JPH0523200A - Highly sensitive detection method of non-a non-b type hepatitis virus using oligonucreotide primer and oligonucleotide primer - Google Patents

Abstract

PURPOSE:To obtain a new oligonucleotide primer used for diagnosis excellent in specificity and sensitivity of non-A non-B hepatitis. CONSTITUTION:An oligonucleotide primer, e.g. TAGATTGGGT or GTGCGCGCGA, having a base sequence specific to non-A non-B hepatitis virus RNA and obtained by nucleotide system.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting non-A non-B hepatitis virus (hereinafter abbreviated as "NANB hepatitis virus") at a gene level with high sensitivity, and an oligonucleotide primer used for the method.

[0002]

2. Description of the Prior Art Hepatitis B, hepatitis A, D, etc. have been established for hepatitis derived from viruses because DNA and RNA of the virus itself have already been identified, and methods for their diagnosis and prevention have been established.
There are hepatitis C and hepatitis E. However, with regard to non-A non-B hepatitis (hereinafter referred to as “NANB hepatitis”), which is mainly hepatitis transmitted through blood and does not belong to the above classification, many researchers in the world have searched for the causative virus. Despite this, the virus itself was not elucidated.

In 1988, Chiron became a NAN.
As the causative virus of hepatitis B, an RNA virus genome named hepatitis C virus (hereinafter abbreviated as "HCV") was successfully cloned, and its nucleotide sequence was announced. Based on this, an HCV antibody measurement system has been developed, and is now being used for screening transfused blood and diagnosing patients. This HCV antibody measurement system is certainly found to be partially related to NANB hepatitis. However, there are still serious problems such as the capture rate of carriers and chronic hepatitis of only about 70%, and the inability to detect antibodies in the acute phase.
Coping with hepatitis B is still unsolved. On the other hand, a method for detecting hepatitis B virus has been established, and as a result, 95% or more of post-transfusion hepatitis in Japan is NANB.
It is hepatitis, and it is estimated that about 280,000 cases occur annually.
The course of NANB hepatitis is poor, and most cases are expected to become carriers and progress to chronic hepatitis. Further, since it progresses to liver cirrhosis over 10 to 20 years at a fairly high rate and further to liver cancer, it is desired to elucidate the virus itself and develop a diagnostic agent as soon as possible.

[0004]

[Problems to be Solved by the Invention] HC of Chiron Corporation
There are many NANB hepatitis that cannot be diagnosed by the V antibody detection kit.
Since it has been revealed that the ANB hepatitis virus is considerably different in nucleotide sequence from HCV elucidated by Chiron, it is indispensable to develop a NANB hepatitis diagnostic method which is further excellent in specificity and sensitivity. The object of the present invention is to
It is intended to provide a method for detecting NB hepatitis virus at a gene level with high sensitivity, and an oligonucleotide primer used for the method.

[0005]

The present inventors have found that NANB
For the purpose of elucidating the gene body of hepatitis virus, RNA of NANB hepatitis virus was isolated from sera of humans and chimpanzee carriers, and cDNA covering the entire gene region including the non-coding region of the virus was cloned to obtain its nucleotide sequence. Were determined. As a result, the inventors
It was clarified that there is a highly conserved region of NANB hepatitis virus and a region having many mutations among virus strains, and the present invention was completed based on this.

The gist of the present invention is as follows. The NANB hepatitis virus RNA of the present invention was obtained as follows, and its nucleotide sequence was determined. Five types of samples were obtained from human and chimpanzee plasma. HC-J1, HC-J5, HC-J
6 was obtained from a Japanese blood donor, HC-J7 was obtained from a Japanese renal dialysis patient, and all were obtained from specimens judged to be positive for HCV antibody. HC-J4 was derived from a sample obtained from a chimpanzee confirmed to have NANB hepatitis infectivity, but this was (-) in the HCV antibody from Chiron.
RNA was extracted from these 5 types of sample plasma. Furthermore, each R
The homology of the nucleotide sequence between NAs was examined, and the nucleotide sequences of about 1900 to 2500 on the 5'end side and about 1100 on the 3'end side of the extracted RNA were determined as shown in the examples. Among them, the nucleotide sequences of HC-J1 and J4 and primers based on them are already described in Japanese Patent Application No. 2-1534.
We disclosed some of them in 02, but this time we newly added HC-J5,
The present invention has been completed by researching the non-coding region of all strain genes including J6 and J7 strains, the region encoding the structural protein, and a part of the region encoding the non-structural protein.

As shown in Example 1, HC-J1, HC
RNA from each of the -J4, HC-J5, HC-J6, and HC-J7 strains is 340 to 341 from the 5'end.
It has a 5'untranslated region consisting of 4 nucleotides, followed by a region coding for a structural protein (1149 bases)
, Including the region encoding nonstructural proteins,
It has 1885 to 2551 nucleotides.
Based on this finding, the polynucleotides shown in SEQ ID NOS: 1 to 5 were obtained.

Further, as shown in Example 3, HC-J
In 1, J4, J5, J6, and J7, it was found that the 3'-terminal side of RNA has a non-coding region gene of 77 bases or more including T-stretch following 1096 bases encoding a non-structural protein, Based on this, the polynucleotides shown in SEQ ID NOS: 6 to 10 were obtained.

European Published Patent No. 388232
When compared with the sequence of HCV shown in No. No. (Chiron), the nucleotide sequence of the present invention has J1, J4, J6, J with respect to 5477 terminal 2477 bases containing a non-coding region.
In each of the 7 strains, 95.2%, 80.5%, 72.
It has homology of 5% and 71.6%, and is 1 in HC-J5 strain.
It shows only 76.8% homology with 468 bases. This indicates that the HC-J1 strain is an American type virus relatively closely related to the virus strain identified by Chiron, whereas the J4, J5, J6, and J7 strains are slightly different from this, and the Japanese type virus is slightly different. It was suggested that.

In order to detect a virus by the polymerase chain reaction, which has high specificity and high sensitivity, the present inventors have prepared primers based on the cDNAs of SEQ ID NOs: 1 to 10 and SEQ ID NOs: 11 to 42. A method using primers was established. The 5'non-coding regions of the five viral RNAs elucidated by the present inventors all consist of 340 to 341 bases, and the nucleotide sequence is highly conserved. For example, the base sequence difference is 8 bases between HC-J1 and HC-J4, 17 bases between HC-J1 and HC-J5, and the most distantly related HC-J1 and HC.
-J7 has only 20 bases and is closely related to HC-J5 and HC
-In J6, 340 bases were exactly the same. It is surprising that the nucleotide sequences of the 5 strains of viral RNA are very well conserved in the 5'non-coding region in consideration of the fact that the strains among the strains in the regions other than those mentioned above vary greatly. Further, in the coding region, the upstream portion following ATG has relatively few mutations. Therefore, oligonucleotides derived from these regions # 23, # 25,
It was found that NANB hepatitis virus RNA can be detected with high sensitivity without being influenced by the strains, that is, regardless of strains, by using # 32, # 33, # 36, and # 48 as primers.

Based on the elucidated nucleotide sequences, when the amino acid sequences at the 5'-terminal side of the two strains J1 and J4 were compared, the homology of the amino acid sequence was higher in the upstream region than in the downstream region, and in the upstream region. Has a high hydrophilicity because it contains many basic amino acids such as arginine. Since basic amino acids are often found in core proteins and give affinity to nucleic acids, the upstream region encodes the viral core protein, the downstream region contains many amino acid substitutions, and the eight sugar chain binding sites are common. It was presumed that the protein encodes an outer coat (envelope) protein.

Similarly, comparing the homology of the amino acid sequence on the 5'-end side between each strain in the upstream region and the downstream region, HC-J1 and HC- in the aa1-191 presumed to be the core region are compared.
High homology between J4 and HC-J6 and HC-J7 is shown with 96.9% and HC-J6 and HC-J7.
It is 90.6% between J6 and 90.6% between HC-J4 and HC-J7. In addition, in aa192 to 383 estimated to be the envelope part, HC-J1 and H
77.6% between C-J4, HC-J6 and HC-J
It is 71.4% between 7 and is considerably lower than the core part. Furthermore, HC-J1 and HC-J
6 showed a very low homology of 60.9%, and HC-J4 and HC-J7 showed a very low homology of 52.6%.

On the other hand, the homology between the strains of the 3'-terminal side sequence was 87.4% between HC-J1 and HC-J4 and 8 between HC-J6 and HC-J7 for 365 amino acids.
While it is 9.6%, it is a little low between HC-J1 and HC-J6, 73.7%. As for 39 bases of non-coding region, 64.1% between HC-J1 and HC-J4 and 8 between HC-J6 and HC-J7.
While it was 2.1%, it was considerably low at 30.8% between HC-J1 and HC-J6, reflecting the difference between American type and Japanese type and that there are many mutations among Japanese type. Was assumed to be.

Based on the above findings, the present inventors
As a suitable oligonucleotide primer used for highly sensitive detection of ANB hepatitis virus at the gene level, the following oligonucleotides having the nucleotide sequences of SEQ ID NOs: 11 to 42 were found, and all of them were synthesized to complete the present invention. . Each of these oligonucleotides can be used as a primer. The nucleotide sequences of the oligonucleotide primers of the present invention, whether sense: antisense, and the virus strains detected by each primer are as follows. # 23 (SEQ ID NO: 11; +; all strains) # 25 (SEQ ID NO: 12;-; all strains) # 32 (SEQ ID NO: 13; +; all strains) # 33 (SEQ ID NO: 14; +; all strains) # 36 (SEQ ID NO: 15;-; all strains) # 38 (SEQ ID NO: 16; +; J5 strain) # 41 (SEQ ID NO: 17;-; J5 strain) # 48 (SEQ ID NO: 18;-; all strains) # 50 (sequence No. 19; +; J5, J6, J7 strains) # 51 (SEQ ID NO: 20;-; J5 strain) # 54 (SEQ ID NO: 21;-; J5, J7 strain) # 55 (SEQ ID NO: 22; +; J5 strain) # 57 (SEQ ID NO: 23; +; J4 strain) # 61 (SEQ ID NO: 24; +; J4 strain) # 63 (SEQ ID NO: 25;-; J5 strain) # 78 (SEQ ID NO: 26; +; J4 strain) # 80 (SEQ ID NO: 27; +; all strains) # 90 (SEQ ID NO: 28; +; J6 strain) # 91 (SEQ ID NO: 29; +; J1 strain) # 9 7 (SEQ ID NO: 30; +; J6 strain) # 100 (SEQ ID NO: 31; +; J1 strain) # 122 (SEQ ID NO: 32;-; J6, J7 strain) # 123 (SEQ ID NO: 33; +; J7 strain) # 124 (SEQ ID NO: 34; +; J7 strain) # 127 (SEQ ID NO: 35;-; J7 strain) # 128 (SEQ ID NO: 36;-; J6 strain) # 129 (SEQ ID NO: 37; +; J7 strain) # 136 ( SEQ ID NO: 38;-; J7 strain) # 145 (SEQ ID NO: 39; +; J7 strain) # 148 (SEQ ID NO: 40; +; J1 strain) # 149 (SEQ ID NO: 41; +; J6 strain) # 150 (SEQ ID NO: 42; +; J7 strain) In the present invention, any of these oligonucleotides can be used as a primer (claim 5).
Section).

As described above, the J1 strain is an American type virus, while the J4, J5, J6, and J7 strains are Japanese type viruses. Therefore, these Japanese type NANB hepatitis viruses can be detected or their strains can be detected. In order to carry out the identification of # 38, # 41, # 5 among the oligonucleotides of the present invention,
0, # 51, # 54, # 55, # 57, # 61, # 6
3, # 78, # 90, # 91, # 97, # 100, # 1
22, # 123, # 124, # 127, # 128, # 1
29, # 136, # 145, # 148, # 149, # 1
It is particularly useful to use 50 as the primer.

In the present invention, as described above, each oligonucleotide having any of the nucleotide sequences of SEQ ID NOS: 1 to 10 is a part of the 5'-terminal non-coding region of NANB hepatitis virus RNA and / or the coding region. Was found to be specific to a part of the oligonucleotide, and based on this, these oligonucleotides were synthesized as the primers of the present invention (claim 4).

The present invention is also applicable to oligonucleotides containing a small number of substitutions derived from mutations between strains, as long as oligonucleotides specific for NANB hepatitis RNA are used, and these are also within the scope of the present invention. Included.

When the NANB hepatitis virus is detected by using the PCR method, a more preferable effect can be obtained by using the primer of the present invention in combination. A suitable example for this combination is
# 23 and # 25, # 32 and # 36, # 33 and # 48, #
38 and # 41, # 50 and # 54, # 50 and # 63, # 5
0 and # 128, # 54 and # 55, # 127 and # 129,
Each of the primer pairs # 136 and # 145 may be mentioned (claim 6).

The present invention also provides claims 4 to 6.
It is an invention of a NANB hepatitis virus detection system having the primer according to item (claim 7).

As described above, the present invention provides a method for detecting NANB hepatitis virus with high sensitivity using an oligonucleotide primer having a nucleotide sequence specific to a part of the coding region and the non-coding region of NANB hepatitis virus RNA. It is the invention (claim 1).

Furthermore, the present invention uses NANB hepatitis virus RNA with a pair of oligonucleotide primers.
Is an invention of a method for detecting the viral RNA with high sensitivity by amplifying the cDNA of Escherichia coli by polymerase chain reaction (Claim 2).

In that case, it is preferable to perform cDNA amplification twice using two pairs of oligonucleotide pairs in order to detect NANB hepatitis virus with high sensitivity (claim 3).

[0023]

As described above, the method of the present invention can detect the NANB hepatitis virus at the gene level with high sensitivity, and the oligonucleotide primer of the present invention is
It can be used for highly sensitive detection of ANB hepatitis virus.

[0024]

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

Example 1 The 5'-terminal base sequence and amino acid sequence of NANB hepatitis virus were determined as follows. (1) RNA extraction Specimens (HC-J1, J5, J6) determined to be positive for HCV antibodies (according to the Ortho HCVAb ELISA test of Ortho Diagnostic Systems Co., Ltd.) obtained from plasma of Japanese blood donors, Japan Samples obtained from human renal dialysis patients and also determined to be positive for HCV antibodies (HC-J
7), RNA was extracted from the HCV antibody (-) sample (HC-J4) obtained from chimpanzees confirmed to have NANB hepatitis infectivity as follows. 1.8
1 ml Tris-HCl buffer (10 mM, p
H8.0) was added, and the mixture was centrifuged at 68 × 10 ³ rpm for 1 hour. 200mM NaCl, 10m on the obtained pellet
Tris-hydrochloric acid buffer solution (50 mM, pH 8.0) containing M EDTA, 2% (w / v) sodium dodecyl sulfate (SDS) and 1 mg / ml proteinase K was added, and the temperature was 60 ° C.
After heating for 1 hour and extracting with phenol / chloroform, ethanol precipitation was performed to obtain RNA.

(2) cDNA synthesis RNA extracted from HC-J1 plasma was heated at 70 ° C. for 1 minute, and 10 units of reverse transcriptase (c
DNA Synthesis SystemPlus,
Amersham Japan) and oligonucleotide primer (20-mer) 20 pmol 42
CDNA was obtained by reacting at 1.5 ° C. for 1.5 hours. As the primer, # 8 (5 ') synthesized with reference to the nucleotide sequence of HCV shown in European Patent Application No. 8831022.5.
-GATGCTTGCGGAAGCAATCA-3 ')
Was used.

(3) Amplification of cDNA by polymerase chain reaction (PCR) was performed as follows.
DNA Thermal Cycler (Perkin-Elmer
-Cetus) using the Gene Amp DNA amplification reagent kit (Perkin-Elmer-Cetus), the method of Saiki et al. (Science 239, 48)
7-491.1988) for 35 cycles of cDNA
A amplification was performed.

(4) Determination of base sequences of J1, J4 and 5'ends by constructing a cDNA library HC-J1 and HC-J4 using a cDNA library
As shown in FIGS. 1 and 2, analysis of the nucleotide sequence on the 5'end side of the genome was performed to analyze the clone obtained by inserting the cDNA into bacteriophage λgt10 and to analyze the clone obtained by amplifying the cDNA by PCR. Both of the results were determined together. 1 and 2 show the 5'end of the NANB hepatitis virus genome together with a restriction enzyme cleavage site, and also show the positions of the primers used. In the figure, the solid line shows the range of the base sequence determined by the clone of the bacteriophage λgt10 library, and the dotted line shows the range of the base sequence determined by the clone by PCR. HC-J1 nt454 ~
The 1656 bases of 2109 is the cD obtained from primer # 8.
NA to λgt10 phage vector (Amersha
It was determined by the clone φ41 obtained by inserting into m). Next, nt8 synthesized based on this sequence
24-843 new primer # 25 (5'-TCC
Using CTGTTGCATAGTTCACG-3 '), four cDNA clones φ60, φ61, φ66, φ75 were sequentially obtained from the HC-J4 cDNA library, and the nucleotide sequences of upstream nts 18 to 843 were determined. HC
-For J4, antisense primer # 36 at nt 246-265 to identify the 5'end further upstream.
(5'-AACACTACTCGGCTAGCAGT-
3 ') was used to synthesize cDNA, and then terminal deoxynucleotidyl transferase was used to synthesize cDNA.
DA is added to the 5'end of A, and two-step one-Si
dedPCR amplification was performed, that is, the first time was oligo dT primer (20-mer) and nt188-207.
Antisense primer # 48 (5'-GTTGAT
PCR amplification for 35 cycles was performed using CCAAGAAAGGGACCC-3 ′), and the second time using the PCR product as a template, oligo dT primer (20-mer) and n were used.
Antisense primer # 109 from t140 to 160
(21-mer; 5'-ACCGGATCCGCAGA
30 cycles of PCR using CCACTAT-3 ')
I went. The obtained PCR product was subcloned into the M13 phage vector. Six independent clones, C896, believed to have the full length 5'end sequence.
2, C8968, C8970, C8974, C903
4, C9036 was obtained, and the nucleotide sequences of nt1 to 17 were determined.

On the other hand, the upstream sequence of HC-J1 is
For nt 18-843, primer # 44 (5'-
GGCGACACTCCACCCATGAAT-3 ') and # 25 (5'-TCCCCTGTTGCATAGTTCA
Determined by clones C2503, C2508, and C2510 obtained by PCR with CG-3 '). At the 5'end further upstream, 16 clones C8931, C were obtained by the same method as HC-J4.
8932, C8935, C8937, C8942, C8
944, C8949, C8950, C8951, C89
54, C8955, C9023, C9026, C903
0, C9031, C9032 were used to determine the nucleotide sequence of nt1-37. Next, in order to determine the nucleotide sequence downstream of the 5'end of HC-J1, primer # 148 (5'-
TGCACCTGGGATGAACTCAAC-3 ') and # 146 (5'-AGTAGCATCATCCACAA)
30 cycles of PCR were performed using GCA-3 '). The obtained clones C11444, C11450,
The nucleotide sequence of nt 1984 to 2560 was determined by C11451. On the other hand, nt738-1900 of HC-J4
Downstream sequence up to 1163 bases is primer # 3
0 (5'-CTCATGGGGTACATTCCGCT
-3 ') and # 42 (5'-TCGGTCGTCCCCCA
3 clones C2821, C3173, C3192 obtained by PCR with CCACAAC-3 ')
Determined by Furthermore, in order to determine the nucleotide sequence downstream of HC-J4, primer # 57 (5'-TATT
GCTTCACCCCAAGCCC-3 ') and # 146
(5'-AGTAGCATCATCCACAAGCA-
PCR was performed for 30 cycles using 3 '). Obtained clones C11462, C11463, C11464
The nucleotide sequence of nt1860 to 2560 was determined by.
From the above analysis results, HC-J1 and HC-J4 are 5 '
It was determined to have the nucleotide sequences shown in Sequence Diagrams 1 and 2 on the terminal side (SEQ ID NOS: 1 and 2), respectively.

(5) Determination of base sequence of J5, 5'end by construction of cDNA library The base sequence of 5'end of HC-J5 genome is shown in FIG.
As shown in (C), it was determined from the analysis result of the clone obtained by amplifying the cDNA by PCR. RNA was extracted from the HC-J5 sample in the same manner as above, and its sequence was determined. nt6 to 1868 was determined from each clone obtained by PCR with the following primer combinations. nt24-265 # 32 (5'-ACTCCACCATAGATCACTCC-3 ') # 36 (5'-AACACTACTCGCGCTAGCAGT-3') clone; C3534, C3536, C3537 nt63-508 # 33 (5'-TTCACGCAGAAAGCGTCTAG5'51 #). '-GACCGCTCCGAAGTCTTCCT-3') clone; C4658, C4659, C4660 nt 467-843 # 23 (5'-TAGATTTGGGTGTGCGCGCGCA-3 ') # 25 (5'-TCCCTGTTGCATAGTTCACG-3') 24, 53, 53C53, 53C51; 50 (5'-GCCGATCTCATGGGGTACAT-3 ') # 63 (5'-CTGG GTTCTTAACCTGGAC-3 ') clone; C5592, C5593, C5594 nt867-1354 # 55 (5'-ATTTTCTTGCTGGCCCTGCT-3') # 54 (5'-ATCGCGTACGCCCAGGACATCAT-3 ') clone; 5'-TGCAATTGTTTCTATCTACCC-3 ') # 41 (5'-CAGGGCTTGGGGGTGAAGCAA-3') clone; C3722, C3748, C3753 nt1842 to 1976 # 4 (5'-AGTGGTGTGTGTGTGCTGTATA-3'GCTGAG5CCGGTATA-3 ') CGT5G (CCGGTATA-3'GCTGAT5G). ) Clones; C3952, C3969, C3990

In order to further identify the 5'-terminal side, 3 cDNA clones C8995, C8997 and C8998 were obtained by the same method as in Example 1- (4), nt1.
The base sequence of ~ 160 was determined.

From the above analysis results, HC-J5 was 5 '
It was determined to have the base sequence shown in SEQ ID NO: 3 on the terminal side. In the non-coding region on the 5'side in HC-J5, 5 consecutive nts 6 to 10 in HC-J1.
1 of the 3 cytosines is missing, for a total of 340
It has become clear that it has non-coding region bases.

(6) Determination of base sequence of J6, 5'end by construction of cDNA library The base sequence of 5'end of HC-J6 genome was amplified by PCR as shown in FIG. It was determined from the analysis results of the obtained clones. RNA was extracted from the HC-J6 sample by the same method as above, and its sequence was determined. nt24 to 2551 was determined from the clones obtained by PCR with the following combinations of primers. nt24-826 # 32 (5'-ACTCCACCATAGATCACTCC-3 ') # 122 (5'-AGGTTCCCTGTTGCATAATT-3') clone; C9397, C9388, C9764 nt732-1907 # 50 (5'-GCCCACCTCATGGGGTACAT5 '#). '-TCGGTCGTGCCCACTACCAC-3') clone; C9316, C9752, C9753 nt 1847-2571 # 149 (5'-TCTGTGTGTGGGCCCAGTGTA-3 ') # 146 (5'-AGTAGCATCATCCACACAAGCA-3'21, 116C, 11C, 11C, 11C;

In order to further identify the 5'-terminal side, 13 cDNA clones, C9577, C9579, C9581 and C958, were prepared in the same manner as in Example 1- (4).
4, C9587, C9590, C9591, C959
5, C9606, C9609, C9615, C961
6, C9619 was obtained, and the base sequence of nt 1-160 was determined.

From the above analysis results, HC-J6 was 5 '
It was determined to have the base sequence shown in SEQ ID NO: 4 at the end. In the 5'non-coding region of HC-J6, 5 consecutive nts 6 to 10 in HC-J1.
1 of the 3 cytosines is missing, for a total of 340
It was revealed that it has a non-coding region base.

(7) Determination of base sequence of J7, 5'end by construction of cDNA library The base sequence of 5'end of HC-J7 genome was amplified by PCR as shown in FIG. It was determined from the analysis results of the obtained clones. RNA was extracted from the HC-J7 sample by the same method as above, and its sequence was determined. nt24-2498 was determined from each clone obtained by PCR with the following primer combinations. nt24-826 # 32 (5'-ACTCCACCATAGATCACTCC-3 ') # 122 (5'-AGGTTCCCTGTTGCATAATT-3') clone; C10621, C10622, C10623 nt732-1354 # 50 (5'-GCCGACCTCATGGGGACAT5 '#). '-ATCGCGTACCGCCAGGATCAT-3') clone; C10461, C10463, C10615 nt 1309-1625 # 129 (5'-CGCATGGCATGGGGATATGAT-3 ') # 127 (5'-ATGTGCCAACTGCCGTTTGGT-111, 111'83, 111'83, C111T85GT, C111, C111, C111, C111, C111, C111, C111. 145 (5'-CAGGATATCAGTCTAAT CAA-3 ') # 136 (5'-ACTGGGGCTGGGAGTGAAACA-3') clone; C11361, C11364, C11374 nt1833-2518 # 150 (5'-ATCGTCTCTGCGTAAGACGGT-3 ') # 146 (5'-AGTAGCATCACATCACACAAG). C11535, C11540, C11566

In order to further identify the 5'-terminal side, 8 cDNA clones were prepared in the same manner as in Example 1- (4).
C10513, C10515, C10521, C105
54, C10558, C10568, C11231, C
11232 was obtained, and the base sequence of nt 1-160 was determined.

From the above analysis results, it was determined that HC-J7 had the base sequence shown in SEQ ID NO: 5 on the 5'end side. HC-J1, J4, J disclosed by this example
Unlike the HCV sequence described in the above-mentioned European Patent Publication No. 388,232, the 5'-terminal side nucleotide sequences of 5, J6 and J7 were first disclosed by the present invention.

Example 2 (1) Determination of 3'-terminal side nucleotide sequence by construction of cDNA library The nucleotide sequence of 3'-terminal side of each genome of HC-J1, J4, J5, J6 and J7 is as shown in FIG. In addition, it was determined by the analysis result of the clone obtained by amplification by PCR. HC-
93 of nt1 to 938 of J1, J4, J5, J6, and J7
For the 8-base sequence, use each sample with primer # 80
(5'-GACACCGCTGTTTTTGACTC-
3 ') and # 60 (5'-GTTTCTTACTGCC
It was subjected to PCR using CAGTTGAA-3 ') and determined from the nucleotide sequence of each amplification product. The resulting clone is shown below. HC-J1 C7391, C7343, C7377 HC-J4 C5584, C5585, C5586 HC-J5 C7212, C7257, C7261 HC-J6 C9760, C9234, C9761 HC-J7 C9928, C11125, C11141 nt939 base downstream 3'terminal side sequence. Was determined according to the following method. That is, RNA was extracted from each sample according to the method described in Example 1, and poly (A) was added to the 3'end of the RNA using poly (A polymerase. CDNA synthesis was performed using oligo (dt) ₂₀ as a primer. The obtained cDNA was subjected to PCR using the template as a first step, and as a first step, a 20-mer oligonucleotide specific to each strain cDNA was used as a sense primer and oligo (dt) ₂₀ was used as an antisense primer.
And then the obtained cDNA was subjected to the second step P
The specific sense primer corresponding to the downstream of the first step as CR is oligo (d
t) ₂₀ was used. The T4 DNA polylase was allowed to act on the amplification product obtained by the two-step PCR to make both ends blunt, and then the 5'end was phosphorylated with T4 polynucleotide kinase, and Hin of the M13mp19 phage vector was used.
The base sequence was determined by subcloning into the cII site.

For each strain, the two types of primers used for PCR and the clones obtained are shown below. HC-J1 # 100 (5′-AAGGCTGCCATATGTGGCAA-3 ′) # 91 (5′-GCCATATTGGGCAAGTACCT-3 ′) Clone: C9707, C9714, C9719, C9724, C9726, C9730, C9737, C9738, C9747, C9742, C9742 , C9936, C9943, C9945, C9949) HC-J4 # 61 (5'-TTGCGAGTCTGGAGACCATCG-3 ') # 78 (5'-TGTCCCGCGCTAAGCTACTGT-3') Clone: C8761, C8764, C8776, C8784, C8783, C8796. C8811, C8812, C8819, C8825, C8849, C8851) HC-J5 # 97 ( '-AGTCAGGGCGTCCCTCATCT-3') # 90 (5'-GCCGTTTTGCGGCCGATATCT-3 ') Clone: C10820, C10827, C10829, C10843, C1 0844, C10848, C10849, C10864, C10865, C10872) HC-J65'5 #. -AGTCAGGGCGTCCCTCATCT-3 ') # 90 (5'-GCCGTTTGCGGCCGATATCT-3') cloned: C10311, C10313, C10314, C10320, C1 0322, C10323, C10326, C10328, C103 30, C10333, C10334, C10336, C10337, C10345, C10346 , C10347, C10349, C1035 , C10357) HC-J7 # 123 (5'-CTTAGAGCGTGGAAGAGTCG-3 ') # 124 (5'-GCCATCTGTGGCCGTTACCT-3') cloned: C10801, C10804, C10807, C10809, C1 0811, C10812, C10814, C10815, C108 18) From the above analysis results, HC-J1, J4, J5, J
6, J7 are SEQ ID NOS: 6, 7, 8 and
It was determined to have the nucleotide sequences shown in 9 and 10. HC-J1, J4, J5, J6 disclosed by the present embodiment,
The 3'-terminal nucleotide sequence of J7 overlaps at nt 7938-8866 (929 nucleotides) of the HCV sequence described in the above-mentioned European Patent Publication No. 388,232, but the sequence of the present invention is distinctly different from that of the present invention. It was first disclosed by.

Example 3 Synthesis of primer based on 5'untranslated region and core protein coding region and establishment of assay system (1) Synthesis of oligonucleotide primer of HC-J1 and HC-J4 strains determined in Example 1 Based on the nucleotide sequences of the 5'translated region and the core protein coding region,
An oriconucleotide primer (20mer) was synthesized. Also, European Patent Application No. 88310922.5
The oligonucleotide primer was also synthesized with reference to the nucleotide sequence of HCV shown in No. The synthesis is performed by the 380B DNA synthesizer (Applied Biosystems Jap).
an). The synthesized primer is # 3,
4, 5, 6, 9, 10, 11, 12, 16, 17, 2
1, 22, 23, 25, 32, 33, 34, 35, 3
Table 6 shows the positions and nucleotide sequences from the 5'-end of each primer.

[0042]

[Table 1]

(2) NANB hepatitis virus R from the sample
90 ml of NA blood extract 1 ml with TL-100 (Beckman) centrifuge
Centrifuge at Krpm for 15 minutes and collect the resulting pellet for 20 minutes.
0 mM NaCl, 10 mM EDTA, 2% (W / V)
Sodium dodecyl sulfate (SDS), Tris hydrochloric acid (50
mM, pH 8.0), 1 mg / ml proteinase K
Was dissolved in a solution containing and heated at 60 ° C. for 1 hour. After extracting twice with an equal amount of a phenol / chloroform solution, ethanol precipitation was performed at -20 ° C for 3 hours or more. The precipitate was centrifugally washed with 70% ethanol, vacuum-dried and then 5
It was dissolved in μl of distilled water.

(3) Synthesis of cDNA RNA extracted from 1 ml of plasma in (2) was
It was denatured at 0 ° C. for 1 minute, rapidly cooled on ice, and subjected to cDNA synthesis. Reverse transcription was performed using 100 pmol of antisense primers # 5, 6, 11, 12, 16, 17, 25, and
35 nm, 36 nm, and 48 nmol of 10 kinds of four deoxyribonucleotide triphosphates (Takara), 10 unit
RNase inhibitor (Takara), 10 units of Avianemeloblastosis virus
Add reverse transcriptase (Boehringer Mannheim) to 10μ
1 Tris-hydrochloric acid (50 mM pH 8.4), 8 mM M
gCl ₂ , 30 mM KCl, 1 mM dithioth
In reitol solution, react at 42 ° C for 90 minutes to give cDN
A was synthesized. The obtained cDNA was purified by extraction with phenol / chloroform.

(4) Amplification by PCR DNA thermal cycler (Perkin)
-Elmer Cetus) and DNA amplifi
Cation reagent kit (Perkin
-Elmer Cetus) using the usual method (Sa
iki et al 1988). The reaction cycle consisted of denaturation (94 ° C for 1 minute), primer annealing (5
5 ° C 1 min 30 sec) and primer extension (72 ° C 3
Step), and this was repeated 35 times. The reaction products were 1-1, 5% Nusieve and 1-1, 5% Se.
Electrophoresed on a Kakem mixed agarose gel (FMC). After staining with ethidium bromide, bands were confirmed by irradiation with ultraviolet rays.

(5) Amplification by secondary PCR The PCR product obtained by PCR with the first set of primers (for example, # 32 and # 36) is further subjected to the second PCR amplification, if necessary. The first one as a primer
The inner primer of the pair of primers (eg # 33 and #
48) was selected and 30 cycles were repeated for 5 μl of PCR product. The reaction conditions were denaturation (94 ° C., 1 minute) annealing (55 ° C., 1.5 minutes) and extension (72 ° C., 2 minutes), and the obtained PCR product was electrophoresed in the same manner as (4). Analyzed.

Example 4 Selection of Primer Pair Effective for Use in Detection System of NANB Hepatitis Virus by PCR 10 specimens already determined to be anti-HCV positive, that is, plasma specimen No. of Japanese blood donors. 1, 3, 5, 7,
9 and serum sample Nos. Of NANB hepatitis patients. 2, 4, 6,
Table 2 shows the results of PCR using two sets of primers for 8 and 10.

[0048]

[Table 2]

The target nucleotide sequence region is HC shown in European Patent Application No. 88310922.5.
V non-structural protein NS5 to 2 region, NS3 to 2 region,
PCR amplification was attempted on two regions of the region extending upstream from NS1 and two regions of the core region revealed by the present inventors, a total of 10 regions from the 5'untranslated region to two regions. As a result, when using the primer pair # 32 / # 36, # 33 / # 48 from the 5'untranslated region and the primer pair # 23 / # 25 from the core region, 10
145 bp in 3 regions for all samples,
242 bp, and N of the expected size of 377 bp
The ANB cDNA band could be detected. But the other 7
In the region, 2 to 9 out of 10 samples were only amplified, while the presence of HCV RNA was confirmed. From this fact, as a pair of primers for detecting a wide range of HCV RNA, # 32 / from the 5'untranslated region was detected.
# 36 and # 33 / # 48 and # 23 / # 2 in the core area
It was concluded that 5 was efficient. In some cases, only the primary PCR may be sufficient to detect NANB hepatitis RNA, but it is preferable to further perform the secondary PCR to increase the sensitivity. For example, after performing cDNA synthesis using the # 36 primer, PCR amplification (primary P
CR) was performed, and for a sample in which the expected band of 242 bp was not observed, # 33 using PCR-amplified RNA as a template
And the # 48 primer were used for re-amplification (secondary PCR). (Double PCR)

Example 5 10 specimens of chronic hepatitis patient and 12 blood serum of a normal donor of ALT
RNA detection by PCR was performed on the sample. The results are shown in Table 3.

[0051]

[Table 3]

NANB type 32 specimens were previously
-As a result of measuring HCV, 20 samples were positive and 12 samples were negative. The 10 type B patients and 12 normal blood donors were all anti-HCV negative. NANB type 3
Of the two specimens, 20 specimens of anti-HCV (+)
15 cases of primary PCR and 5 cases of secondary PCR and 100% RN
A could be detected. 12 which was designated as anti-HCV (-)
Regarding the example, 7 cases in the first PCR and 4 cases in the second PCR,
92% RNA detection was possible. In addition, 10 cases of B type and 12 cases of blood donors performed the secondary PCR,
It was negative. From this data, NANB virus R
As a NA detection system, a PCR method using an oligonucleotide primer is excellent, and particularly, according to the double PCR method performed up to the secondary PCR, the sensitivity is extremely high (50% or more higher than that of HCV antibody). It was confirmed that the detection method has high specificity in 96.9%, which corresponds to almost all NANB samples.

Example 6 Detection Sensitivity of NANB Hepatitis Virus by cDNA / Double PCR Method The detection sensitivity of NANB hepatitis virus RNA by the cDNA / double PCR method of the present invention will be described below.
The results are shown in Table 4.

[0054]

[Table 4]

Plasma of known infectious titer 10 ⁷ CID /
A 10-fold dilution series was prepared using ml, and the same measurement was performed 3 times. 10 in 2 out of 3 measurements in primary PCR
The detection of a band of 242 bp predicted to 0 CID / ml and 10 CID / ml for the remaining one time was confirmed.
By performing the secondary PCR, 10 CID can be obtained in two measurements.
/ Ml, a band of 145 bp predicted to 1 CID / ml was observed in one measurement. Also, 1 CID / m
No band was observed at dilutions below 1 and negative plasma. Mean titer of NANB hepatitis patient is believed to ¹⁰ 2 to 4 CID / ml per plasma, NANB
It was confirmed to have clinically effective detection sensitivity in the diagnosis of hepatitis C patients.

[0056]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for detecting NANB hepatitis virus, which has a high degree of nuisance and is specific to the virus. Therefore,
It can play an important role in preventing post-transfusion hepatitis by accurately diagnosing hepatitis patients or screening blood donations.

[Brief description of drawings]

The figure which shows the method of nucleotide sequence determination of NANB hepatitis virus.

FIG. 1 is the HC-J1 strain.

FIG. 2 is the HC-J4 strain.

FIG. 3 is the HC-J5 strain.

FIG. 4 shows HC-J6 strain.

FIG. 5: HC-J7 strain.

FIG. 6 is a method for determining the base sequence of the 3'end of each genome. In the figure, the solid line shows the range in which the base sequence was determined by the clone of the bacteriophage λgt10 library, the dotted line shows the range in which the base sequence was determined by the PCR clone, and the clone name was shown next to the line.

[Sequence list]

Claims (7)

[Claims] 1. A method for detecting non-A non-B hepatitis virus with high sensitivity using an oligonucleotide primer having a base sequence specific to non-A non-B hepatitis virus RNA. 2. The method according to claim 1, wherein a cDNA of non-A non-B hepatitis virus RNA is amplified by polymerase chain reaction using a pair of oligonucleotides to detect the viral RNA. 3. The method according to claim 2, wherein the amplification of cDNA is performed twice using two pairs of oligonucleotide primers. 4. An oligonucleotide primer specific for non-A non-B hepatitis virus RNA having the nucleotide sequences of SEQ ID NOs: 1 to 10. 5. Oligonucleotide primers # 23 and # 2 having the nucleotide sequences of SEQ ID NOs: 11 to 42.
5, # 32, # 33, # 36, # 38, # 41, # 4
8, # 50, # 51, # 54, # 55, # 57, # 6
1, # 63, # 78, # 80, # 90, # 91, # 9
7, # 100, # 122, # 123, # 124, # 12
7, # 128, # 129, # 136, # 145, # 14
8, # 149, # 150. 6. A primer pair selected from the oligonucleotide primers according to claim 5 by the following combination. # 23 and # 25 # 50 and # 63 # 32 and # 36 # 50 and # 128 # 33 and # 48 # 54 and # 55 # 38 and # 41 # 127 and # 129 # 50 and # 54 # 136 and # 145 7. A non-A, non-B hepatitis virus detection system comprising the primer pair according to claim 4.