JPH0751080B2 - A simple method for detecting the human hepatitis C virus genome - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for detecting a viral genome
Related to the polymerase chain reaction, especially from biological samples.
Samples for use are simply prepared and polymerase chain reaction is used.
To detect Te, simple of human hepatitis C virus genome
According to Do detection method.

[0002]

2. Description of the Related Art Recently, as one of diagnostic methods for viruses and genetic diseases, Polymerase Chain Rea
ction: hereinafter abbreviated as “PCR”) method has come to be used. The PCR method is a method that amplifies the target DNA gene region based on the specificity of the primer by a factor of 1,000,000 or more (when the viral genome is RNA or when a mutation in mRNA is detected, RNA is extracted from the RNA prior to PCR. It is necessary to convert it into DNA), and in principle, it is a high-sensitivity detection method that can amplify only one copy of viral genome or target mRNA. Therefore, the PCR method is
As a method of detecting A mutation and the virus itself, and after infection,
The usefulness is recognized in that a biological sample before the antibody is produced can be used as a specimen.

In order to perform this PCR reliably, the step of extracting the desired DNA or RNA from the biological sample and converting it into DNA using reverse transcriptase in the case of RNA should be performed reliably and as much as possible. The point is whether or not it can be done simply. However, when the mRNA or viral genome is RNA, it is difficult to extract, and because the PCR method is a highly sensitive detection method, it may be detected as “positive” even by slight contamination. It's a problem. This contamination
The scattering of mRNA and viral genome-containing substances generated by opening and closing the lid of the microtube, pipetting, etc., and the amplified target gene attached to the laboratory equipment and the hands and clothes of the measurer carry-over. -It is caused by

It is known that viruses are roughly classified into a DNA virus having a DNA genome and an RNA virus having an RNA genome. Among RNA viruses, there are many viruses that require diagnosis, such as retroviruses such as adult T-cell leukemia virus and AIDS virus, and influenza virus and hepatitis C virus. On the other hand, mRNA expressed in various genetic diseases
It is known that the disease is caused by the production of an abnormal protein due to the mutation.

[0005] For example, Lesh-Nyhan (LN) Mr. syndrome Non-port xanthine phosphoribosyltransferase (HPRT)
It is a genetic disease caused by mutations in the gene locus
It is said that there is a mutation in the mRNA. Among RNA viruses, human non-A non-B hepatitis virus accounts for 90-95% of hepatitis caused by blood transfusion, and the treatment method has not yet been established. There is a strong demand for the development of a treatment agent, a prophylactic vaccine, etc. for detoxifying human non-A, non-B hepatitis virus, which is sometimes present in blood for transfusion. In recent years, human non-A non-B hepatitis virus itself has been studied, and it has been reported that single-stranded RNA may be involved in infection (Japanese Patent Laid-Open Nos. 62-24999 and 63-63).
No. 91328), also cloned liver or et cDNA Chimpanzee Chiron US were infected with human non-A, non-B hepatitis virus in recently, this vector λgt11
When expressed in Escherichia coli incorporated into E. coli, the product was found to react with the sera of patients infected with human non-A non-B hepatitis virus. Ultimately this is a single-stranded RNA viruses with about 10,000 nucleotides proven virus close to flavivirus, said Chiron, which human hepatitis C virus (Hepatitis C Viru
s: while designated hereinafter abbreviated as "HCV"), published a cDNA salt <br/> group sequence of have about 7,000 nucleotides Donitsu of which (European Patent Publication 318,216).

[0006] Furthermore, the above Chiron company has developed a method for measuring the antibody in the serum of HCV-infected patients using the polypeptide expressed in yeast using this part of the gene as an antigen. In this measurement method, anti-HC
Since the V antibody appears, it is currently considered to be suitable for the differentiation and diagnosis of viral chronic liver diseases and the screening of blood for transfusion.

[0007]

[Problems to be Solved by the Invention and Object of the Invention] According to conventional techniques, RNA virus genome and
The method for preparing a sample that extracts mRNA, converts it into DNA, and allows the PCR reaction as it is has not been established.

Conventionally, in order to extract and separate the viral genome and mRNA, treatment with a protein denaturant such as guanidine isothiocyanate, phenol, urea and chloroform and a surfactant such as sodium dodecyl sulfate is carried out. centrifugation for deproteinization, or recombinant Shi moved to another tube and collect viral genome or mRNA-containing solution layer, drying causes further precipitation of the viral genome and mRNA by alcohol to remove the treating agent from the solution layer is there.

Since these operations require time and labor, they are not suitable for processing a large number of samples, and also increase the probability of occurrence of contamination. Moreover, the viral genome and m
When the amount of RNA is very small, the recovery rate by alcohol precipitation is low (50% or less), and in addition to the inability to obtain quantitative results, it is necessary to redissolve the precipitate and then convert RNA into DNA by reverse transcriptase. There is a big problem where there is.

The object of the present invention is to obtain human C-type liver in a biological sample.
Simplify this if you have a flu virus (HCV)
The method can cause contamination in a short time.
With or without extracting the RNA virus genome,
Is converted into DNA to prepare a PCR sample, and this sample is used.
PCR of the human hepatitis C virus genome is performed.
It is to provide a simple detection method .

The present inventors have simplified HCV RNA from biological samples.
Extract in stool, convert this to DNA, and immediately perform PCR
We first examined the possible methods, and treated biological samples with proteolytic enzymes in the presence of nonionic surfactants , and then with reverse transcriptase, to obtain PCR samples.
And reach the pre-stage of the object of the present invention.
It came to be achieved . Therefore, in addition to PCR primers
As a result of diligent studies, it was considered suitable for detection of HCV
A primer set that can be found was found.

In the extraction method according to the present invention, the biological sample may be serum, plasma and excised tissue, and nonidet p-40 or the like may be used as the nonionic surfactant. Further, proteinase K or the like can be used as the proteolytic enzyme.

Therefore, according to the present invention, the above-mentioned object is not
Processing the biological sample by 蛋 white degrading enzyme in the presence of an ionic surfactant, the possible presence in the extract obtained Oh
Human hepatitis C virus RNA is converted to DNA, and 5'-GAGTGCCGCTCACACCTTTCCTT
A primer having a base sequence of ACATCG-3 'and 5'-AAGCCTGCTAGATACTGTATCC
A reaction solution containing a primer having a base sequence of CGC-3 'and Taq polymerase was added, the first polymerase chain reaction (PCR) was performed on the above DNA, and then 5'-CTTCCTTACATCGAACAAGGA-
A primer having a base sequence of 3 ', 5'-TTCCACATGTGCTTCGCCCAG-
3 'consists of the reaction mixture containing the primers and Taq polymerase having a base sequence by adding a second round of polymerase chain reaction performed (PCR), then it corresponds to the product of the viral genome to be detected more electrophoresis dynamic van <br/> de of 176bp are you characterized by checking whether the detected human
It is achieved by a simple detection method of the hepatitis C virus genome.

The inventors of the present invention have diligently studied the reaction conditions, etc., and found that when serum or plasma is used as a biological sample, the amount of the sample is within 1-20 μl, and when biological tissue is used, it is 10 mg. If it is kept within the range, neither complicated removal treatment of denatured protein nor alcohol precipitation operation is required, and tube exchange is not required. Extraction of viral genome and RNA in one reaction tube are not required. It was found that the reverse transcription reaction to the DNA and the PCR reaction can be performed.

The detection method according to the present invention is carried out by the conventional method of Kumzdinsky et al. Using guanidine isothiocyanate ("A nalytical Biochemistry ", 162 , 156-159, 1).
987), and the detection sensitivity was about 1% compared to the case where the viral genome product was detected using the PCR method.
I understand also that you are 0 times better, of several peak in a biological sample
It was found that RNA was sufficient to detect it.

[0016]

EXAMPLE etc.] following the real施例further and specifically described in detail the present invention by the test examples and comparative test examples.

[0017] microtube extraction 0.5ml capacity of the viral genome from Example (a) human biological sample, a sample derived from a living body
Blood whey 10μl of HCV patients injected Te, to which 1μl of solution [500 mM tris (hydroxymethyl) aminomethane - HCl buffer (pH 8.0), 250 mM potassium chloride, 30 mM magnesium chloride, 0.5% Nonidet p-40, and 10mg / Add ml Proteinase K] and mix. Then 35 ° C
1 hour to obtain a subsequently incubated by Ri RNA U Irusugenomu extract to 10 minutes at 94 ° C. in.

When the volume of plasma as a biological sample is 10 μl or less, a solution obtained by diluting 10 times the same solution as the above-mentioned solution except that it does not contain nonidet p-40 and proteinase K is added. Adjust the volume to 10 μl. When a tissue is used as a sample, 10 μl of a 10-fold diluted solution of the same solution as above except that it does not contain nonidet p-40 and proteinase K per 10 mg of tissue.
Add and incubate as well.

[0019] (b) amplifying the extracted exudates 11μl primer (described later) of 20pmol to the DNA by transformation and PCR to DNA from RNA, the reaction solution containing reverse transcriptase 14 units [50 mM tris (hydroxymethyl) aminomethane - Hydrochloric acid buffer (pH8.0), 25m
M potassium chloride and 3 mM magnesium chloride, 250 μM each
Four deoxyribonucleotides (dATP, dGTP, dC
TP and dTTP), containing 2.5 mM dithiothreitol and 40 units of RNasin] 14 μl at 40 ° C.
Complementary and by Ri RNA to be incubated for 1 hour
The specific D NA was synthesized.

A reaction solution containing 5 units of Taq polymerase in this mixed solution containing this RNA and complementary DNA
[50 mM Trishydroxyaminomethane-hydrochloric acid buffer (pH
8.0), 50 mM Potassium Chloride, 1.5 mM Magnesium Chloride, 0.001% Gelatin] Add 75 μl, 94 ℃ for 30 seconds, 55 ℃
The first PCR consisting of 20 cycles was performed with 1 minute at 72 ° C and 1 minute at 72 ° C. Furthermore, the first PC
Transfer 10 μl of R reaction solution to another 0.5 ml microtube,
Reaction solution containing 20 pmol of primer (see below) and 5 units of Taq polymerase [10 mM Trishydroxyaminomethane-hydrochloric acid buffer (pH 8.0), 50 mM potassium chloride, 1.5
mM magnesium chloride, 200 μM each of 4 kinds of deoxyribonucleotides (dATP, dGTP, dCTP, dTTP)] 90 μl was added,
The second PCR of 30 cycles consisting of the same system as the first PCR was performed. Then, after electrophoresis, it was stained with ethidium bromide, and it was confirmed that a band having a predetermined base pair length corresponding to the HCV product was present. HCV
In the detection of the above, a part of the cDNA sequence of HCV shown in FIG. 1 and cloned by the present inventors (Japanese Patent Application No. 1-344876) was used as a primer.

That is, in the first PCR, the 22nd to 49th sequences in SEQ ID NO: 1 of the sequence listing were selected.
That 5 '- GAGTGCGCCTCACACCTTCCTT
ACATCG-3 ' with a primer having a nucleotide sequence of 203 to 227
5'-AAGCCTGCTAGATACTGTATCC which is the sequence up to and including the sequence up to
CGC - 3 'becomes a primer having the nucleotide sequence are used, also in the second round of PCR, 37 in SEQ ID NO: 1
CTTCCTTACATCGAACAAGGA - 5 'Ru array der from up to 57-th-th -
A primer having a base sequence of the 3 ', from 1 74th 194
Is the sequence of SEQ complementary strand to turn first 5 '- TTCCACATGTGCTTCGCCCAG -
A primer having a 3 'becomes nucleotide sequence was used. still,
The base pair length corresponding to the HCV genomic product is 176 bp.

Test Example Setting of concentration range of proteinase K required for extraction of viral genome To determine the concentration range of proteinase K required for extraction of viral genome, 1 μ of the lysate mentioned in the examples was determined.
and the same varying concentrations of proteinase K in l
Virus genomic products were detected by PCR using plasma of HCV patients as a sample . The results are shown in Figure 1.

[0023] The concentration of proteinase Inaze K 0 - 10mg / m l
Fig. 1 shows the results of detection of viral genome products by setting to 1
Gas collector in migration patterns of lanes No. 5 - as indicated by 11, 2.5 - H of interest in the range of 10mg / ml
A band of the CV genomic product was detected and thus the protein
It was found that a concentration of the casease K of 5 mg / ml or more is sufficient . Comparative Test Example Comparison of detection sensitivity between the method of the present invention and the conventional method The viral genome extraction method adopted in the method of the present invention
When the extraction method such as com Sophora root skis using conventional guanylyl Jin isothiocyanate [ "Analytical Biochemistry",
Vol 162, page 156 -. 159 (1987)] the plasma from HCV patients as a sample to compare the detection sensitivity in the case of using, to thereby detect the viral genome product by P CR.

[0024] Figure 2 extracts the HCV genome in both methods described above, it is to be shown the results of detection by the PC R. Book
In the case of the method of the invention, the desired HC was obtained by using 10 μl of plasma.
V Genome product was detected.
If 10 μl plasma is used for genome extraction in
It is impossible to product detection, since the detection is required plasma on 100μl about or more than, compared to the conventional method
The present invention method has been found to be superior about ten times in the sensitivity detect when.

Figures 3 and 4 show 12 cases including HCV patients.
Using plasma to perform genome extraction by both methods described above and PCR
It is also to indicate the result of detection of genomic products by. Figure
3 the extraction method used in the present invention a method blood whey 10 [mu] l
A case applied to, while Figure 4 is co Mujinsuki over such
When the extraction method described above was applied to 100 μl of plasma (Fig. 3
In 4 and 4, the common lane number is the same HCV patient.
Or, the result of using plasma derived from a healthy person as a sample
The result is shown) .

From these results, in both methods, the band of the desired PCR product was detected in the same patient plasma used in lane numbers 1, 3, 4, 5, and 7 of the electrophoretic pattern, and the virus was detected by the method of the present invention. It has been revealed that even if the genome is extracted, it is possible to detect with a reliability equal to or higher than that of the conventional method.

[0027]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 240 base pairs Sequence type: Nucleic acid Number of strands: single Topology: linear Sequence type: genomic DNA Origin organism name: human PROPERIES: human hepatitis C virus CAGAGATGAGTGATGAGGAGTGTTG TCACACTTCCTCTACATCGA ACAAGGAATG 60 CAGCTCGCCG AGCAATTCAA GCAGAAGCGCG 90 CTCGGGCTGCC TGCAGTAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGTGCCCCGTAGCTAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCGAGCTAGCTAGCTAGCTACT T CACGGGTAGA 210 CAGTATCTAG CAGGCTTGTC CACCCTGCCT 240 SEQ ID NO: 2 Sequence length: 240 base pairs Sequence type: Nucleic acid Number of strands: single Topology: linear Sequence of origin: GatCruGitC ROGitCruGitRuGitRuGuRuGitRuGuRuGuRuGuRuGuRuGuRuIuRuGuRuGuRuTaiGuRuGuRuIuRuRuIuRuIuRuIuRuIuRuIuRuIuRuIuRuIuRuIuRuhuman. ATGAGATGGA GGAGTGCGCT 30 TCACACCTCC CTTACATCGA ACAGGGGAATG 60 CAGCTCCGCCG AGCAATTCAA GCAGAAGGGCG 90 CTCGGGCTGT TGCAGAGCAGCGAGCGCGCGCGCGCGCGCGCGCGCGCGCT TCTTGAGAC CTTCTGGGCG 180 AAGCACATGT GGAATTTCAT CAGCGGGATA 210 CAGTACCTAG CAGGCTTGTC CACTCTGCCT 240 SEQ ID NO: 3 sequence length of the: 240 base type pairs sequence: the number of nucleic acid strands: single Topology: linear sequence type: genomic DNA source organism name: human PROPERTIES: human hepatitis C virus CA
AGAGTTCG ACGAAATGGA AGAGTGCGCC 30 TCACACCTCC CTTACATCGA ACAGGGAATG 60 CAGCTCGCCG AGCAATTCAA GCAGAAAGCG 90 CTCGGTTTGC TGCAGACAGC TACCAAGCAA 120 GCGGAGGCTG CAGCCCCCGT GGTGGAGTCC 150 AAGTGGCGGG CCCTTGAGAC TTTCTGGGCG 180 AAGCACATGT GGAATTTCAT CAGCGGGATA 210 CAATACTTAG CAGGCTTATC CACTTTGCCT 240 SEQ ID NO: 4 sequence Length: 240 Type of base pairs SEQ: nucleic acid strands Number of: single Topology: linear Sequence type: genomic DNA Origin organism name human PROPERTIES: human hepatitis C virus CCGGCGTTCG ATGAAATGGA AGAGTGTGCC 30 TCACACCTCC CTTACATCGA ACAAGGAATG 60 CGGCTCGCCG AACAATTCAA GCAGAAGGCG 90 CTCGGGTTGC TGCAAACGGC CACCAAGCAA 120 GCGGAGGCTG CCGCTCCCGT GGTGGAGTCC 150 AAGTGGCGTA CCCTTGAGGC CTTCTGGGCG 180 AAGCACATGT GGAATTTCAT CAGCGGGATA 210 CAATACCTAG CAGGCTTGTC CACTCTGCCT 240 SEQ ID NO: 5 SEQ length of: 240 base Pairs Sequence type: Nucleic acid Number of strands: ingle Topology: linear sequence type: genomic DNA source organism name: human PROPERTIES: human hepatitis C virus CAGGAGTTTG ATGAGATGGA GGAGTGCGCC 30 TCACACCTTC CTTACATCGA ACAAGGAATG 60 CAGCTCGCCG AGCAATTCAA GCAGAAGGCG 90 CTCGGGCTGC TGCAAACGGC CTCCAAGCAA 120 GCGGAGGCTG CTGCTCCCGT GGTAGAGTCC 150 AAGTGGCAAG CCCTTGAGGC CTTCTGGGCG 180 AAGCACATGT GGAATCTCAT CAGCGGGATA 210 CAGTATCTAG CAGGCTTGTC CACCCTGC T 240

[0028]

EFFECTS OF THE INVENTION According to the method for extracting mRNA and virus genome of the present invention, the removal treatment of denatured proteins and the precipitation treatment operation with alcohol are not required. Therefore, when detecting specific mRNAs and viral genomes from biological samples, the reaction of the samples with proteolytic enzymes and nonionic surfactants begins and RNA to DNA is performed as necessary.
The process from reverse transcription to polymerase chain reaction (PCR) can be performed continuously in a single reaction tube, which simplifies the operation and greatly establishes contamination. Can be reduced to

In the case of the present invention, the time required for extraction of mRNA and viral genome was shortened to within 2 hours, which is less than half the time required by the conventional method, and the human hepatitis C virus
In the case of detecting (HCV) as an HCV genome product using the PCR method, it took 2 days according to the conventional method, but it can be performed within 8 hours by using the method of the present invention. Since the present invention is simple, detection can be automated or semi-automated.

[Brief description of drawings]

FIG. 1 shows the results of detection of viral genome products using the plasma of HCV hepatitis patients in the same manner as described in Example 2 in order to examine the concentration of proteinase K required for extraction of viral genomes. It is shown and is a copy of the electrophoretic pattern.

FIG. 2 is a copy of an electrophoretic pattern showing the results of extracting the viral genome by the method of the present invention and the conventional method, detecting the viral genome product by PCR, and comparing the detection sensitivities.

[Figure 3]

FIG. 3 and FIG. 4 show the method according to the present invention and the method of Komkudinsky et al. Using guanidine isothiocyanate [Analytical Biochemistry 162,156-159, (1987)].
Was applied to the plasma of HCV patients, but the amount of plasma used was 1/10 in the case of the present invention, and the viral genome was extracted by applying it, and then the viral genome was detected in the same manner as described in Example 2. The results are shown, and the respective electrophoretic patterns are copied.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12Q 1/68 ZNA A 9453-4B (72) Inventor Takahito Shiromori Higashitobori-cho, Higashi-ku, Nagoya-shi, Aichi 35, Sanwa Chemical Research Institute, Inc. (72) Inventor Haruo Takahashi, Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Prefecture 35, Inc. 35, Machi Sanwa Chemical Research Institute Co., Ltd. (72) Inventor Eiji Suzuki, 35, Higashitoboricho, Higashi-ku, Nagoya-shi, Aichi Prefecture Sanwa Chemical Research Laboratories, Inc.

Claims (4)

[Claims] 1. A non-ionic in the presence of a surfactant treated biological sample by proteolytic enzymes, the possibility of Ah Ruhito hepatitis C virus RNA present in the extract thus obtained was DNA
Convert to 5'-GAGTGCCGCCTCACACCTTCCCTT
A primer having a base sequence of ACATCG-3 'and 5'-AAGCCTGCTAGATACTGTATCC
A reaction solution containing a primer having a base sequence of CGC-3 'and Taq polymerase was added, the first polymerase chain reaction (PCR) was performed on the above DNA, and then 5'-CTTCCTTACATCGAACAAGGA-
A primer having a base sequence of 3 ', 5'-TTCCACATGTGCTTCGCCCAG-
3 'consists of the reaction mixture containing the primers and Taq polymerase having a base sequence by adding a second round of polymerase chain reaction performed (PCR), then it corresponds to the product of the viral genome to be detected more electrophoresis dynamic van <br/> de of 176bp are you characterized by checking whether the detected human
Simple test Deho of the hepatitis C virus genome. Wherein characterized in that the biological sample is selected from serum, plasma and excised tissue claim
Simple inspection Deho of human hepatitis C virus genome according to 1. 3. Oh proteolytic enzyme is in proteinase K
You characterized by that, the human C according to claim 1 or 2
Simple test Deho types of hepatitis virus genome. When using serum or plasma as wherein the biological sample, the amount of the sample 1 - within 20 [mu] g, also in the case of using a biological tissue you characterized in that keep within 10 mg, claim 1 , 2 or 3 human hepatitis C
Simple test Deho of Irusugenomu.