011
Forward CLIP Primer: (SEQ ID NO:10) 5'Cy5.5-5'TAT GAY ACC CGC TGY
TTY GAY TC-3' Reverse CLIP Primer: (SEQ ID NO:11) Cy5-5'-VGT CAT
RGC ITC YGT RAA GGC TC-3'
[0122] The necessary number of PCR strip tubes and caps is
assembled in a tray and place in a cold block. A 0.5 mL tube is
labeled for each sample and control and place in cold block. CLIP
reagents (except Thermo Sequenase enzyme) are removed from the
freezer, and thawed at room temperature. Each component (except
Thermo Sequenase enzyme) is vortexed and and quick spin to recover
volume. Using a 10 .mu.L adjustable pipet, 3 .mu.L of the
appropriate CLIP terminator mixes is transferred directly to the
bottom of the respective column of wells in each of the PCR
tubes.
[0123] A 1:10 dilution of Thermo Sequenase enzyme in Thermo
Sequenase Dilution Buffer is made. The CLIP Master Mix is prepared
using reagent volumes calculated according to the following
formula: TABLE-US-00012 HCV NS5b CLIP Master Mix Final Volume CLIP
reagent Concentration per sample Nuclease free water 6.50 .mu.L
7-Deaza-dGTP Cy5/Cy5.5 Dye Primer 1X 2.50 .mu.L Cycle Sequencing
Kit (100 tests) Thermo Sequenase Dilution Buffer (520 .mu.L) Thermo
Sequenase Enzyme (57 .mu.L) Sequencing Buffer (320 .mu.L) A
Termination Mix (375 .mu.L) C Termination Mix (375 .mu.L) G
Termination Mix (375 .mu.L) T Termination Mix (375 .mu.L) Stop
Loading Dye (2750 .mu.L) M-NS5B Forward CLIP Primer 0.4 .mu.M 2.50
.mu.L M-NS5B Reverse CLIP Primer 0.1 .mu.M 2.50 .mu.L DMSO 8% 2.00
.mu.L Themo Sequenase enzyme 1:10 dilution 0.512 U/.mu.L 4.00 .mu.L
TOTAL CLIP reagent 20.00 .mu.L
[0124] 20 .mu.L CLIP Master Mix are aliquoted into each labeled
tube and transferred to tubes containing CLIP reagents. The PCR
amplicon (2 .mu.L) is added to appropriate tubes containing the
CLIP Master Mix, vortex briefly and microcentrifuged to collect
volume at bottom of tube. The CLIP mix (5 .mu.L) is added to each
of the 4 terminator tubes in the cold block
[0125] The CLIP amplification/sequencing reaction is performed on a
thermocycler in accordance with the following protocol:
TABLE-US-00013 HCV NS5b CLIP # Cycles Time Temp .degree. C. Process
1 2 min 94 Initial denaturation 25 20 sec 94 Denaturation 45 sec 45
Annealing 1 min 68 Extension 15 25 sec 94 Denaturation 2 min 70
Annealing and extension 1 2 min 72 Final extension 1 forever 4
Hold
[0126] Tubes are removed from the thermocycler and add Stop Loading
Dye (6 .mu.L) is added to each tube, and vortexed to mix. The tubes
are returned to the thermocycler and subjected to denature
conditions, as follows: TABLE-US-00014 HCV NS5b Denature # Cycles
Time Temp .degree. C. Process 1 2 min 80 Denaturation 1 Forever 4
Hold
[0127] Tubes are remove from the thermocycler and stored or
subjected to gel electrophoresis, as described below.
[0128] Gel Electrophoresis is performed using a Long-Read Tower
Sequencer, as described by the manufacturer, using the following
sequencer control settings: TABLE-US-00015 Gel Temperature
(.degree. C.) 60.degree. C. Gel Voltage (V) 1800 V Laser Power (%)
50% Run Clock (Sampling Interval) 0.5 sec Run Clock (Run Duration)
50 min
[0129] Assays are assigned with sample and control information.
EXAMPLE 2
Evaluation of Performance Characteristics of HCV NS5b Genotyping by
Sequencing
[0130] The performance characteristics of HCV NS5b genotyping assay
were evaluated, based on genotyping assays substantially as
described above in Example 1, using the following reagents.
TABLE-US-00016 HCV NS5b Specific Reagents Description Lot #
M-NS5b-FF1 Oct. 27, 2004 M-NS5b-FF2 Dec. 27, 2004 M-NS5b-RR1 Oct.
27, 2004 M-NS5bSes-Cy5.5 Oct. 27, 2004 M-NS5bSeq-Cy5 Oct. 27,
2004
[0131] TABLE-US-00017 General Purpose Reagent's Description Lot #
PCR Buffer II E10896 25 mM MgCl2 E10900 dNTP 100 mM 36227107050
Random Hexamers F06662 RNase Inhibitor F07872 SuperScript III RT
1232439 AmpliTaq Gold E11258 DMSO R19030 SureFill 6% Sequencing Gel
0294B94 MicroCel 500 2624 Cy5.5/5 Cycle Sequencing Kit containing
the 01609 following: Sequencing Buffer ThermoSequenase Enzyme
ThermoSequenase Dilution Buffer
[0132] The following samples were used: TABLE-US-00018 Samples HCV
Genotype (LiPA HCV Genotype HCV TMA (TRUGENE Genotype Viral Load
LiPA) 5'NC) (NS5B) Specimen ID (c/mL) Vendor 1 Acrometrix 1
Acrometrix 2b Acrometrix 2 Acrometrix 3a Acrometrix 3 Acrometrix 4a
Acrometrix 4 Acrometrix 5a Acrometrix 5 Acrometrix 6a Acrometrix 6
Acrometrix 1a 1a 1 MP 1 Millenium 1a 1a 1a MP 2 Millenium 1a 1a 1a
MP 3 Millenium 1a 1a 1a MP 4 Millenium 1a 1a 1a MP 5 Millenium 1b
1b 1b MP 6 Millenium 1b 1b 1b MP 7 Millenium 1b 1b 1b MP 8
Millenium 1b 1b 1 MP 9 Millenium 1b 1b 1b MP 10 Millenium 2 2b 2 2b
MP 11 Millenium 2 2b 2 2b MP 12 Millenium 2 2b 2 2b MP 13 Millenium
2 2b 2 2b MP 14 Millenium 2 2b 2 2b MP 15 Millenium 3a 3a 3a MP 16
Millenium 3a 3a 3a MP 17 Millenium 3a 3a 3d MP 18 Millenium 1a 1a
1a MP 19 Millenium 4 4 4a MP 20 Millenium 4c/4d 4c/4d 4a MP 21
Millenium 3a 3a 3a MP 22 Millenium 4h 4 4a MP 23 Millenium 5a 5a 5a
MP 24 Millenium 5a 5a 5a MP 25 Millenium 1A GP1A-C2 10,000 Bayer
Clinical Affairs 1B GP1B-A2 10,000 Bayer Clinical Affairs 2A
9810067 2,292,000 Bayer Clinical Affairs 2A GP2A-A2 10,000 Bayer
Clinical Affairs 2A GP2A-B2 10,000 Bayer Clinical Affairs 2A
GP2A-C2 10,000 Bayer Clinical Affairs 2B GP2B-A2 10,000 Bayer
Clinical Affairs 3A 3A 3-3A 30,043,850 Bayer Clinical Affairs 4A
GP4A-A2 10,000 Bayer Clinical Affairs 4A GP4A-B2 10,000 Bayer
Clinical Affairs 4A GP4A-C2 10,000 Bayer Clinical Affairs 5A
(Sequetech) 1-5A 9,745,338 Bayer Clinical Affairs 6A GP6A-A2 10,000
Bayer Clinical Affairs 6A GP6A-A3 5,000 Bayer Clinical Affairs 6A
2-6A (24485) 11,168,451 Bayer Clinical Affairs 6A (Sequetech)
GP6A-C2 10,000 Bayer Clinical Affairs 6A (Sequetech) GP6A-C3 5,000
Bayer Clinical Affairs 4A 14682 1,326,000 Teragenix 5A 15038
2,522,000 Teragenix 6A 20759 8,112,000 Teragenix 7C HCVGTP-004c #1
17,628,000 Teragenix 9B HCVGTP-004c #2 10,660,000 Teragenix 8C
HCVGTP-004c #3 45,240,000 Teragenix 6B HCVGTP-004c #4 1,237,000
Teragenix 7C HCVGTP-004c #5 31,824,000 Teragenix 10A HCVGTP-004a #1
7,852,000 Teragenix 8C HCVGTP-004a #2 25,584,000 Teragenix 10A
HCVGTP-004a #3 19,032 Teragenix 9B HCVGTP-004a #4 14,248,000
Teragenix neg neg M60825 negative diluent Bayer Clinical Affairs
neg neg HCV negative control (Jan. 29, 2004) Bayer Clinical Affairs
neg M60789 negative diluent Bayer Clinical Affairs 1b positive
control Cntrol
[0133] Analytical Accuracy Analysis
[0134] In order to determine the analytical accuracy of the methods
and reagents of the invention, an analysis was performed using a 25
member panel comprising genotypes 1-5, a 6 member panel comprising
genotypes 1-6, a 12 member panel comprising genotypes 4-10, and 15
additional clinical samples comprising genotypes 1-6 were analyzed
and results compared to previous genotype.
[0135] Samples were genotyped using the methodology described in
Example 1, above, and were compared with the genotype of the same
samples, as previously characterized by either LiPA or TruGene 5'NC
genotyping or another NS5b method as detailed in the samples
section, to determine analytical accuracy. The results of this
analysis are summarized in the following table: TABLE-US-00019
Analytical Accuracy Data Expected Genotype BRTL NS5B Sample ID (new
nomenclature) Genotype MP 1 1a 1a MP 2 1a 1a MP 3 1a 1a MP 4 1a 1a
MP 5 1a 1a MP 6 1b 1b MP 7 1b 1b MP 8 1b 1b MP 9 1b 1a MP 10 1b 1b
MP 11 2b 2b MP 12 2b 2b MP 13 2b 2b MP 14 2b 2b MP 15 2b 2b MP 16
3a 3a MP 17 3a UG rpt 3a MP 18 3a 3a MP 19 1a 1a MP 20 4a 4a MP 21
4a 4a MP 22 3a UG (hets) rpt 3a MP 23 4a 4a MP 24 5a 5a MP 25 5a 5a
Acrometrix 1 1b 1a Acrometrix 2 2b 2b Acrometrix 3 3a 3a Acrometrix
4 4 4a Acrometrix 5 5a 5a Acrometrix 6 6a UG HCVGTP-004c #1 7c (6f)
UG HCVGTP-004c #2 9b (6i) 6i HCVGTP-004c #3 8c (6n) 6n HCVGTP-004c
#4 6b 6b HCVGTP-004c #5 7c (6f) UG HCVGTP-004a #1 10a (3k) 3k
HCVGTP-004a #2 8c (6n) 6n HCVGTP-004a #3 10a (3k) 3k HCVGTP-004a #4
9b (6i) 6i 14682 4a 4a 15038 5a 5a 20759 6a UG GP1A-C2 (10,000
c/mL) 1a 1a GP1B-A2 (10,000 c/mL) 1b 1a rpt 1a 9810067 (2,292,000
c/mL) 2a 2a GP2A-A2 (10,000 c/mL) 2a 2a rpt 2a GP2A-B2 (10,000
c/mL) 2a 2a rpt 2a GP2A-C2 (10,000 c/mL) 2a 2a GP2B-A2 (10,000
c/mL) 2b 2b rpt 2b 3-3A (30,043,850 c/mL) 3a 3a GP4A-A2 (10,000
c/mL) 4a 4a GP4A-B2 (10,000 c/mL) 4a 4a 2-6A (24485) (11,168,451
c/mL) 6a UG GP4A-C2 (10,000 c/mL) 4a 4a 1-5A (9,745,338 c/mL) 5a 5a
GP6A-A2 (10,000 c/mL) 6a UG GP6A-C2 (10,000 c/mL) 6a UG
[0136] A total of 58 accuracy samples analyzed yielded 51 NS5b
genotype results. 51 of 51 or 100% of samples genotyp able with
NS5b yielded a genotype concordant with previously determined
genotype at type level. 48 of 51 samples were concordant at both
type and subtype level, and 3 of 51 samples were concordant at type
level only (all 3 were 1b by LiPA and 1a by NS5b). 7 samples (5
genotype 6a and 2 genotype 7c) failed amplification and were unable
to be genotyped by NS5b.
[0137] The above data demonstrates that the accuracy of the
genotyping assay of the invention is superior to other commercially
available assays. The assay showed 100% concordance with previous
genotype result at the type level for 51 samples that were
genotypable with NS5b. Seven samples that were ungenotypable with
NS5b were excluded from this phase of the evaluation. All 7
ungenotypable samples were repeated in Phase 2 of this
evaluation.
[0138] An alternate set of RT-PCR primers is in development to
amplify genotype 6a and 7c, was subsequently designed and analyzed,
as described below in connection with the Phase 2 analysis.
[0139] Reproducibility Analysis
[0140] In order to determine the reproducibility of the HCV NS5b
genotyping assay and reagents of the invention, a total of 22
samples comprising genotypes 1-10 were analyzed by two operators on
two separate runs and results compared for concordance at the type
and subtype level. The results of positive controls on each of the
eight runs was also compared. TABLE-US-00020 Operator to Operator
Reproducibility Data Operator 1 NS5b Operator 2 NS5b Sample ID
Genotype Genotype HCVGTP-004c #1 UG UG HCVGTP-004c #2 6i 6i
HCVGTP-004c #3 6n 6n HCVGTP-004c #4 6b 6b HCVGTP-004c #5 UG UG
HCVGTP-004a #1 3k 3k HCVGTP-004a #2 6n 6n HCVGTP-004a #3 3k 3k
HCVGTP-004a #4 6i 6i 14682 4a 4a 15038 5a 5a 20759 UG UG Acrometrix
1 1a 1a Acrometrix 2 2b 2b Acrometrix 3 3a 3a Acrometrix 4 4a 4a
Acrometrix 5 5a 5a Acrometrix 6 UG UG GP4A-C2 4a 4a 1-5A 5a 5a
GP6A-A2 UG UG GP6A-C2 UG UG
[0141] TABLE-US-00021 Run to Run Reproducibility Data Sample ID
Expected genotype BRTL NS5b Genotype Positive control run 1 1b 1b
Positive control run 2 1b 1b Positive control run 3 1b 1b Positive
control run 4 1b 1b Positive control run 5 1b 1b Positive control
run 6 1b 1b Positive control run 7 1b 1b Positive control run 8 1b
1b
[0142] 22 samples were analyzed separately by two operators,
yielding 16 NS5b genotypes. The above data demonstrates that the
NS5b genotyping assay resulted in 16/16 or 100% concordance of NS5b
genotypes at both type and subtype level between two operators, as
well as between two runs.
[0143] 6 samples (4 genotype 6a and 2 genotype 7c) were unable to
be amplified or genotyped by both operators. Run to Run
reproducibility: 100% concordance among 8 replicates of positive
control from 8 runs.
[0144] Sensitivity Analysis
[0145] In order to determine the level of sensitivity of the
genotyping assay, a dilution panel of genotypes 1-10 was also
prepared as detailed below TABLE-US-00022 HCV NS5b Sensitivity
Dilution Panel # replicates Genotype nominal c/mL # replicates
tested genotypable 1a 5,000 3 3/3 1a 1,000 6 3/6 1a 500 6 0/6 2b
5,000 3 3/3** 2b 1,000 6 6/6** 2b 500 6 4/6 3 1,000 3 3/3 4 1,000 3
1/3 5 1,000 3 3/3 6 1,000 3 0/3 6b (004c #4) 2,423* 3 0/3 7c (004c
#1) 258* 3 0/3 7c (004c #5) 101* 3 0/3 8c (004c #3) 182* 3 0/3 8c
(004a #2) 665* 3 0/3 9b (004c #2) 283* 3 0/3 9b (004a #4) 1,372* 3
0/3 10a (004a #1) 1,743* 3 3/3 10a (004a #3) 1,000 3 0/3 4a (14682)
571* 1 0/1 5a (15038) 368* 1 0/1 6a (20759) 343* 1 0/1 *Sensitivity
panel dilutions made based on Certificate of Analysis values
supplied by vendor. Subsequent viral loads determined by bDNA were
used to recalculate the nominal value for these sensitivity panel
members. **This sample is confirmed as a 2b by LiPA and HCV 5'NC
TruGene genotyping and genotypes as a 1b with NS5b geenotyping. It
will be sent to a reference lab for additional testing to resolve
genotype discrepancy.
[0146] The above data indicate that the HCV NS5b genotyping assay
meets criteria for acceptable sensitivity for genotypes 1-5, as HCV
genotype 1-5 samples were accurately and reproducibly genotyped at
1,000 c/mL (192 IU/mL).
[0147] The HCV NS5b genotyping assay did not, however, consistently
genotype samples for HCV genotype 6. HCV genotype 6 samples were
consistently ungenotypable at 1,000 c/mL (192 IU/mL). Accordingly,
due to issues in preparing the sensitivity dilutions for genotype
6-10, sensitivity at 1,000 c/mL can not be determined from this
data. The NS5b genotyping assay is therefore accurate at 1,000 c/mL
for genotypes 1-5 with variable consistency.
EXAMPLE 3
Evaluation of Performance Characteristics of HCV NS5b Genotyping by
Sequencing
[0148] Analytical Accuracy Analysis
[0149] A subset of the phase 1 validation accuracy samples
consisting of a 6 member panel comprising genotypes 1-6, a 12
member panel comprising genotypes 4-10, and 4 additional clinical
samples comprising genotypes 4, 5 and 6 will be analyzed and
results compared to previous genotype. Samples were previously
characterized by either LiPA or TruGene 5'NC genotyping or another
NS5b method as detailed in the samples section. TABLE-US-00023
Analytical Accuracy Data Sample ID Expected Genotype BRTL NS5b
Genotype HCVGTP-004c #1 7C (6f) 6f HCVGTP-004c #2 9B (6i) 6i
HCVGTP-004c #3 8C (6n) 6n HCVGTP-004c #4 6B 6b HCVGTP-004c #5 7C
(6f) 6f HCVGTP-004a #1 10A (3k) 3k HCVGTP-004a #2 8C (6n) 6n
HCVGTP-004a #3 10A (3k) 3k HCVGTP-004a #4 9B (6i) 6i 14682 4A 4a
15038 5A 5a 20759 6A 6a Acrometrix 1 1B 1a Acrometrix 2 2B 2b
Acrometrix 3 3A 3a Acrometrix 4 4 4a Acrometrix 5 5A 5a Acrometrix
6 6A 6a GP4A-C2 4A UG (no amp) rpt 4a 1-5A 5A 5a GP6A-A2 6A 6a
GP6A-C2 6A 6a
[0150] The above data indicates that the accuracy of the HCV NS5b
genotyping assay is 100% concordant at the type level for all
samples, including two 7c samples and four 6a samples that had
previously failed.
[0151] Reproducibility Analysis
[0152] The results of positive controls on each of the four runs
was compared, as follows: TABLE-US-00024 Reproducibility Data
Sample ID Expected Genotype BRTL NS5bGenotype Positive control run
1 1b 1b Positive control run 2 1b 1b Positive control run 3 1b 1b
Positive control run 4 1b 1b
[0153] The above data shows 100% concordance among 4 replicates of
positive control from 4 runs
[0154] Sensitivity Analysis
[0155] A dilution panel of genotype 1-6 was prepared as detailed
below: TABLE-US-00025 Sensitivity Data # replicates # replicates
Genotype ID Nominal c/mL tested genotypable 1a 5,000 5,000 3 3/3 1a
1,000 1,000 3 1/3 2b 5,000 5,000 3 3/3* 2b 1,000 1,000 3 2/3* 3
1,000 1,000 3 3/3 4 1,000 1,000 3 3/3 5 1,000 1,000 3 3/3 6 1,000
1,000 3 0/3 7c 004c #1 5,000 3 3/3 9b 004c #2 5,000 3 3/3 8c 004c
#3 5,000 3 1/3 6b 004c #4 5,000 3 3/3 7c 004c #5 5,000 3 3/3 10a
004a #1 5,000 3 3/3 8c 004a #2 5,000 3 3/3 10a 004a #3 1,000 3 1/3
9b 004a #4 5,000 3 1/3 6a GP6a A3 5,000 3 1/3 6a GP6A C3 5,000 3
1/3 6a 20759 5,000 3 3/3 *This sample is confirmed as a 2b by LiPA
and HCV 5'NC TruGene genotyping and genotypes as 1b with NS5b. It
will be sent to a reference lab for additional testing to resolve
genotype discrepancy.
[0156] This assay resulted in accurate and reproducible genotypes
at 1,000 c/mL (192 IU/mL) for genotypes 1-5, as well as accurate
and reproducible genotypes at 5,000 c/mL (962 IU/mL) for genotype
6.
Sequence CWU 1
1
12 1 23 DNA Human cytomegalovirus 1 tatgayaccc gctgyttyga ytc 23 2
23 DNA Human cytomegalovirus modified_base (10)..(10) modified_base
(10)..(10) i misc_feature (10)..(10) n is a, c, g, or t 2
vgtcatrgcn tcygtraagg ctc 23 3 25 DNA Human cytomegalovirus 3
tggggttckc gtatgayacc cgctg 25 4 25 DNA Human cytomegalovirus
modified_base (11)..(11) i misc_feature (11)..(11) n is a, c, g, or
t modified_base (23)..(23) i misc_feature (23)..(23) n is a, c, g,
or t 4 tggggttckc ntatgayacy mgntg 25 5 26 DNA Human
cytomegalovirus modified_base (18)..(18) i misc_feature (18)..(18)
n is a, c, g, or t 5 gartayctvg tcatrgcntc ygtraa 26 6 25 DNA Human
cytomegalovirus misc_feature (11)..(11) n is a, c, g, or t
misc_feature (23)..(23) n is a, c, g, or t 6 tggsbttykc ntaygayacy
mgntg 25 7 25 DNA Human cytomegalovirus 7 tggggttckc gtatgayacc
cgctg 25 8 25 DNA Human cytomegalovirus modified_base (11)..(11) i
misc_feature (11)..(11) n is a, c, g, or t modified_base (23)..(23)
i misc_feature (23)..(23) n is a, c, g, or t 8 tggggttckc
ntatgayacy mgntg 25 9 26 DNA Human cytomegalovirus modified_base
(18)..(18) i misc_feature (18)..(18) n is a, c, g, or t 9
gartayctvg tcatrgcntc ygtraa 26 10 23 DNA Human cytomegalovirus 10
tatgayaccc gctgyttyga ytc 23 11 23 DNA Human cytomegalovirus
modified_base (10)..(10) i misc_feature (10)..(10) n is a, c, g, or
t 11 vgtcatrgcn tcygtraagg ctc 23 12 1944 DNA Human cytomegalovirus
12 cccgactccg acgttgagtc ctattcttcc atgccccccc tggaggggga
gcctggggat 60 ccggatctca gcgacgggtc atggtcgacg gtcagtagtg
gggccgacac ggaagatgtc 120 gtgtgctgct caatgtctta ttcctggaca
ggcgcactcg tcaccccgtg cgctgcggag 180 gaacaaaaac tgcccatcaa
cgcactgagc aactcgttgc tacgccatca caatctggtg 240 tattccacca
cttcacgcag tgcttgccaa aggaagaaga aagtcacatt tgacagactg 300
caagttctgg acagccatta ccaggacgtg ctcaaggagg tcaaagcagc ggcgtcaaaa
360 gtgaaggcta acttgctatc cgtagaggaa gcttgcagcc tggcgccccc
acattcagcc 420 aaatccaagt ttggctatgg ggcaaaagac gtccgttgcc
atgccagaaa ggccgtagcc 480 cacatcaact ccgtgtggaa agaccttctg
gaagacagtg taacaccaat agacactacc 540 atcatggcca agaacgaggt
tttctgcgtt cagcctgaga aggggggtcg taagccagct 600 cgtctcatcg
tgttccccga cctgggcgtg cgcgtgtgcg agaagatggc cctgtacgac 660
gtggttagca agctcccctt ggccgtgatg ggaagctcct acggattcca atactcacca
720 ggacagcggg ttgaattcct cgtgcaagcg tggaagtcca agaagacccc
gatggggctc 780 tcgtatgata cccgctgttt tgactccaca gtcactgaga
gcgacatccg tacggaggag 840 gcaatttacc aatgttgtga cctggacccc
caagcccgcg tggccatcaa gtccctcact 900 gagaggcttt atgttggggg
ccctcttact aattcaaggg gggaaaactg cggctaccgc 960 aggtgccgcg
cgagcagagt actgacaact agctgtggta acaccctcac tcgctacatc 1020
aaggcccggg cagcctgtcg agccgcaggg ctccaggact gcaccatgct cgtgtgtggc
1080 gacgacttag tcgttatctg tgaaagtgcg ggggtccagg aggacgcggc
gagcctgaga 1140 gccttcacgg aggctatgac caggtactcc gccccccccg
gggacccccc acaaccagaa 1200 tacgacttgg agcttataac atcatgctcc
tccaacgtgt cagtcgccca cgacggcgct 1260 ggaaagaggg tctactacct
tacccgtgac cctacaaccc ccctcgcgag agccgcgtgg 1320 gagacagcaa
gacacactcc agtcaattcc tggctaggca acataatcat gtttgccccc 1380
acactgtggg cgaggatgat actgatgacc cacttcttta gcgtcctcat agccagggat
1440 cagcttgaac aggctctcaa ctgcgagatc tacggagcct gctactccat
agaaccactg 1500 gatctacctc caatcattca aagactccat ggcctcagcg
cattttcact ccacagttac 1560 tctccaggtg aaattaatag ggtggccgca
tgcctcagaa aacttggggt cccgcccttg 1620 cgagcttgga gacaccgggc
ctggagcgtc cgcgctaggc ttctggccag aggaggcaag 1680 gctgccatat
gtggcaagta cctcttcaac tgggcagtaa gaacaaagct caaactcact 1740
ccgataacgg ccgctggccg gctggacttg tccggctggt tcacggctgg ctacagcggg
1800 ggagacattt atcacagcgt gtctcatgcc cggccccgct ggttctggtt
ttgcctactc 1860 ctgcttgctg caggggtagg catctacctc ctccccaacc
gatgaagatt gggctaacca 1920 ctccaggcca ataggccatt ccct 1944
* * * * *