JPH03259099A - Detection of hepatitis c virus and relating nucleic acid and oligo-dna set for primer - Google Patents

Abstract

PURPOSE:To enable the detection of the subject material in high sensitivity by amplifying a specific DNA and coloring with a color-developing agent or by hybridizing with a labeled and amplified nucleic acid. CONSTITUTION:A nucleic acid prepared from serum or liver tissue made to react with a template-dependent DNA polymerase (e.g. Taq DNA polymerase) to obtain a synthetic cDNA (A). Separately, an oligo-DNA set (B) for amplification primer of hepatitis C virus and its relating nucleic acid is prepared by selecting each one DNA from the DNA group 1 of formula I and the DNA group 2 of formula II. The component A is amplified with the component B and the obtained DNA is separated by agarose gel electrophoresis to obtain amplified DNA (C). The component C is stained with ethidium bromide, etc., and irradiated with ultraviolet radiation to effect the color development and detect the hepatitis C virus and its relating nucleic acid. As necessary, the component C is hybridized with hepatitis C virus labeled with biotin, etc., and the desired nucleic acid is detected.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention is directed to the hepatitis C virus (industrial application) in serum or liver tissue.
The present invention relates to a method for detecting HCV (hereinafter abbreviated as HCV) and its related nucleic acids. More specifically, cDNA is synthesized using a specific primer DNA set and/or hexaoligomer DNA, and the DNA is amplified and colored with a DNA coloring agent or hybridized with a labeled IEHcV nucleic acid to detect HCVa-linked DNA. The present invention relates to a method for detecting nucleic acids and an oligo DNA set for the primers.

<Prior art and problems to be solved by the invention> Viral hepatitis is type A, type B, delta (D), non-A, non-B.
It is estimated that there are two types of non-A, non-B hepatitis viruses: a casually transmitted type and an orally transmitted type. Among these, chronic hepatitis is known in hepatitis B and parenterally transmitted non-A, non-B hepatitis, and preventive treatment of such types of viral hepatitis is currently an important issue.

In hepatitis B, the causative virus has already been identified and diagnosed.
Prevention methods have almost been established, and it is thought that the number of new cases will decrease considerably in the future. On the other hand, non-A
Non-B hepatitis is mainly transmitted through blood transfusions, and currently 95% of the 15-17% of blood recipients (estimated to be 150,000-300,000 people per year in Japan) are infected with this virus. It is considered to be a parenterally transmitted type of non-A, non-B hepatitis.Furthermore, about 50% of these non-A, non-B hepatitis patients progress to chronic hepatitis, and 20% of patients develop chronic hepatitis after 10 to 20 years. It is estimated that non-A, non-B hepatitis can result in liver cirrhosis and liver cancer.In addition to blood transfusions, non-A, non-B hepatitis may require vaccination using a nail, or administering blood or body fluids to multiple people. The number of non-A, non-B hepatitis virus carriers (estimated at 4 million in Japan) and chronic hepatitis patients (estimated at 900,000 in Japan) is increasing due to blood transfusions. There is a need for an effective method to prevent the occurrence of new patients with non-A, non-B hepatitis.One way to do this is to improve the conventional standard of transaminase GPT value of 35 or less as a guideline for the safety of blood that can be used for transfusion. value 25
The following was done. However, even if only blood with a GPT value of 25 or less is safe, 15% of transfused patients will develop serum hepatitis, so a more effective method for determining safety is required.

In 1987, Chiron Corporation of the United States developed a blood transfusion non-/l:B
European patent 0318 to announce hepatitis virus
216. Published May 31, 1989).

According to the announcement, nucleic acids extracted from the serum of chimpanzees that had developed transfusion-induced non-A, non-B hepatitis were isolated and amplified using genetic engineering techniques, and proteins were expressed that reacted with the serum of patients with non-A, non-B hepatitis. Through selection, the causative virus, hepatitis C virus (hereinafter abbreviated as "HCV"), is identified, and those that react with serum using a specific antigen protein are determined to have hepatitis C. Furthermore, the specific hepatitis C diagnostic kit published by Chiron relies on a system that uses ICV nonstructural protein antigen to detect hepatitis C virus antibodies present in patient serum. Even though you are already an HCV carrier at the early stage of infection, there is a possibility that you will be determined to be negative for CV antibodies.For such serum, blood may be transfused if it is determined to be negative for HCV antibodies even though it contains HCV. The publication states that the genomic nucleic acid of HCV is single-stranded RNA, and a portion of its base sequence is also described, and that it is safe to use for the diagnosis of hepatitis C. It also describes some specific base sequences.

However, since the base sequences of viruses with heavy chain RNA genomes often vary considerably, and it is thought that the same is true for HCV, methods for detecting HCV-related nucleic acids with high sensitivity are still insufficient. I have no choice but to.

As is clear from the above description, the specific antigen and base sequence announced by Chiron cannot be considered effective for diagnosing all types of non-A, non-B hepatitis, and it is clear that further investigation is necessary. be.

<Object of the Invention> As described above, the present invention provides a highly sensitive method for detecting hepatitis C virus and its related nucleic acids, and an excellent oligo DNA set for primers used for the detection. Another object of the present invention is to provide a suitable combination of a primer oligo DNA set, a template-dependent DNA polymerase, a labeling substance, and the like. Also, oligo D for primer
The objective is to provide a relative fixed mouth formula by utilizing the difference in detectability of NA sets.

<Structure of the Invention> The present inventors have arrived at the present invention as a result of intensive studies in view of the above situation. That is, the present invention relates to a method for detecting a nucleic acid related to HCv, which is a type of non-A, non-B hepatitis that is transmitted parenterally.

More specifically, a template-dependent DNA polymerase is used to synthesize c[)NA of nucleic acids in serum or liver tissue, and a DNA-dependent DNA polymerase is then used to synthesize c[)NA.
The present invention relates to a method for highly sensitive detection of HCV-infected nucleic acids by amplifying DNA and coloring the amplified DNA or hybridizing it with labeled HCV nucleic acids.

That is, a template-dependent DNA polymerase is used to synthesize C[)NA of a nucleic acid prepared from serum or liver tissue, and then a DNA-dependent DNA polymerase is used to synthesize D
cDNA was generated using oligo DNA sets selected from NA group 1 and DNA group 2 as primers.
This method detects hepatitis C virus and its related nucleic acids by amplifying the hepatitis C virus and then coloring it with a DNA coloring agent, or by hybridizing it with a labeled amplified nucleic acid of a known hepatitis C virus.

DNA group 1: Oligo DNA 1 (5'-GGTCATAGTGGG
CAGGGTCGTCTT-3'), oligo DNA3
(5'-ACCTGGTAGCGTACCAAGC
C-3'>, oligo DNA5 (5'-CTGCT
TGTGGATGATGCTAC-3') DNA group 2: Oligo DNA2 (5'-CCAGGCAGCGTT
GACAAGCCCGCCAAG-3'), oligo D
NA4 (5'-TATGCTTCGCCCAGAA
GGTC-3'>, oligo DNA6 (5'-TC
CACACGTGCAGTTGCGCT-3') In the above detection method, when synthesizing cDNA of a nucleic acid prepared from serum or liver tissue, an oligo DNA set and/or one selected from each of DNA group 1 and DNA group 2 is used. Kisa oligomer DNA can be used.

Template-dependent DNA polymerases include RNA-dependent D
There is NA polymerase or DNA-dependent DNA polymerase. Reverse transcriptase is an RNA-dependent DNA polymerase. Taq DNA polymerase and Escherichia coli DNA polymerase as DNA-dependent DNA polymerases 1. Examples include T4 DNA polymerase.

These can be used to synthesize cDNA of nucleic acids prepared from serum or liver tissue.

Preferably, the reverse transcriptase is derived from Moloney-Mulleen-Leukemia virus.

The labeled amplified nucleic acid of known hepatitis C virus is preferably a nucleic acid that does not contain at least one base sequence of the oligo DNA sets of DNA group 1 and DNA group 2.

Digoxignin or biotin can be used as labels.

It goes without saying that the above-mentioned use of hexa-oligomers, template-dependent DNA polymerases, labeling substances, etc. can be used in combination.

The present invention also provides the following DNA group 1 and UDNA DNA group 2.
It includes oligo DNA sets for amplification primers for hepatitis C virus and related nucleic acids selected from the above.

DNA group 1: Oligo DNAI (5'-GGTCATAGTGGG
CAGGGTCGTCTT-3'), oligo DNA3
(5'-ACCTGGTAGCGTACCAAG
CC-3'), oligo DNA5 (5'-CTGC
TTGTGGATGATGCTAC-3') DNA
N2O 2rigoDNA2 (5' -CCAGGCAGCGTT
GACAAGCCCGCCAAG-3'), oligo D
NA4 (5'-TATGCTTCGCCCAGAA
GGTC-3'), oligo DNA6 (5'-TC
CACACGTGCAGTTGCGCT-3') This method uses a nucleic acid prepared from the serum or liver tissue of a subject whose presence of HCV-related nucleic acids is to be diagnosed. The nucleic acids are obtained by treating crushed liver tissue with protease in serum or an appropriate buffer that does not cause decomposition and denaturation of nucleic acids, followed by treatment with phenol or a phenol/chloroform mixture and, if necessary, isoamyl alcohol. Deproteinize with a mixed solution to which about 1/10 to 1/20 was added, collect the aqueous phase, add an appropriate salt such as sodium acetate, and then add 2 to 3 times the volume of ethanol or the equivalent volume of isopropanol. It can be obtained as a precipitate by high-speed centrifugation. In the present invention, nucleic acids can be prepared by simply removing phenol from the aqueous phase obtained after the above protein removal operation using a poorly water-soluble solvent such as chloroform or diethyl ether.

In the present invention, complementary DNA (cD
NA) is synthesized. There is no particular limit to the amount of oligo DNA to be removed as long as it does not interfere with subsequent operations. Also c
When synthesizing DNA, the above oligo DNA set and/or oligo DNA with a random 6 base sequence (for example, a hexaoligomer, Boehringer Mannheim Yamanouchi)
cD using "random" p(dN)s)
NA can be synthesized. Zaraani corresponding C[)NA
The above-mentioned template-dependent DNA polymerase is used in the synthesis. General commercially available reverse transcriptases can be used and there are no specific restrictions, but examples include AMV (Avian myeloblastosis virus) reverse transcriptase, R8V (Rous sarcoma virus) reverse transcriptase, and M-ML.
V (Moloney Mullin Leukemia Virus) reverse transcriptase can be used. Among these, M-MLV reverse transcriptase is preferably used because it has no endonuclease activity on synthesized cDNA and has low RNASeH enzyme activity. These reverse transcriptases can be used to prepare the cDNA of the present invention according to the manufacturer's instructions for each enzyme. If necessary, M. Taiho DNA polymerase I is added to the thus obtained cDNA to make it into a double-stranded DNA without producing it. When the above-mentioned oligo DNA having a random 6-base sequence is used when synthesizing CD N A, it is preferable to remove excess hexa-oligomer by a commonly known operation such as alcohol precipitation or gel filtration.

In the present invention, cDNA is then amplified using DNA-dependent [)NA polymerase. DN to use
As the A-dependent DNA polymerase, Escherichia coli DNA polymerase 1. T4 DNA polymerase, TaQ DNA polymerase, etc. can be used.

Each of these DNA polymerases may be used according to the supplier's instructions. Among these DNA polymerases, T
Most preferably, a(l[)N polymerase is used. The TaQ DNA polymerase used is proa+e.
ga, boxy brown, bethesda research.
Those sold by Laboratories, Takara Shuzo, etc. can be used according to the manufacturer's instructions. Among these companies, those made by Promega and Boxui Brown are more preferred because they have high enzyme activity. Taq
[) Amplification of itc DNA using NA polymerase was carried out by the method of 3aiki R, K, et al. (Science,
230.1350-1354 (1985).

Nature, 324.163-166 (1
986)) has been developed and can be implemented according to the supplier's documentation.

In the present invention, TaQ DNA polymerase is used to generate DNA.
An oligo DNA set is used as a primer for amplifying A. As the oligo DNA set, a combination set of one of oligo DNA 1.3.5 of the DNA group 1 and one of DNAN2O2 oligo DNA 2.4.6 is used. This selection combination is important because the detection situation changes depending on which primers are selected, and in some cases, detection becomes impossible.

As oligo DNA sets, it is preferable to use a set of oligo DNAs 1 and 4, a set of oligo DNAs 2 and 3, and a set of oligo DNAs 5 and 6 because of their high detectability. Further, by utilizing these differences in detectability, the relative concentration of the virus and its related nucleic acids can be determined. Such quantification results in alanine aminotransferase (ALT
) can be used as a means of diagnosing hepatitis by comparing the correlation with liver function measurements and the relationship with changes over time.

In the present invention, the DNA amplified as described above is subjected to agarose gel electrophoresis, then stained with ethidium bromide, and then irradiated with UV heat to develop color and detect a specific nucleic acid band. The operations of agarose gel electrophoresis of DNA, staining with ethidium bromide, and detection of colored specific nucleic acid bands by UV heat irradiation can be carried out by methods generally known in the art.

When a specific DNA band is not observed by color development, Southern hybridization is performed with a known amplified HCV nucleic acid labeled with a specific compound.

As the probe used for hybridization, an amplified HCV nucleic acid labeled with digoxigenin or biotin is used. The amplified nucleic acid used here can be prepared by the same method as described above, but it suffices if it has a partial sequence of nucleic acid in the same region as the one used to prepare the amplified DNA to be detected. It is preferable that the length is at least 15 bases and that it does not contain at least one base sequence in the oligo DNA set used for amplification. To add digoxigenin as a label to the amplified nucleic acid used as a hybridization probe, use the Boehringer Mannheim DNA Labeling Detection Kit.
Alternatively, biotin can be added as a label using a biotin labeling kit manufactured by Bethesda Research Laboratory, following the manufacturer's instructions.

<Effects of the Invention> Unlike hepatitis C determination using an HCV antigen-antibody system, the method of the present invention amplifies and detects HCV-related nucleic acids. Therefore, HCV-related nucleic acids can be detected with high sensitivity even in the early stages of HCV infection or when HCV antibodies are extremely low. This is a useful method because it can detect nucleic acids.

Furthermore, by utilizing the fact that detection accuracy varies depending on the combination of oligo DNA sets, it has become possible to provide a method for relative quantification of )-1cV and its related nucleic acids in a specimen sample.

<Examples> The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

(Examples 1 to 3 and Comparative Examples 1 to 2) Diagnosed with non-A, non-B chronic hepatitis and using Chiron HCV
Using an antibody detection kit, ultracentrifuge 1 d of antibody-positive serum at 50,000 G for 5 hours, and centrifuge the resulting pellet at 10 aM of 70 μρ.
Tris HCJ (pH7,5>/0.IM
Na(J/1mM EDTA/0.1%
2-Mercaptoethanol was dissolved in 10.1% BSA buffer (hereinafter referred to as TNE buffer), this was layered on TNEil buffer containing 30% sucrose, and the suspension was ultracentrifuged at 50,000 yen for 5 hours to obtain a suspension of virus particles. Made by M. The suspension was 0.5
Proteinase was treated at 50°C for 2 hours in the presence of %SO8, and after phenol/chloroform extraction, DNA was precipitated with ethanol, and 3 μp of 10 m Tris
HCf/1 mM EDTA (pH 7,5) (4
unit/μρ of RN asin-containing) solution. To the RNA solution obtained in this way, 50mM Tri
s HCj (pH 8,3) /75 mM K(J
/101M dithiothreitol 15mM MgCa2/4
mMRI! 1-lJ μm/1 mM dNT
Ps R compound (N = A, C, G, T) / 1.6 μ9 random primer (Boehringer Mannheim no.
1034731), 0.2 u/μJ2 AMV reverse transcriptase (total amount 20 μm), and cDN at 42°C for 1 hour.
A was synthesized. Next, this solution was diluted to 251M
Tris H(J (pt-+ 8.3>/100m
M KCN/ 51MMg (Jz 15mM D
TT/200 μm dNTPs mixture and further 1
00 u/dE, RNaseH (final concentration) of coli DNA polymerase I/20 u/7 was added (R final volume 100 μp) at 12°C for 1 hour.
The mixture was reacted at 22° C. for 1 hour to obtain double-stranded DNA.

0.0 for the cDNA solution thus obtained.
5M K(J, 0.IM Tris HCf,
A solution of 15 mM MCI C1z, 0.1% gelatin, 0.1% Tritton
XTaq DNA polymerase solution) 5 μl, sterile water 29 μl, dNTPs mixture (dATP, dCTP
, dGTP, dTTP (1.251 M solution each)
Sentence 1 Oligo DNA set in Table 1 (each oligo DNA△(11
degree 0.1μg/μm) and 2.5u each! l), and further Ta
(I DNA polymerase <promega no. 101423) 1.25 units and 50 ufl of liquid paraffin were added, and the mixture was incubated at 95°C for 1 minute, then at 57°C.
C. for 2 minutes, then 72.degree. C. for 3 minutes, and this three-step temperature/time change operation was repeated 30 times, and finally a polymerase reaction was performed at 72.degree. C. for 7 minutes. After the reaction, 5 μg of the DNA was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and the Uv heat irradiated DNA band was observed. The observed DNA bands were as shown in Table 1.

Table 1 Oligo DNA 1: 5'-GGTCATAGTGGG
CAGGGTCGTCTT-3'oligo DNA2 5'-CCAGGCAGCGTTGACAAGCCC
GCCAAG-3'oligo DNA3:5'-ACCT
GGTAGCGTACCAAAGCC-3'oligo DNA
4: 5' (ATGCTTCGCCCAGAAGG
T(,-3'oligo DNA5:5'-CTGCTdingG
TGGATGATGCTAC-3'oligo DAN6:5
' , TCCACACGTGCAGTTGCGCT-3
'It can be seen that the l-1cV nucleic acid can be easily detected by performing a DNA polymerase reaction using the oligo DNA set specific to this example.

Furthermore, the oligo DNA set of the present invention also has a difference in detectability, and this difference can be used to relatively determine the amount of the virus and its related nucleic acids in the sample.

(Example 4 and Comparative Example 3) Patients diagnosed with non-A, non-B chronic hepatitis and using Chiron HC
250 μρ of serum from a son who was positive for antibodies using the V antibody detection kit was treated with proteinase at 50°C for 2 hours in the presence of 0.5% SDS, and after 3 phenol/chloroform extractions and 1 chloroform extraction, the resulting aqueous phase After adding 1/10 volume of 3 mol of sodium acetate and 3 times the volume of ethanol, let stand at -80°C for 30 minutes and further at 40,000 rpm.
The DNA was precipitated by ultracentrifugation for 1 hour, and 10 μfl (
7) Dissolved in 10 mM Tris H (A/1 mM Tris H (pH 7,5) (containing 4 units of RNasin). In this solution, 50111M Tris H
Cf (pH 8,3) /75 mM KCffi/
10mM dithiothreitol/5mM IV
DI CF2/4 1Mgi acid 1'J'7mu/1
mM dNTPs mixture (N-Δ, C, G, T)
/i, 6μ7 random primer (manufactured by Boehringer Mannheim no. 1034731), 0.2u /μ
x M-MLV (Moloney Mulleen Leukemia Virus) reverse transcriptase (Bethesda Research)
(manufactured by Laboratory Co., Ltd.) was added thereto [total reaction solution volume: 20 μm], and cDNA synthesis was performed at 42° C. for 1 hour. This solution was then diluted and 251M Tris HCf (1)
H8,3) /l00mM K(J15 Tsukuda M M
(1(Jz 15 wM DTT/ 200MMdN
TPs mixture and further 100 u/d E.

coli DN△polymerase I/20u/d R
Add NaseH (final concentration) (final volume 100
μJ2), reacted at 12°C for 1 hour and at 22°C for 1 hour,
It was made into double-stranded DNA.

0.0 for the cDNA solution thus obtained.
5M KCL O, IM Tris H
Cf, 15 mM (1 (Jz, 0.1% gelatin, 0.1% Triton X100) solution (10
XTaq DNA polymerase solution) 5μ polish, sterile water 29μρ, dNTPs mixture (dATP, dCT
8 of 1.25mM solution of P, dGTP, dTTP each)
μp, the same oligomer DNA set as in Example 1 (2.5μ of each oligo DNA (0.1ug/μM at a time)
fl ), and Taq DNA polymerase (P r0
111efIa notO? 423) Add 1.25 units of liquid paraffin and add 50 μl of liquid paraffin, then 95°C for 1 minute, 57°C for 2 minutes, and 72°C for 3 minutes. Repeat these three steps of temperature time change operation 30 times, and finally
Polymerase reaction was carried out for 7 minutes at °C. 2 5 μm after reaction
% agarose gel electrophoresis, staining with ethidium bromide, UV irradiation, and observing DNA bands. As a result 5
A DNA band of 70 bρ was observed.

As a comparative example, reaction production was observed in the same manner as in Example 4 except that AMV-derived reverse transcriptase was used instead of Ill-MLV-derived reverse transcriptase, but no DNA band was clearly visible. From this, it can be seen that M - M L, V is better.

Claims (1)

[Claims] 1. Using a template-dependent DNA polymerase to synthesize cDNA of a nucleic acid prepared from serum or liver tissue, and then using a DNA-dependent DNA polymerase to synthesize DNA.
cDNA is amplified using oligo DNA sets selected from Group 1 and DNA Group 2 as primers, and then colored with a DNA coloring agent, or amplified nucleic acid of a known hepatitis C virus that has been labeled is used. A method for detecting hepatitis C virus and its related nucleic acids by hybridization. DNA group 1: Oligo DNA 1 (5'-GGTCATAGTGGGCA
GGGTCGTCTT-3'), oligo DNA3 (5'
-ACCTGGTAGCGTACCAAGCC-3')
, oligo DNA5 (5'-CTGCTTGTGGATG
ATGCTAC-3') DNA group 2: Oligo DNA 2 (5'-CCAGGCAGCGTTGA
CAAGCCCGCCAAG-3'), oligo DNA4 (5'-TATGCTTCGC
CCAGAAGGTC-3'), oligo DNA6 (5'
-TCCACACGTGCAGTTGCGCT-3') 2. An oligo DNA set for amplification primers for hepatitis C virus and related nucleic acids selected from DNA group 1 and DNA group 2 below. DNA group 1: Oligo DNA 1 (5'-GGTCATAGTGGGCA
GGGTCGTCTT-3'), oligo DNA3 (5'
-ACCTGGTAGCGTACCAAGCC-3')
, oligo DNA5 (5'-CTGCTTGTGGATG
ATGCTAC-3') DNA group 2: Oligo DNA 2 (5'-CCAGGCAGCGTTGA
CAAGCCCGCCAAG-3'), oligo DNA4 (5'-TATGCTTCGC
CCAGAAGGTC-3'), oligo DNA6 (5'
-TCCACACGTGCAGTTGCGCT-3')