JPH06319563A - Hepatitis c viral gene, oligonucleotide and method for determining hepatitis c viral genotype - Google Patents

Abstract

PURPOSE:To obtain a gene of a newly elucidated hepatitis C virus (HCV), a specific oligonucleotide, a method for detection thereof and a method for determining the genotype of the HCV. CONSTITUTION:The polynucleotide has a base sequence described in sequence Nos. 1 to 5. The oligonucleotide is described in sequence No 6. The methods for detecting an HCV gene and determining the genotype use the polynucleotide and oligonucleotide as a primer, a probe, etc. Furthermore, the polypeptide is described in sequence Nos. 15 to 19. Thereby, the HCV gene of the newly found genotype can be detected and the HCV genotype Can simultaneously be determined over a wide range.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cDNA gene of hepatitis C virus (hereinafter abbreviated as “HCV”), a specific oligonucleotide constituting the same, a protein derived from the same, a peptide, and HCV using these. It relates to a genotyping method.

[0002]

2. Description of the Related Art Since a part of the HCV gene was elucidated and announced in 1988, many techniques applicable to HCV diagnosis have been developed and put into practical use. Until now, HCV
An antibody test method for detecting HCV antibody that appears in the blood of a patient due to infection and a method for detecting the HCV gene existing in the body have been developed and widely used. Further, a method for determining the genotype of HCV has been studied and developed. The present inventors have been deeply involved in this. The most important issues at the present time in these diagnostic techniques are to achieve high sensitivity and high specificity for each genotype. In order to solve this technical problem, there is an urgent need to identify a gene sequence or a specific antigen specific to each genotype of HCV and establish a diagnostic technique based on the identification. In fact, for some HCV strains, the entire gene sequence has been elucidated, and for other strains, part of the gene sequence has been elucidated to identify the sequences of HCV-specific genes or HCV-specific amino acid sequences. Was used. As a result, recent diagnostic methods developed based on this information have higher specificity and sensitivity than conventional testing methods, and based on this, it becomes possible to adopt an appropriate treatment policy. began. However, on the other hand, it has been found that there are cases of HCV disease that cannot be captured even when these test methods are used, and development of a diagnostic method having higher specificity and sensitivity is desired.

[0003]

[Problems to be Solved by the Invention] HCV has not yet been confirmed as a virus itself because the gene sequence has not been elucidated for the first time. Moreover, the number of HCV strains for which the entire gene sequence has been elucidated is still small, and it cannot be said that the genetic characteristics of HCV have been completely elucidated. Therefore, it is not clear whether the gene sequences published to date provide common information to all HCVs.
In order to complete a complete HCV diagnostic method, it is essential to elucidate the genetic characteristics of HCV that cannot be captured by current diagnostic methods and to construct a diagnostic method that reflects that information. The object of the present invention is to clarify the gene sequence of an HCV strain which has not been sufficiently characterized by the HCV test methods to date, and to elucidate the gene characteristics thereof, thereby making it possible to obtain a genotype having an accurate and wide range of application. It is to provide a determination method and an oligonucleotide or the like used for the determination method.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have found that HCV which has not been sufficiently characterized by various HCV examinations to date.
For the purpose of elucidating the gene body of the strain, intensive research was conducted, and as a result, RNA was isolated from a human specimen that was HCV-RNA positive but could not be typed by conventional genotyping methods, and Used to determine the entire gene sequence of an unknown HCV strain. Furthermore, as a result of comparing the novel gene sequence with the previously reported known HCV gene sequences of each genotype, the inventors found that the HCV strain of the present invention did not correspond to any of the known genotypes. , A completely different genotype (the inventors tentatively named this genotype 1c type). Based on this
The present inventors have identified a gene sequence specific for the 1c type genotype and not present in other genotypes. By using an oligonucleotide having this sequence as a primer or a probe, 1c type genotyping became possible. Furthermore, by identifying a gene sequence common to all genotypes including known genotypes, and utilizing the oligonucleotide having this sequence and the oligonucleotide of the present invention having a sequence specific to 1c type as a primer The inventors have found that genotype determination can be realized with a single test, and completed the present invention. The plumer and probe comprising the polynucleotide and the oligonucleotide of the present invention exert an action by specifically binding to a gene common to HCV genotypes or a type-specific gene sequence. It is well known that the binding of polynucleotides and oligonucleotides is not affected even if there is a slight difference in sequence, and therefore, a polynucleotide having a slight substitution for the polynucleotide or oligonucleotide of the present invention Also, oligonucleotides are naturally included in the scope of the present invention.

That is, the present invention is an HCV which is a HCV having a new genotype 1c type different from known HCV.
-The invention of the G9 cDNA gene and the invention relating to a polynucleotide or an oligonucleotide having a nucleotide sequence which constitutes a part of its specific gene sequence or has a nucleotide sequence complementary thereto, and specifically, SEQ ID NO: 1 It is an invention of a polynucleotide or an oligonucleotide having all or a part of the nucleotide sequence described in 1 to 5 or a nucleotide sequence complementary thereto. The present invention is an invention of a primer, probe, or labeled probe comprising the above-mentioned oligonucleotide, and is also an invention of a primer or probe having all or part of the nucleotide sequence of SEQ ID NO: 6. The present invention is an invention relating to a mixed primer which can detect HCV gene and determine genotype by using the above primer in combination with a primer comprising an oligonucleotide having the gene sequence of SEQ ID NO: 7 to 14. is there.
The present invention is also an invention of a method for detecting an HCV gene or a method for determining a genotype by using the above primers or probes alone or simultaneously.

As shown in Example 1, the present inventors extracted RNA from a plurality of HCV antibody-positive human specimens that could not be typed by the conventional genotyping method according to a predetermined method, and then extracted HC- For G9, specify the entire gene sequence,
The HCV is designated as HC-G9, the partial sequences of the remaining two specimens are specified, and the specimens are designated as S117 and SR0.
It was named 37. Its base sequence is as shown in SEQ ID NOS: 1 to 5. The entire gene sequence of HC-G9 consists of 9440 bases excluding the T stretch part found at the 3'end, an untranslated region of 341 bases at the 5'end, followed by 9033 bases of 3011 amino acids. It was found that the coding region was further composed of an untranslated region consisting of 66 bases at the 3'end.

As shown in Example 2, the present inventor compared the nucleotide sequences of the above-mentioned strains, which are the subject of the present invention, with the nucleotide sequences of 14 strains belonging to four known genotypes, and as a result,
20 between the nucleotide sequence of the above strain and the known genotype sequence.
It was found that the gene and the polynucleotide of the present invention have a homology of not less than%, and are novel ones that cannot be classified into any known genotype. Furthermore, the present inventors have also compared the gene sequences of the genes and polynucleotides of the present invention to HCV strains in which only some gene sequences are known. As a result, it was found that the gene and polynucleotide according to the present invention are classified as an independent genotype that is a different type from HCV whose only partial sequence is known. The present inventors tentatively named the novel HCV genotype of the present invention as 1c type.

As shown in Example 2, the present inventors
The core region was identified as the optimal gene region for genotyping of type HCV strains. From this region, an oligonucleotide of SEQ ID NO: 6 having a 1c-type specific sequence was obtained.

The present inventors have noted that the region to be used for HCV gene detection and genotyping is in the core region in the 1c type genotype as in the case of other genotyping. Polynucleotides that can be used in combination with the polynucleotide of the present invention described in 6 were searched from among known primers. As a result, SEQ ID NO: 7
The sequence of the known oligonucleotide described is well conserved in the 1c type, and it was found that this can be used as a common primer in HCV gene detection and genotyping. It has been found that the 1c-type gene can be specifically amplified by using the 1c-type specific oligonucleotide primer of SEQ ID NO: 6 in combination with the oligonucleotide primer of SEQ ID NO: 7, which is a known common primer.

In the present invention, polymerase, thiein, reaction method (PCR method) can be preferably used as a gene amplification method. At that time, as the primer pair, a combination of the oligonucleotide shown in SEQ ID NO: 6 and the primer pair shown in SEQ ID NO: 9 is also preferable.

In the PCR method, the region common to each genotype is amplified as the first step, and type-specific amplification is performed as the second step, whereby the detection of the HCV gene and the gene are performed with higher sensitivity. The type is determined. Suitable oligonucleotide pairs used in the PCR method in this manner include:
The oligonucleotide of SEQ ID NO: 7 and the oligonucleotide of SEQ ID NO: 8 for the first step, and the oligonucleotide of SEQ ID NO: 6 and the SEQ ID NO: 9 for the second step
The described oligonucleotides can be exemplified.

Further, according to the method of the present invention, the novel oligonucleotide (SEQ ID NO: 6) of the present invention is combined with each known genotype-specific oligonucleotide primer of SEQ ID NO: 7 to SEQ ID NO: 14 or a common oligonucleotide primer. When used at the same time, the PCR method or the known two-step PCR method can be used to detect the HCV gene and determine the genotype by a single operation.

Further, the present inventors obtained the polypeptide of the present invention by incorporating it into a known host for expression and chemically synthesizing it by a conventional method.

The amino acid sequence of the polypeptide of the present invention has a high homology with the polypeptide of known genotype HCV in the core region, but the homology is low in the envelope (E1) and E2 / NS1.
It is shown to be type-specific. E1, E2 / NS1
Is considered to be a protein present on the surface of virus particles, and a number of cases having antibodies against this part are also found. Therefore, the polypeptide of the present invention and its partial oligopeptide can be used for preparing a detection system for anti-envelope antibody specific to 1c type and for use in HCV vaccine. Further, the NS2 to NS5 regions are considered to encode nonstructural proteins such as proteases, and the polypeptide and its partial oligopeptide of the present invention can be used for the development of a nonspecific antibody detection system and HCV growth inhibitor. .

[0015]

The present invention has a broader genotype of H than the conventional one, including HCV of the newly found genotype 1c type.
CV can be detected with high sensitivity, and it can be specifically determined simultaneously with each genotype of HCV. The gene, polynucleotide, oligonucleotide primer, protein, and peptide of the present invention can be used for CV gene detection and genotyping.

[0016]

EXAMPLES Examples of the present invention will be described below.
Of course, the present invention is not limited to these examples.

Example 1 A plurality of novel HCV strains that were not classified into conventional genotypes were found, and the entire nucleotide sequence and a part of the nucleotide sequences were determined as follows.

(1) Extraction of RNA Commercially available HCV antibody test agent and antibody test method developed and patent pending by the present inventors (Japanese Patent Application No. 2-15340).
1) and HCV infection has been confirmed by the HCV detection method using an oligonucleotide primer, which was separately filed by the present inventors (Japanese Patent Laid-Open No. 5-23200), but the present inventors have filed a separate patent application. HC
Method for V genotyping (Japanese Patent Application No. 3-30729)
6, 4-093960), blood samples (HC-G9, YS11) derived from hepatitis patients whose genotype could not be determined.
7, and SR037), RNA was extracted as follows. An appropriate amount of Tris buffer (10 m
M, pH 8.0) and added at 90 × 103 rpm for 15
Minute centrifugation was performed. 200m on the obtained pellets
M NaCl, 10 mM EDTA, 2% (weight / volume)
Tris buffer (50 mM, pH) containing sodium dodecyl sulfate (SDS) and 1 mg / ml proteinase K
8.0) was added, and the mixture was heated at 60 ° C. for 1 hour, and phenol /
After extraction with chloroform, ethanol precipitation was performed to obtain RNA.

(2) Preparation of cDNA RNA obtained from each sample was heated at 70 ° C. for 1 minute and then rapidly cooled to obtain template RNA. 100 units of reverse transcriptase (Superscript) was added to this template RNA sample.
t; GIBCO, BRL) and 20 pmol of the oligonucleotide primer were added and reacted at 42 ° C. for 1 hour to obtain cDNA.

(3) Amplification of cDNA by polymerase chain reaction (PCR) FIG. 1 shows the single-stranded cDNA obtained by the above operation.
Amplification was performed by using a primer pair consisting of a sense-side oligonucleotide primer and an antisense-side oligonucleotide primer set for each region shown in Table 1 for each region shown in Table 1. Mafuku is the goal of DNA thermal cycler (Perkin-Elmer. Cetus)
ene Amp DNA amplification reagent kit (Perkin
-Elmer Cetus) using the method of Saiki et al. [Science, Vol. 239, p487-49
1 (1988)], the PCR method consisting of 35 amplification cycles.

[0021]

[Figure 1]

[0022]

[Table 1]

(4) Determination of nucleotide sequences of HC-G9, YS117, and SR037 by construction of cDNA library Each region gene derived from each sample amplified by PCR was subjected to T4 polynucleotide kinase (New England).
Biolabs), T4 DNA polymerase (Tak)
ara Biochemicals) and then M13
It was inserted into a phage vector and cloned. Nucleotide sequence determination was done with the deoxy chain termination
Ion method, Sequencense seque
encekit ver 2.0 (United Sr
are Biochemicals) or Aut
oRead Sequencing kit (Pha
rmcia). For each sample, 3 clones for each region were obtained, the sequence was determined for each, and the sequence was determined by using a base common to each clone or a base with a high concordance rate. For HC-G9, the entire region was determined, and for YS117 and SR037, the sequence from the 63rd base to the 1867th base and the sequence from 8259th to 9440th from the 5'end were determined. HC-G9 in Sequence Listing 1, YS117 in Sequence Listing 2,
5'side, YS117 in Sequence Listing 3 and 3'side, Sequence Listing 4
The nucleotide sequence of 5'side of SR037 is shown in the sequence table, and each nucleotide sequence of 3'side of the nucleotide sequence of SR037 is shown in Sequence Listing 5. Table 2 shows HC-G9, Y
The homology between the sequences of S117 and SR037 is shown. As a result, the nucleotide sequences of these three specimens are 95% homologous, and the HCVs of the above three specimens are classified into the same genotype. FIG. 2 shows the nucleotide sequence homology between HC-G9 and 14 HCV strains whose nucleotide sequences have been elucidated so far. As a result, it was revealed that HC-G9 showed 20% or more heterology with the nucleotide sequences of any known genotype strains. From this, HCV derived from 3 specimens including HC-G9
Confirmed that they are classified into new genotypes different from the conventional genotypes. From the estimation of the phylogenetic classification based on the gene sequence, this newly found genotype belongs largely to the classification called type 1, and there are already type I and type II in these classifications, which are called 1a and 1b. Therefore, it was decided to temporarily call it the 1c type in accordance with this.

[0024]

[Fig. 2]

[0025]

[Table 2]

Example 2 1c type HCV genotyping method (1) Selection of primer suitable for genotyping 1c type HCV Example 1 was selected in order to select a primer used for genotyping 1c type HCV. Based on the sequence revealed by the above, a gene region having the most conserved nucleotide sequence in 1c type HCV and having low homology with the existing genotype HCV strain was searched. As a result, it was revealed that the conserved region suitable for genetic determination is the core region like other genotypes. Based on this, an oligonucleotide primer having a sequence suitable for amplification of a gene fragment specific for 1c type from the core region was selected. As a result, # 186 described in Sequence Listing 7 and Sequence Listing 8 was used as the oligonucleotide primer used for the first-stage gene amplification.
And # 256 listed in Sequence Listing 9 as oligonucleotide primers to be used in the subsequent second-stage amplification.
104 and the # 321 (Sequence Listing 6) primer of the present invention were selected. The oligonucleotide primers of # 186, # 256 and # 104 have already been reported by the present inventors (Japanese Patent Application Nos. 3-307296 and 4-09).
3960) Although it is an oligonucleotide primer used in a known genotyping method, it was determined to be applicable in the present invention based on the sequence of 1c type HCV clarified in Example 1. These oligonucleotide primers are used in the present invention to amplify a conserved nucleotide sequence specific to a genotype. On the other hand,
The # 321 oligonucleotide primer of the present invention is an oligonucleotide primer having specificity only for the 1c type clarified in Example 1.

(2) HCV using # 321 of the present invention
Gene detection and genotyping of 1c type HCV HCV strains of each genotype (HC-J1: type I, HC-J
4: II type, HC-J6: III type, HC-J8: IV
Type) and HC-G9-derived RNA, cDNA was obtained using the # 186 primer. Then cDNA
P using primers # 256 and # 186
A first stage amplification was performed using CR. First stage P
The CR was carried out for 35 cycles, with denaturation at 94 ° C for 1 minute, primer binding reaction at 55 ° C for 1 minute 30 seconds, and primer extension reaction at 72 ° C for 2 minutes as one cycle. The second-stage amplification is performed by using type-specific oligonucleotide primers # 296, # 133, and # 13 described in SEQ ID NOS: 11 to 14.
4, # 135 and # 321 primer of the present invention, and PCR using the type-common oligonucleotide primer # 104 primer described in SEQ ID NO: 9. PCR was carried out using a 1/50 dose of the amplification product amplified in the first step as a sample for the second step amplification. The reaction conditions were a denaturation reaction at 94 ° C. for 30 seconds, a primer binding reaction at 60 ° C. for 30 seconds, and a primer extension reaction at 72 ° C. for 30 seconds.
Repeated times. After the completion of the second step, the amplified product was converted to 1.5% NuSieve and 1.5% SeaKem (FM
C Bioproducts, U.S.A. S. After completion of agarose electrophoresis using A), the DNA was stained by ethidium bromide staining after completion, and the genotype was determined from the migration position of each band. The mobility of the amplification product of each sample is the mobility predicted from the position of the primer set for each genotype, that is, 49, 144, 174, 123, 200b.
It was confirmed that each genotype could be distinguished by migrating to the position of p (type I, type II, type III, type IV, type 1c). In addition, only the band of the expected genotype was seen in the electrophoretic image of each sample, and the band corresponding to another type did not appear. Therefore, it was proved that the HCV genotyping method according to the present invention has sufficiently high specificity for all genotypes including 1c type. The migration pattern is shown in FIG.

[0028]

[Figure 3]

Example 3 Occurrence Frequency of Type 1c HCV in Each Country Using the method of Example 2 of the present invention to determine the frequency of type 1c in HCV patient specimens from Japan, China, Thailand, Indonesia and New Zealand. I looked it up. As a result, as shown in Table 3, 1c-type HCV was found only in Indonesia with a frequency of 9.9%, and it was revealed that it has a localized locality.

[0030]

[Table 3]

[0031]

EFFECTS OF THE INVENTION The genotypes of HCV, which were conventionally classified into 4 types, were newly classified into 5 types by the present inventors, and it was found that they each have a characteristic nucleotide sequence. The present invention provides an oligonucleotide having a type-specific sequence or having complementarity to the type-specific sequence. As a matter of course, these oligonucleotides can be used as primers in gene amplification for detection of HCV gene and type determination at the gene level. In addition, it can be used as a probe by adding an appropriate label, or can be used for type determination at the gene level by combining with a gene amplification. By using the oligonucleotide primer newly set according to the present invention, the genotype of 1c-type HCV, which could not be determined by the conventional method, can be determined, and it is considered that a more complete determination technique has been established. To be In addition, 1 which has been clarified by the present invention
The type-specific antigenic position can be estimated from the gene sequence of c-type HCV, and the protein or polypeptide of the present invention can also be used for the development of therapeutic drugs using gene products.

[0032]

[Sequence list]

[0033]

[0034]

[0035]

[0036]

SEQ ID NO: 6 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method of Characterization: E Sequence (# 321) AACCTCCGCC GGGGATCAGA 20

SEQ ID NO: 7 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method of Characterization: E Sequence (# 186) AYGTACCCCA YGAGRTCGGC 20 (Y is T or C; R is G or A)

SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to genomic RNA Method for characterizing: E sequence (# 256) CGCGCGMCNA GGAARRCTTC 20 (M is A or C. N is A, T, C or G. R is A or G)

SEQ ID NO: 9 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method for Characterization: E Sequence (# 104) AGRAARRCTT CSGAGCGRTC 20 (R is G or A. S is C or G.)

SEQ ID NO: 10 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method of Characterization: E Sequence (# 132) YRCCTTGGGC ATAGGCTGAC 20 (Y is T or C. R is G or A.)

SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to genomic RNA Method for characterizing: E sequence (# 133) GARCCAWCCT GCCCAYCCYA 20 (R is G or A. W is T or A. Y is C or T.)

SEQ ID NO: 12 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method of Characterization: E Sequence (# 134) CCAARAGGGA CGGGARCCTC 20 (R is G or A.)

SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Sequence type: cDNA to genomic RNA Method for characterizing: E sequence (# 135) RCCYTCGTTT CCRTACAGRG 20 (R is G or A. Y is C or T.)

SEQ ID NO: 14 Sequence Length: 20 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: cDNA to genomic RNA Method of Characterization: E Sequence (# 296) GGATAGGCTG ACGTCTACCT 20

[0046]

[0047]

[0048]

[0049]

[0050]

[Table 1]

[Table 2]

[Table 3]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Diagram showing amplification regions used for HCV gene sequencing. FIG. 2: Nucleotide sequences of HC-G9 and 14 HCV strains whose entire nucleotide sequences have been elucidated to date. The figure which shows homology. FIG. 3: Electrophoresis photograph showing the migration pattern of the electrophoresis test performed in Example 2. The judgment by the conventional method is shown in (a). Type 1c is not detected. # 321 and # 10 in (b)
The judgment in 4 is shown. No other than 1c type is detected. 1 is H
C-J1, 2 is HC-J4, 3 is HC-J6, 4 is HC
-J8, 5 is HC-G9, 6 is YS117, 7 is SR0
37 is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12Q 1/68 A 7823-4B G01N 33/576 Z 8310-2J // G01N 33/53 D 8310- 2J C07K 99:00

Claims (22)

[Claims] 1. A hepatitis C virus gene cD having the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence complementary thereto.
NA / HC-G9. 2. A polynucleotide YS117 having the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence complementary thereto.
・ 5 '. 3. A polynucleotide YS117 having the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence complementary thereto.
・ 3 '. 4. A polynucleotide SR037 having the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence complementary thereto.
・ 5 '. 5. A polynucleotide SR037 having the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence complementary thereto.
・ 3 '. 6. A polynucleotide or an oligonucleotide which constitutes a part of the nucleotide sequences of SEQ ID NOs: 1 to 5 or a part of a nucleotide sequence complementary thereto. 7. A primer or a primer pair comprising the oligonucleotide according to claim 6. 8. An oligonucleotide # 321 having the nucleotide sequence of SEQ ID NO: 6. 9. An oligonucleotide primer having the base sequence of SEQ ID NO: 6. 10. A primer pair comprising at least one of an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide primer having the nucleotide sequence of SEQ ID NOs: 7 to 9. 11. A primer pair comprising at least one of an oligonucleotide primer having the nucleotide sequence of SEQ ID NO: 6 and an oligonucleotide primer having the nucleotide sequence of SEQ ID NOs: 10 to 14. 12. A hepatitis C virus genotyping method using the oligonucleotide primer or the oligonucleotide primer pair according to claim 7. 13. A method for detecting a gene for hepatitis C virus, which uses the oligonucleotide primer pair according to any one of claims 7 to 10. 14. The method according to claim 12, wherein the cDNA is amplified by the polymerase chain reaction method.
The hepatitis C virus genotyping method according to claim 13 or the hepatitis C virus gene detection method according to claim 13. 15. The method for detecting a hepatitis C virus gene according to claim 13, or the method according to claim 14, wherein a plurality of sets of the oligonucleotide primers according to claim 11 or 12 are used to carry out amplification a plurality of times. Hepatitis C virus genotyping method. 16. The labeled probe according to claim 6 or 9. 17. A hepatitis C virus genotyping method using the labeled probe according to claim 16. 18. A polypeptide HC according to SEQ ID NO: 15.
-G9Peotein, as well as its partial peptides. 19. The polypeptide YS of SEQ ID NO: 16.
117.5 'Peptide, and its partial peptides. 20. The polypeptide YS of SEQ ID NO: 17.
117-3 'Peptide, and its partial peptides. 21. Polypeptide SR according to SEQ ID NO: 18
037.5 'Peptide, and its partial peptides. 22. Polypeptide SR according to SEQ ID NO: 19
037.3 'Peptide, and its partial peptides.