WO2001046469A1

WO2001046469A1 - Method of judging hepatitis c virus genotype

Info

Publication number: WO2001046469A1
Application number: PCT/JP2000/009144
Authority: WO
Inventors: Moritoshi Kinoshita; Yusuke Ishida; Makoto Kuboki
Original assignee: Otsuka Pharmaceutical Co., Ltd.
Priority date: 1999-12-22
Filing date: 2000-12-22
Publication date: 2001-06-28

Abstract

A method of judging hepatitis C virus genotype characterized by determining the bases at least at the 167-, 163- and 161-positions of the 5'-NCR of HCV gene and then judging the genotype 1 and subtypes 2a, 2b and 3a on the basis of the base data thus obtained; a primer comprising the base sequence represented by SEQ ID NO:1; and a method of judging hepatitis C virus genotype for providing data concerning the amount of HCV-RNA and the genotype thereof in a specimen which is characterized by involving the following steps: (a) the step of amplifying the 5'-NCR of hepatitis C virus gene by the PCR method and quantifying the hepatitis C virus-RNA; and (b) the step of determining the base sequence of the PCR amplification product obtained in the above step (a) and judging the hepatitis C virus-RNA genotype.

Description

Specification

Method for determining genotype of hepatitis C virus

The present invention relates to a method for determining the genotype of hepatitis C virus (hereinafter abbreviated as “HCV”). Background art

Gene mutations are frequently found in HCV (showing remarkable base diversity), and typing has been performed based on differences in gene sequences. At present, these genotypes categorize HCV into six genotypes, from type 1 to type 6, with subtypes a, b, c, etc. added (P. Si Marauders onds, et al., Journal of General Virology, 74, 66, 668 (1993)). Typical genotypes widely distributed in the world include 1a, lb, 2a, 2b, and 3a. Five genotypes are known.

In recent years, the detection of HCV-RNA genome in the serum of hepatitis patients has been considered to be a direct proof of HCV infection and is clinically useful as a marker for viral proliferation. As a prediction, genotype information as well as the amount of HCV-RNA in patient sera is regarded as important indicators.

In addition, if the genotype of HCV is determined, it will be possible to analyze the degree of progression of hepatitis, cirrhosis, the rate of transition to liver cancer, the characteristics of symptoms, etc. for each type, and to predict prognosis and guide treatment. It can also be used to elucidate the route of infection. Therefore, genotyping of HCV is extremely important for diagnosis, treatment, and prediction of prognosis. Conventionally, as a method for determining the type of HCV, Okamoto et al. Prepared seven types of primers targeting the core region of HCV, combined these primers, and performed RT-PCR using reverse transcriptase. (H. Okamoto, et al., Journal of General Virology, 73, 673-679 (1992)), selected the NS5 region of HCV by Chayama et al. A method of judging the type of the liver has been reported (Kazuaki Chayama et al., “Liver”, Vol. 33, pp. 805-806, 1992). Furthermore, an attempt was made to cut the gene amplification product of the 5 'untranslated region of HCV (hereinafter abbreviated as "5'-NCR") with a restriction enzyme to determine the type (F. McOmis, et al., Transfusion, 33). , 7-13 (1993); Japanese Unexamined Patent Publication No. 9-75100, etc.) and a determination method based on base information obtained by directly sequencing the amplification product (Genner JJ, et al., J. Clin. Microbiol., 37 (8), 2625-2630 (1990); Holland J., et al., Patholgy, 30 (2), 192-195 (1998); Doglio A., et al, Res. Virol .,] 49 (4), 219-227 (1998)).

However, the various methods for determining HCV genotypes reported above are not yet fully satisfactory in terms of determination accuracy, determination efficiency, complexity of operation, time required for determination, cost, and the like. Development of a method that can measure HCV simply and quickly clinically is desired, including establishment of a method for determining HCV. Disclosure of the invention

An object of the present invention is to provide an improved HCV genotype determination method that meets the above-mentioned demands.

In view of such circumstances, the present inventors have determined that the nucleotide sequence of 5′-NCR, which is one of the PCR-amplified regions in a method for qualitatively or quantitatively measuring HCV-RNA, in a large number of patient sera. When these were determined and used as a database for creating an algorithm for genotyping, several bases at specific positions in the 5'-NCR nucleotide sequence were specific to the HCV genotype, and at least three Base information (— 16 The 7th position,-16 3 position and-16 1 position) can reliably determine the HCV genotype type 1 and subtypes 2a, 2b and 3a, and furthermore, the base information at position 199 By reference, it was found that the subtypes _1a and 1b can be determined, and that the quantification of HCV-RNA in the clinical sample and the genotype determination can be performed simultaneously with a simple operation and in a short time. Was completed.

That is, the present invention determines the bases at least at positions −167, 166-3 and −161 of the 5′-NCR of the HCV gene, and determines genotype type 1 and type It is intended to provide a method for determining the genotype of HCV, which comprises determining subtypes 2a, 2b and 3a.

Further, the present invention determines the bases at least at positions 9.9, 1167, 1163 and -161 of the 5'-NCR of the hepatitis C virus gene, and based on the base information, And genotype subtypes 1a, 1b, 2a, 2b and 3a.

The present invention also provides a primer consisting of the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a method for providing information on the amount of HCV-RNA in a sample and its genotype, comprising the following steps (a) to (mouth):

(Ii) 5'-NCR of HCV gene was amplified by PCR method, and HCV-

A step of quantifying the amount of RNA,

(Mouth) a step of purifying the PCR amplification product prepared in step (a), and

(8) Determine the nucleotide sequence of the PCR amplification product purified in step

Determining the genotype of the RNA,

And a method for determining the genotype of HCV. Further, the present invention provides the following steps (a) to (mouth) using a sample containing hepatitis C virus collected from a hepatitis C patient:

(B) Amplify 5'-NCR of hepatitis C virus gene by PCR A step of quantifying the amount of hepatitis virus-RNA,

(Mouth) a step of purifying the PCR amplification product prepared in step (b), and

(C) determining the nucleotide sequence of the PCR amplification product purified in step (mouth), and determining the genotype of Hepatitis C virus-1 RNA;

The present invention provides a method for predicting the effect of interferon treatment in the hepatitis C patient. BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, the abbreviations for amino acids, peptides, nucleotide sequences, nucleic acids, restriction enzymes, etc. are referred to by the nomenclature of IUPAC and IUPAC-IUB or their definitions, and Guidelines for the preparation of specifications, including specifications ”(March 1997, Examination Standards Office, JPO Coordination Division). In addition, the nucleotide sequence of the 5′-NCR of the HCV gene in the present invention, as a representative example of HCV genotype 1b, is a K-14 (DDBJ) registered in the National Institute of Genetics Nucleic Acid Database (DDBJ). D31602), the A in the start codon (ATG) of the translation region is 0, the base in the 5'-NCR immediately preceding this A is the first, and this is the base point toward the 5 'upstream. Are shown with consecutive negative numbers.

According to the present invention, as described above, only three bases at specific positions in the 5′-NCR of the HCV gene (ie, —positions 167, 1-163 and 1-161) and optionally a base at position 199 are added. Just by identification, the HCV genotype can be accurately determined. In addition to the above four locations, further information on the base in the 5'-NCR, preferably, -159, -155, -132, -128, -124, -124 The HCV genotype can be determined more accurately by referring to information on at least one base selected from the group consisting of positions 122 and 119.

A specific position in the 5′-NCR of the above HCV gene confirmed by the present inventors, The association with HCV genotype is as shown in Table 1 below.

table 1

As shown in Table 1 above, when the 1st 167th of HCV 5'-NCR is T-163rd is Α and the 1-161th is G, the HCV genotype can be identified as type 1. This is also the case where 1 1 59th is C or Ding, 1 1 55th is (:, — 13 2nd is G, 1 1 2 8 is A or T — 1 24 is C or G 1 122 is C , G or T and at least one of the base information where the 19th is A or G. Furthermore, to identify the subtype (la and lb) of type 1 The base information may be referred to. When the base is G, it can be identified as subtype 1b, and when it is A, it can be substantially identified as subtype 1a.

When the 167th position is T, the 163rd position is G and the 161st position is G, the HCV genotype can be identified as 2a. This is A for the 159th and 155 for the References at least one of the base information where the order is T, the -132 is A, the 128th force T, the -124th force C, the 122nd is C, and the 119th is C or T. .

If the same 167th is T, -163 is G and -161 is —, the HCV genotype can be identified as 2b. This is the base information where the 1st 159th is A, the 155th is T, the -132th is Α, the -128th is Τ, the -124th is Τ, the 122nd is C and the 119th is Τ. Can refer to at least one of

If the same is the 167th power SC, the 163rd is G and the 161st is G, the HCV genotype can be identified as 3a. This is also the base information of base information where T is the 159th, C is the 155th, C is the 132th, G is the 132nd, A is the 128th, C is the 124th, C is the 122nd and G is the 119th. At least one can be referenced.

The HCV genotyping method of the present invention uses the base information at a specific position of the 5′-NCR of the HCV gene as an index as described above, but the preparation of the gene used for type determination and the base sequence thereof There are no particular limitations on the method for determining the value, and various methods that are known or that can be obtained in the future can be widely used.

The genotype of HCV in the present invention can be determined by the following steps (a) to (8).

(B) Preparation of PCR amplification product

As a gene to be used for genotyping, a PCR amplified product obtained by amplifying the 5'-NCR region of HCV-RNA in a sample by RT-PCR or the like is used. Such a PCR amplification product may be one which gives a base length corresponding to the entire region of 5′-NCR of HCV, but one containing base information usable for genotyping of HCV of the present invention, ie, at least It is not particularly limited as long as it is a DNA fragment containing nucleotide numbers 1 167 to 1 161 and preferably -167 to 199, including DNA fragments. Not. The RT PCR can be performed according to a conventional method (for example, refer to the above-mentioned literature concerning the known determination method), and is designed to specifically amplify an appropriate target region including a specific position of 5′-NCR of HCV. What is necessary is just to use the primer set suitably.

For the preparation of such PCR amplification products, known methods used in HCV-RNA quantitative measurement can be used as they are, and if they are used, the desired method for use in HCV genotyping of the present invention can be used. This is particularly useful because it enables the preparation of PCR amplification products and the measurement of the amount of HCV-RNA in a sample.

The method of quantification of HCV-RNA is not particularly limited as long as the 5'-NCR of HCV is used as a PCR amplification product, and various methods known or that can be obtained in the future can be widely used. it can.

Examples of a method for quantitatively measuring HCV-RNA that can be used in the method for genotyping HCV of the present invention include, for example, “Amplicor ^™ HCV monitor (for measuring hepatitis C virus (HCV) RNA)” (Roche-Diagno Sticks), “Amplico ™ HCV Monitor V2.0 (for measuring hepatitis C virus (HCV) RNA)” (Roche Diagnostics), etc., and the latter kit In the quantification using, a desired PCR amplification product consisting of the nucleotide sequence from position 274 to position 31 of HCV 5'-NCR is prepared for quantification.

As described above, the PCR amplification product prepared at the time of quantitative measurement of HCV-RNA using the above-described assay kit can be extremely effectively used for the determination of the HCV genotype of the present invention. Of course, it is extremely useful in that both information on the amount of HCV-RNA and its genotyping can be obtained simultaneously.

The PCR amplification product prepared in the above-described HCV-RNA quantitative measurement method may be used as a type II, if necessary, using a primer appropriately set in the region so as to amplify a part of the region. Further PCR amplification is possible.

(Mouth) Purification of PCR amplification products Next, the gene product amplified by PCR is subjected to an ordinary method, for example, to a purification step such as ordinary column chromatography using a glass carrier to be purified.

In the purification step, commercially available purification columns such as GFX column (Amersham Pharmacia), Centrisep spin column (PE Biosystems), Centrispin 20 column (Princeton Separation) are used. Use can be suitably exemplified.

(8) Base sequence determination and genotype determination

Next, the nucleotide sequence of the purified PCR amplification product is determined, and the genotype is determined by the method described above.

The method for determining the nucleotide sequence is not particularly limited as long as the specific base information according to the determination method of the present invention can be obtained, and various methods that are known or that can be obtained in the future can be widely used as the method. Preferably, a direct sequencing method using an arbitrary sequencing primer (for example, a cycle sequencing method; Masahira Hattori, "Direct nucleotide sequencing using PCR" Experimental medicine (extra number) New PCR and its application, pp. 29-35 (1997), Yodosha, etc.) Here, the sequencing primer is not particularly limited as long as it is set so as to obtain the specific base information according to the present invention, and can be arbitrarily set from the region of the PCR amplification product. As a particularly preferred example of the primer, a primer (named “geno 7”) consisting of the base sequence of SEQ ID NO: 1 corresponding to HCV5′—NCR_210 to 1-189 can be exemplified. . This primer geno 7 can react with any genotype HCV-RNA in the 5'-NCR, which contains genotype-determinable base diversity, which can be used to perform sequencing very well. . For example, in the examples described later, all test samples subjected to sequencing can be sequenced without exception.

The sample used in the HCV genotyping method of the present invention is not particularly limited. For example, a sample containing HCV collected from a subject, that is, various biological samples is used, and a blood sample such as serum is preferable as a clinical index.

In addition, various operations that can be adopted in the present invention, for example, extraction of RNA, synthesis of DNA or DNA fragment, enzymatic treatment for cleavage, deletion, addition or binding, isolation, purification, and replication of RNA or DNA The selection, amplification, etc. can all be performed in accordance with ordinary methods (see, for example, Molecular Genetics Experiments, published by Kyoritsu Shuppan Co., Ltd. in 1983; PCR Technology, Takara Shuzo Co., Ltd., published in 1990). Further, these can be appropriately modified and used according to a conventional method as needed.

Thus, by using the HCV genotyping method of the present invention, the genotype of HCV in a sample can be determined with a simple operation and in a short time, and at the same time, the amount of HCV-RNA can be measured. . On the other hand, serum HCV-RNA levels in patients with hepatitis C, HCV genotype, and the therapeutic effect of hepatitis C with in vitro ferron were significantly more effective in patients with lower serum HCV-RNA levels. Also, it has been reported that type 1b is more ineffective than type 2a or 2b ("The practice of interfacial oral therapy", the latest guide for hepatitis C, Minami-Edo Co., Ltd.) , 1994), using the method of the present invention to simultaneously measure serum HCV-RNA levels and HCV genotypes in a series of operations, the therapeutic effect of interferon in hepatitis C patients can be predicted, and it can be tailored to individual patients. Thus, appropriate treatment can be performed. Example

Hereinafter, the contents of the present invention will be specifically described using examples. However, the present invention is not limited to these.

Example 1 HCV—RNA 5′—NCR nucleotide sequence library

(1) Preparation of primer

The base sequences of the primers (geno7, geno5 and geno6) used in this example These are shown in SEQ ID NOs: 1, 2, and 3, respectively.

The primer geno 7 is for sequencing and corresponds to the sequence from position 210 to position 189 when A of the initiation codon ATG of the core protein of HCV-RNA is 0.

The primers geno5 and geno6 are used for PCR and correspond to the same sequence from the # 271 to # 252 and from the same # 64 to -43, respectively.

(2) Sample preparation

HCV-RNA quantitative measurement of HCV-RNA levels in serum of 57 patients whose HCV-RNA genotype was determined by the Okamoto method ("Smitest HCV Dienotype", National Institute for Special Immunology) Using a method kit “Amplicor ^™ HCV Monitor V2.0 (for measuring hepatitis C virus (HCV) RNA)” (Roche-Diagnostics), quantification was performed according to the kit operation method.

The above-mentioned assay kit provides a 244 bp PCR product consisting of the 1274th to 131st sequence of the HCV 5'-NCR. Using this measured PCR product solution, A sample was prepared.

The PCR product was purified using a GFX column (GFX ™ PCR DNA and Gel Band Purification: Amersham Pharmacia).

That is, set the collection tube attached to the kit to the G FX column, add 500 I of the capture buffer attached to the kit to the column, and measure the sample with “Amplico 7 ™ HCV Monitor-1 V2.0”. The PCR product solution 60 / i1 was added and stirred by pipetting 4-6 times. The GFX column was centrifuged at 15,000 rpm for 30 seconds with the collection tube set to remove the solution eluted into the collection tube. The collection tube was set again, and Posh buffer 5001 attached to the kit was added to the GFX column, centrifuged at 15,000 rpm for 30 minutes for 30 seconds, and the collection tube together with the solution eluted in the collection tube was discarded. Next, add a 1.5 ml sample tube to the GFX column. Set, add 201 of 10 mM Tris buffer (pH 8.0) containing 1 mM EDTA to the GFX column, leave it at room temperature for 1 minute, centrifuge at 15,000 rpm for 1 minute, The PCR product eluted in one sample tube was collected. 11 of the recovered PCR product was removed, electrophoresed on a 3% agarose gel, and a 244 bp PCR product band was confirmed by ethidium bromide staining.

If a band was confirmed here, the recovered PCR product was used as a test sample in the sequencing reaction described below.

On the other hand, if the 244 bp band is not confirmed, it is considered that the PCR product to be used for the sequencing reaction is insufficient, and thus PCR amplification was performed using the geno5 primer and the geno6 primer. In other words, 2/1 of the PCR product solution of "Amplicor ™ HC V Monitor-1 v2.0" was added to 10 X PCR buffer (Advanced Biotechnology, Inc.) 21 and 1 OmM dNTPs solution (Packin · Elma 1) 1.6 1 and 25mM MgC 12 (Advanced Biotechnology 1) 1. 2 1 and 10 ΡΠΙΟΙΖ 1 geno 5 primer solution 0.8 1 and 10 pmolZ l of geno 6 primer solution 0.8 zl and rThermostable DNA polymerase ”(Advanced Biotechnology, Inc.) Add 0.5 U and add a total volume of 20 / i 1 at 94 ° C for 3 minutes, then 94 ° C for 30 seconds, 56 ° C 35 cycles of 30 seconds, 72 ° C, and 90 seconds were performed, followed by a reaction at 72 ° C for 10 minutes.

The entire amount of the PCR product thus obtained was similarly purified using a GFX column, and subjected to a sequencing reaction as a sample to be tested.

(3) Sequencing (base sequence determination)

The sequencing reaction was performed according to a direct nucleotide sequencing method (cycle sequencing method) using the geno7 primer.

That is, in 2a1 of the sample (PCR product), “BigDye Terminator Rmix”-Kin'Elma 1) 4 1 and 3.2ΡΠΙΟΙΖ1 geno 7 primer The solution 0.51 and sterile distilled water were added to make a total volume of 10 / X1, and 35 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 60 ° (:, 4 minutes) were performed 35 times. After the sequencing reaction, a deionized formamide dye solution was added to the reaction solution, and the base sequence was determined using an ABI 377 automatic sequencer (Perkin-Elma).

(4) HCV 5'-NCR nucleotide sequence database and typing

(1) The HCV genes in the above 57 patient samples were of three types, 1b, 2a and 2b, according to the Okamoto method. The nucleotide sequence information of the HCV 5'-NCR of these samples was used as a database for creating a genotyping algorithm for determining the genotype. 11 ヮ)

For type 3a, sequence information of existing HCV strains (GenBank No. HCV6311, HCV6318 and CV6323) was used.

By carefully observing the data base, the 1'67, 1163 and 1161 bases of HCV's 5'-NCR can be identified as 1b, 2a, 2 The genotypes of b and 3a are common, and it was found that it is possible to distinguish or identify these genotypes based on the base information of these three places. That is, for the HCV-RNA type 1b, the 5'-NCR of HCV was the 1167th power "T", the 1163rd power "Α", and the 161st power "G".

In the HCV-RNA type 2a, the HCV 5'-NCR had the 1st and 6th force "D", the 1st and 16th force "G", and the 1st and 6th force S "G".

In the HCV-RNA type 2b, the 1'67th of the 5'-NCR of the HCV was "D", the 163rd force "G", and the -16th force "A".

In HCV-RNA type 3a, the HCV 5'-NCR had the 1st and 6th force S "C:", the 1st and 63rd force "G" and the 1st and 16th force "G". .

(2) In the above patient sample, subtype 1a did not exist, and we examined the type determination of 1b. That is, using the serum of a hemophilia patient whose HCV genotype was determined to be 1a or 1b by the Okamoto method, the nucleotide sequence information of the 5′-NCR of HCV was determined according to Example 1. Obtained.

As a result, the 1a type of HCV-RNA is as follows: HCV 5'-NCR, -167 is "D", 1163 is "A", and 1161 is "G"; The results were consistent with the results for the type 1b of HCV-RNA. In addition, in all 11 cases of type 1a, the base at position 99 was “A” in all cases.

Based on the above, it is possible to distinguish or identify the type and subtype of HCV-RNA based on the base information at positions 1 167, 163, 161 and 199 of the 5'-NCR of HCV. I found it.

In other words, if HCV 5'-NCR's 1st 67th force "T", 1st 163rd force "Α" and _ 16th 1st force "G" are identified as HCV-RNA type 1 can do.

Furthermore, in this type 1, if the 199th is “G”, it can be identified as HCV-RNA type 1b, and if it is “A” (about 5% With few exceptions) it can be virtually identified as type 1a.

Example 2 HCV type determination

HCV genotype was determined according to Example 1 using the serum of a hepatitis C patient.

That is, the sample diary from patients sera was determined viral loads in "Amplicor ^TM HC V monitor V 2. 0", the 5 HCV '- to determine the nucleotide sequence of the NCR, the base information shown in Example 1 Based on type determination.

The results of the measurement of the amount of HC V-RNA and the results of genotyping for all 290 samples including the 57 cases in Example 1 are as follows.

HCV-RNA was quantified in 284 of the 290 samples, and the amount of virus in the subjects was determined. By using the PCR product of the quantification method, the nucleotide sequence of the HCV 5'-NCR could be determined in all 290 samples. According to the above, information on both the amount of HCV-RNA and its genotype in a subject can be obtained extremely easily, and this is extremely useful clinically for predicting the therapeutic effect of interferon in the subject.

The results of the above genotyping are as follows.

(Type 1):

The number of cases judged as type 1 was 220 cases, which corresponded to the type judgment result by the Okamoto method (all cases were 1b). In all of these examples, the base at position 167 is “T”, the base at position 163 is “Α”, and the base at position 161 is “G”.

The 159th base was characterized as “C” (however, “C” was detected in eight cases and “C” and “T” were detected in three cases).

The 115th base was characterized as "C".

The 13 th base was characterized as “G”.

The 128th base was characterized as "A" (except that "T" was detected simultaneously in one case).

The 124th base was characterized as "C" (except that "G" was detected simultaneously in one case).

The 122nd base was characterized as "T" (provided that "C" or "G" was detected simultaneously in each case).

-The base at position 19 was characterized as "A" (however, "G" was detected simultaneously in two cases).

(Types 1a and 1b):

Of the 220 cases determined to be type 1 above, none of the samples were determined to be type 1b and type 1a by type determination by the Okamoto method.

In addition, the 199th base in the 220 cases and the sample of the above-mentioned hemophilia patient was as follows. In all 11 cases of type 1a, the 199th base was characterized as "A".

In all 320 cases of type 1b, the 199th base was characterized as "G"

(However, "A" was detected in 16 cases of less than 5%).

(2a type):

The number of cases determined to be type 2a was 42 cases, and in all of these cases, the 167th base was “D”, the 163rd base was “G” and the 161st base Base was “G”.

Of these 42 cases, 41 cases were in agreement with the type determination result by the Okamoto method. The non-matching example is one in which the Okamoto method was regarded as (l b + 2a) mixed type, and it is considered that the main method (type 2a) was determined in the determination method of the present invention.

The 159th base was characterized as "A".

The base at position 155 was characterized as "T".

-The second base was characterized as "Α".

The 128th base was characterized as “として”.

The 124th base was characterized as "C".

The 1 2 2nd base was characterized as "C".

The base at position 119 was characterized as "C" (however, "C" and "T" were detected in two cases in one case).

(2b type):

In all 26 cases determined to be of type 2b, the base at position 167 is `` D '', the base at position 163 is `` G '', and the base at position 161 is `` A '' Was.

All of these 26 cases were in agreement with the results of typing by the Okamoto method.

The 159th base was characterized as "A".

The base at position 155 was characterized as "Ding".

The first 132 base was characterized as "A".

The 128th base was characterized as "Ding". The 124th base was characterized as "Ding".

The base at position 122 was characterized as "C".

The 119th base was characterized as "T".

(3a type):

In these 290 cases, no sample was determined to be type 3a.

(Type 1 + 2 b type) Mixing:

In these two examples: — 167th base “D”, 163rd base “A” and “G”, 161st base “A” and “G”, 1159 “A” and “C” as the 55th base — “C” and “C” as the 55th base, “A” and “G” as the 132nd base, and “A” as the 128th base "And" T "," C "and" C "as the 124th base," C "and" C "as the 122nd base," A "and" C "as the 11th base T ”was detected respectively.

Based on the above, according to Table 1, it was determined to be a mixed type of (type 1 + 2b type), which was consistent with the type determination result by the Okamoto method.

Based on the above, based on the nucleotide information of the HCV 5'-NCR at the 16th, 16th and 16th nucleotides, HCV genotype type 1 and subtypes 2a, 2b and 3 It was found that discrimination or identification of a can be performed extremely easily. In addition, it was found that the above type 1 can distinguish or identify subtypes 1a and 1b based on the 99th base information of the 5′-NCR.

In addition, the bases of the 5'-NCR's 159th, -155th, -132th, 1128th, 1124th, 1122th and 1119th bases are similarly It was found that genotypes can be determined more accurately by further referring to at least one base information as necessary.

The relationship between the base information and the genotype according to the present invention is as shown in Table 1. is there. Tables 2 to 4 below summarize the test results.

Table 2 Comparison of genotyping results of the present invention with typing results by the Okamoto method

Table 3 Comparison of genotyping results of the present invention with group-based measurements (selog looping)

In Table 3, the measurement by group is based on the commercially available kit (IMCHECK, F-HCVGR). "Kokusai"; International Reagents Co., Ltd.). In addition, “Untyped” in the group-based measurement indicates cases where the determination was not possible by the same method (examples in which neither group 1 antibody nor group 2 antibody could be detected) and cases in which determination was suspended (both group 1 antibody and group 2 antibody) Example in which was detected and could not be determined).

According to Table 3, in the group-based measurement, 41 cases out of 290 cases could not be judged or the judgment was suspended, and the judgment information by group as a treatment guideline could not be obtained. It can be seen that this information can be obtained for many samples.

Also, from Table 4, the quantification of the viral load was less than 0.5 (KIU / ml) in 6 of 290 cases, but the genotype determination of the present invention was possible in these samples, and the determination of the present invention was possible. The method was considered to be highly sensitive and useful also in terms of virus detection. Industrial applicability

According to the HCV genotyping method of the present invention, HCV genotypes 1 (1a and 1b), 2a, 2b and 3a are reliably determined based on the base information of 5′-NCR of HCV. It is possible to perform HCV-RNA quantification and genotyping in clinical specimens in a simple and easy operation and in a short time. Therefore, by using the HCV genotyping method of the present invention, it is possible to analyze the degree of progression of hepatitis C, the rate of transition to cirrhosis or liver cancer, the characteristics of symptoms, etc., and predict the therapeutic effect of interferon. It greatly contributes to the decision of a treatment policy suited to each patient.

Claims

The scope of the claims

1. Determine at least the bases at positions 167, 163, and 161 of the 5 'untranslated region of the hepatitis C virus gene. Based on the base information, determine genotype type 1 and subtype 2a. A method for determining the genotype of hepatitis C virus, which comprises determining 2b, 3b and 3a.

2. Determine at least the nucleotides at positions 199, 116, 163, and -161 of the 5 'untranslated region of the hepatitis C virus gene, and determine the genotype subtype la based on the base information. , Ib, 2a, 2b and 3a. A method for genotyping hepatitis C virus.

3. At least one member selected from the group consisting of positions 1 159, 1 155, 1 132, 128, 1 124, 1 122 and 1 119 of the 5 'untranslated region of the hepatitis C virus gene 3. The method according to claim 1 or 2, wherein the base information is further referred to.

4. The method according to any one of claims 1 to 3, wherein the genotype is determined using a PCR amplification product of the 5 'untranslated region of the hepatitis C virus gene.

5. The method according to claim 4, wherein the PCR amplification product is a PCR amplification product of the hepatitis C virus-RNA quantitative assay.

6. The method according to any one of claims 1 to 5, wherein the nucleotide sequence is determined by a direct nucleotide sequencing method using a primer comprising the nucleotide sequence of SEQ ID NO: 1.

7. A primer consisting of the nucleotide sequence of SEQ ID NO: 1.

8. A method for providing information on the amount of HCV-RNA in a sample and its genotype. The following steps (a) to (mouth):

(A) amplifying the 5 'untranslated region of the hepatitis C virus gene by PCR and quantifying the amount of hepatitis C virus-RNA; (Mouth) a step of purifying the PCR amplification product prepared in step (b), and

A method for determining the genotype of hepatitis C virus, comprising:

9. The method according to claim 8, wherein the PCR amplification product prepared in step (a) is a DNA fragment comprising the nucleotide sequence of positions 274 to 131 of the 5 'untranslated region of the hepatitis C virus gene. .

10. The determination method according to claim 8 or 9, wherein the PCR amplification product prepared in step (a) is further subjected to PCR amplification using a primer set in the region as type III.

11. The method according to any one of claims 8 to 10, wherein the purification in the step (mouth) is performed by column chromatography using a glass carrier.

12. The determination method according to any one of claims 8 to 11, wherein the determination of the nucleotide sequence in step (8) is performed by a direct nucleotide sequencing method.

13. Using a sample containing hepatitis C virus collected from a hepatitis C patient, the following steps (a) to (mouth):

(B) amplifying the 5 'untranslated region of the hepatitis C virus gene by PCR and quantifying the amount of hepatitis C virus-RNA;

(Mouth) a step of purifying the PCR amplification product prepared in the step (b), and

(8) determining the nucleotide sequence of the PCR amplification product purified in step (mouth), and determining the genotype of hepatitis C virus-1 RNA;

A method for predicting the therapeutic effect of interferon in said hepatitis C patient.