JPH104971A - Oligonucleotide for detecting gbv-c gene and its detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new oligonucleotide capable of hybridizing with the gene of a blood-carrying hepatitis virus GBV-C different from A, B, C, D and E type hepatitis viruses and useful for the detection of the GBV-C gene, the diagnosis of GBV-C infection, etc. SOLUTION: This new oligonucleotide can hybridize with a nucleic acid having either of base sequences expressed by formulas I, II, III, etc., or with a nucleic acid complementary to the above nucleic acid. The new oligonucleotide can hybridize with the gene of a blood-carrying hepatitis virus GBV-C different from five known A, B, C, D and E type hepatitis viruses and is useful for detecting the GBV-C gene, diagnosing the infection of GBV-C, etc. The oligonucleotide is obtained by extracting the genes of viruses from the serum of a non-A, non-B hepatitis patient by a conventional method, setting a gene- obtaining gene to the region of Helicase estimated to have the highest homology, and carrying out a RT-PCR.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a GBV-C gene from a human sample.

[0002]

2. Description of the Related Art Five viruses of types A, B, C, D and E have been found as viruses causing hepatitis in humans. Among these five types of viruses, hepatitis B virus (HBV) was first discovered as a blood-transmitting hepatitis virus transmitted through blood, and then hepatitis C virus (HCV) was further discovered. This HBV, H
With the development of diagnostic agents for CV and screening of infected blood, blood-borne hepatitis has been greatly reduced.
Although the outbreak of hepatitis due to these two types of hepatitis virus has almost disappeared, it has become clear that there are outbreaks of hepatitis that are not detected by these diagnostic agents.
In addition, the possibility that blood-borne hepatitis virus was present was shown.

On the other hand, in 1967, Deinhardt et al. Infected monkeys with the serum of a jaundiced human and reported that “GB agen
t "(Deinhardt F. et al, J. Exp. Med. 125; 673-687,
1967). This GB agent is a virus,
It has been found that it is different from human hepatitis virus known so far and does not cross with antibodies of other hepatitis viruses. However, this GB agent was not isolated until recently.

[0004] Abbott's Simons last year reported that this GB ag
Representation from Tamarins infected by ent
The gene of GB agent was successfully separated by al Difference Analysis (RDA). (Simons, JN, et a
l, Pros.natl.Acad.Sci. USA, 92; 3401-3405, 1995)
They have two kinds of GB age from this Tamarins
nt were separated and named as GBV-A and GBV-B, respectively. These two types of agents have a total length of about 9 Kb, have an open reading frame (ORF) consisting of about 3000 amino acids, and have been confirmed to be homologous to flavivirus, especially to hepatitis C virus (HCV). Helic, a highly non-structural protein
In the case (NS3) part, about 50% homology was confirmed.

[0005] They expressed this gene as a recombinant antigen in Escherichia coli, and screened human sera by ELISA. As a result, serum reactive with this antigen was observed in the range of 1.5% to 19%.
The VA and GBV-B genes could not be obtained. Therefore, they added GBV-A, GBV
-B or a virus closely related to HCV is considered to exist, and the most homologous region, Helicase (N
A primer for obtaining a gene was set in the region of S3), and RT-PCR was performed. As a result, they succeeded in isolating genes of the GBV-C virus closely related to the above three viruses (Simons, JN, et al, Nature
Medicine, 1: 564-569, 1995).

Also independent of this Abbott group,
Linnen et al. Of Genelabs isolated hepatitis G virus (HGV) from the plasma of non-AE hepatitis patients by immunoscreening. (Linnen, J., et al, Science, 271: 505-
508, 1996) This virus has a gene of about 9 Kb in total length, has an ORF encoding about 2900 amino acids, and is highly homologous to GBV-C and is considered to be the same kind of virus.

[0007]

GBV-C is believed to be a virus that propagates through the blood and is closely related to human hepatitis. The causal relationship between this virus infection and hepatitis has not been rigorously proved, but since GBV-C gene is detected in patients with non-B non-C type liver disease, GBV-C is the cause of hepatitis. It is considered a virus. From these facts, it is considered that diagnosing GBV-C infection and performing appropriate treatment are important in suppressing the occurrence and transmission of hepatitis.

[0008] One of the most common methods for making a diagnosis is to use an enzyme immunoassay such as an enzyme linked immunosorbent assay (ELISA) to detect antibodies to GBV-C in the serum or plasma of an infected person. This is a method for measuring and diagnosing whether or not infection is occurring, but there is no report of an effective antibody measurement method for GBV-C at present. An antigen detection method for directly detecting a viral protein as a pathogen is also an effective diagnostic method, but this method has not yet been reported.

In such a situation, the only method for diagnosing GBV-C infection is considered to be a method for diagnosing the presence or absence of the GBV-C gene. Simons et al. Use helicase (NS
A total of eight genes were separated from the region 3) and Ge
It reports that the gene can be detected by RT-PCR by registering it in nebank and setting primers in regions of high homology.

[0010] GBV-C is considered to be a single-stranded positive-strand RNA virus closely related to HCV. RNA viruses are considered to be mutated at a high speed, as represented by human immunodeficiency virus (HIV) and HCV. Therefore, when detecting a gene of an RNA virus by PCR, hybridization, or the like, sufficient care must be taken in designing primers and probes. That is, it is important to select a region having a small number of mutations. GBV-C also has a high possibility of having a genotype having a different genotype like HCV and other RNA viruses, and a genotype different from a strain isolated so far may be present in Japan.

[0011]

Means for Solving the Problems In order to create an efficient detection system for the GBV-C gene, we isolated the GBV-C gene from the serum of a Japanese GBV-C infected patient, and Oligonucleotides were synthesized based on this, and a highly sensitive gene detection system was constructed.

[0012]

[Specific explanation] Simons et al.
One gene fragment of bp and seven gene fragments of 199 bp are published. The present inventors synthesized the following degenerate primers from these eight gene fragments in order to obtain genes from serum of non-B non-C hepatitis patients. GBC.NS3-1: 5'-TTA TGG GCA TGG (T, C) AT (C, A) CC
(C, T) CT (C, G) GA-3 '(SEQ ID NO: 14) GBC.NS3-4: 5'-(G, C) GA (A, G) GA (G, A, T) AG CGC
(G, A) TC (T, A) GT (C, G, A) GC (A, G) CA-3 '(SEQ ID NO: 15) GBC.NS3-2: 5'-TGC CAT TCC AAG GCG GAG TGC GA−
3 '(SEQ ID NO: 16) GBC.NS3-3: 5'-TCC (GA) TC (TC) TT GAT GAT GGA AC
TGTC-3 '(SEQ ID NO: 17)

Using this primer, nested P
By performing CR, the gene of the NS3 region was isolated from a non-B non-C chronic hepatitis patient sample. When the sequences of about 150b of the isolated gene excluding the primer portion and the sequences of eight published GBV-C genes were compared, they showed a homology of 80.8% to 86.1%. Furthermore, among these eight GBV-C genes, Accession no. U2
The following primers were synthesized based on the sequence of 5538, and about 250 b of the gene containing the sequence of 150 b was isolated from the same patient.

GBC-11: 5'-GCT CGC CTA TGA CTC AGC
ATC CAT CCA-3 '(SEQ ID NO: 18) GBC-14: 5'-GTC ACC TCA ACG ACC TCC TCC ACC ACC
A-3 '(SEQ ID NO: 19) GBC-12: 5'-TGA CTC AGC ATC CAT CCA TAA TTG AGA
-3 '(SEQ ID NO: 20) GBC-13: 5'-CGA CCT CCT CCA CCA CCA ACC CAC AGT
-3 '(SEQ ID NO: 21)

The sequence of 205b is obtained by using Accession no.
When compared to the 38 sequences, they showed 81.0 to 84.1% homology. Furthermore, GBC-1 was obtained from four other non-B non-C chronic hepatitis patients, cirrhosis patients or hepatitis patients.
The gene was isolated with primers of 1, GBC-14, GBC-12 and GBC-13. From one of the patients with chronic hepatitis, about 280 b
The extended gene was isolated. Oligonucleotides were synthesized from these isolated gene sequences, and a highly sensitive gene detection system was constructed. Oligonucleotides used for gene detection can be synthesized by a chemical synthesis method such as the phosphoramidite method.

As the oligonucleotide used for amplification or detection of this gene, any sequence may be selected from any region as long as it can bind to a sequence isolated from a Japanese GBV-C infected patient. The length of the oligonucleotide is GBV
Any length is possible as long as it can specifically bind to the sequence of -C. This length is preferably at least 10 bases, more preferably at least 15 bases, and usually at least 20 bases. The upper limit of the length is preferably 100 bases or less, and usually 50 bases or less. Also, the sequence of the oligonucleotide may or may not exactly match the corresponding portion of the sequence shown in SEQ ID NOs: 1-5. Further, inosine may be contained as a base of the oligonucleotide. Oligonucleotides used for detection may be a mixed primer obtained by mixing several oligonucleotides or a degenerate primer.

The method of amplifying a gene is generally the PCR method, but other methods are also possible. For example, the oligonucleotide can be used for the ligase chain reaction (LCR) method. Oligonucleotides obtained from this gene sequence are also useful for another gene amplification method, the NASBA method. Furthermore, for detection of the GBV-C gene, it can be used as a probe for a conventional method such as Southern hybridization, Northern hybridization, or in situ hybridization.

A feature of the present invention is that a GBV-C gene is isolated from a Japanese GBV-C infected person, and an oligonucleotide most suitable for the detection of a Japanese GBV-C virus gene is selected from its sequence. is there. That is, the most remarkable feature is that the oligonucleotide produced from the sequence obtained according to the present invention can detect the Japanese GBV-C gene as compared with the oligonucleotide designed from the sequence separated by Simons et al. It is now possible.

[0019]

The present invention provides an oligonucleotide primer synthesized from the sequence of GBV-C. The present invention further provides a method of detecting a GBV-C gene.

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples. Embodiment 1 FIG . Oligonucleotide Synthesis The oligonucleotides used in the following examples were synthesized by the following method. Synthetic DNA is model 394 DNA / RNA
The synthesis was performed using a synthesizer (Applied Biosysytems). The phosphoramidite method is used for the synthesis. The detailed protocol followed the synthesizer manual. 28
Oligonucleotides cut with aqueous ammonia
The mixture was heated at 56 ° C. for 6 hours or more to deprotect. Thereafter, the aqueous ammonia was removed by a centrifugal dryer, dried, and 50 μL of a TE solution (Tris-HCl 10 mM, EDTA 1 mM) was used.
Was dissolved.

The synthetic oligonucleotide was purified by excising an acrylamide gel or by gel filtration using a NAP-5 column. Excision by acrylamide gel was performed using 40% acrylamide (190 g acrylamide,
25 g of urea 21 g, 10 × Tris-borate buffer (108 g of tris (hydroxymethyl) aminomethane,
55 g of boric acid, 9.3 g of EDTA · 2Na, 1 L of distilled water
Distilled water is added to 5 mL to make 50 mL. This is 10
% Ammonium persulfate 330 μL, N, N, N′N ′ tetramethylethylenediamine 27.5 μL,
Pour into a 0 mm x 160 mm gel plate. Leave for about 1 hour,
Gelled.

The gel was set in the electrophoresis tank, and loading buffer (formamide 80 mL, tris (hydroxymethyl) aminomethane 0.61 g, boric acid 0.31 g, EDTA · 2Na 0.037 g, bromophenol blue 0.1 g, Xylene cyanol 0.1
g in 100 mL of distilled water) and run at 300 V for about 2 hours. Observe the gel by the shadow casting method and cut out the band with a scalpel. The excised band is finely crushed, and 1 mL of an elution buffer (0.1% SDS, 0.5 M ammonium acetate, 10 mM magnesium acetate) is added, and the mixture is left overnight at 37 ° C.

The elution buffer was recovered and subjected to phenol / chloroform treatment three times and ether treatment three times to obtain 5M
Add 0.2 mL of NaCl and 2.5 mL of ethanol, and add -20.
C. for 1 hour or more. 15000 rpm with centrifuge
The mixture was centrifuged at 4 ° C. for 15 minutes, the supernatant was discarded, and the pellet was rinsed with 70% ethanol. Pellets with sterile distilled water 100pm
ol / μL. Purification using an NAP-5 column was performed according to the manufacturer's protocol, and the concentration was adjusted to 100 with water.
Adjusted to pmol / μL.

Embodiment 2 FIG. Non-B non-C chronic hepatitis patient serum
Isolation of GBV-C gene et (1) RNA extraction ISOGEN-LS (Nippon Gene) 750 [mu] L yeast tRNA (Boehringer Mannheim Yamanouchi Co.,
(5 mg / mL) 1 μL and chloroform 200 μL are added.
250 μL of chronic hepatitis C patient plasma is added to this tube, and the mixture is vortexed for 10 seconds and left on ice for 15 minutes. 1 with a micro high-speed centrifuge (TOMY MR150)
The mixture was centrifuged at 5000 rpm for 15 minutes at 4 ° C. The aqueous layer was separated into an assist tube, an equal amount of isopropyl alcohol was added, and the mixture was kept at -20 ° C for 1 hour or more.

Tubes were placed at 15000 rpm for 15 minutes
Centrifuged at ° C. The supernatant was discarded, 0.5 mL of 75% ethanol was added to the precipitate, and the mixture was centrifuged at 15000 rpm for 5 minutes at 4 ° C. Discard the supernatant and air dry for 5 to 10 minutes. It was dissolved in 10 μL of distilled water treated with diethyl pyrocarbonate.

(2) cDNA synthesis and nested P
5 μL of the extracted RNA is placed in a silicon-treated tube (0.5 mL), heated at 70 ° C. for 3 minutes, and then rapidly cooled in ice. Add 5x RT buffer (250 mM Tris-
HCl (pH 8.3), 375 mM KCl, 15 mM Mg
Cl ₂ ) 2 μL, 100 mM dithiothreitol 1 μL, 2 μL
0.5 mM of 0 mM dNTPs, 0.125 μL of RNase inhibitor (Takara Shuzo, 160 units / μL) No. 1
Antisense primer GBC.NS3-4: 5 '-(G, C) GA
(A, G) GA (G, A, T) AG CGC (G, A) TC (T, A) GT (C, G, A) GC
(A, G) CA-3 '(SEQ ID NO: 15), 100 pmol / μ
L) 0.2 μL, MMLV RTase (BRL, 20
(0 unit / μL), 0.5 μL, and 0.675 μL of diethylpyrocarbonate-treated distilled water were added, and the mixture was kept at 42 ° C. for 1 hour. Heated at 95 ° C. for 5 minutes to inactivate the enzyme. This was used as cDNA.

The nested PCR is LA PCR Kit ver.2
And according to the attached protocol. 5 μL of 10 × LA PCR buffer is added to 10 μL of synthesized cDNA.
L, first sense primer (GBC.NS3-1: 5'-TTA TG
G GCA TGG (T, C) AT (C, A) CC (C, T) CT (C, G) GA -3 '
(SEQ ID NO: 14), 100 pmol / μL) 0.2 μL,
TaKaRa LA Taq (Takara Shuzo, 5 units / μL)
0.5 μL was added to make up to 50 μL with sterile distilled water. One drop of mineral oil (Sigma) was layered on this, and the mixture was stirred and lightly centrifuged. This is Perkin Elmer Cetus DNA Th
The PCR reaction was carried out by setting the thermocycler PJ2000. The reaction profile was DNA denaturation at 94 ° C, 0.5
This reaction was repeated for 35 cycles at annealing, 55 ° C. for 1 minute, extension reaction at 72 ° C. for 1 minute. After the completion of the 35 cycles, the temperature was kept at 72 ° C. for 7 minutes and cooled to 4 ° C.

10 × LA was added to 2.5 μL of this PCR product.
5 μL of PCR buffer, 8 μL of 2.5 mM dNTPs,
Second sense primer (GBC.NS3-2: 5'-TGC CAT TC
C AAG GCG GAG TGC GA-3 '(SEQ ID NO: 16), 10
0 pmol / μL) 0.2 μL, second antisense primer (GBC.NS3-3: 5′-TCC (GA) TC (TC) TT GAT GATGGA
ACT GTC-3 '(SEQ ID NO: 17), 100 pmol / μ
L) Add 0.2 μL, 0.5 μL of TaKaRa LA Taq (Takara Shuzo, 5 units / μL), and add 50 μL with sterile distilled water.
Make up to μL. This was reacted with the same profile as the first PCR.

10 μL of the second PCR product was subjected to agarose electrophoresis. Agarose is in 1 × TAE buffer (40 mM
NuSi in Tris-acetate, 1 mM EDTA)
Eve 3: 1 agarose (FMC) was added to 3% to prepare, and electrophoresis was performed using a Mupid-2 electrophoresis tank (Cosmo Bio Inc.). 100V,
After electrophoresis for about 30 minutes, the agarose gel was stained with ethidium bromide and photographed with a polaroid camera under ultraviolet light. A PCR product of about 190 bp was obtained.

In the same protocol as above, primers for cDNA synthesis were used with GBC-14: 5'-GTCACC TCA ACG ACC TCC
Using TCC ACC ACC A-3 '(SEQ ID NO: 19), the first PCR was performed using primers for cDNA synthesis and GBC-11: 5'-
GCT CGC CTA TGA CTC AGC ATC CAT CCA-3 ′ (SEQ ID NO: 18) was used to perform the second PCR on GBC-12: 5′-TGA CTC AGC.
ATC CAT CCA TAA TTG AGA-3 '(SEQ ID NO: 20)
GBC-13: 5'-CGA CCT CCT CCA CCA CCA ACC CAC AGT
-3 '(SEQ ID NO: 21) with two primers,
A PCR product of about 300 bp was obtained.

(3) Cloning of PCR Product into Vector and Sequencing The PCR product was excised from the gel, and Gene Clean was
Purify using the II kit (Bio 101) and add 10 μL of T
E solution (10 mM Tris-HCl (pH 7.5), 1 mM
(EDTA). Add 5 μL of collected DNA to p
1 µL of GEM-T vector (Promega) (50 ng / µ
L) 1 μL of 10 × T4 DNA ligase buffer,
1 μL of T4 DNA ligase (Promega: 1Weiss un
its / μL) and 2 μL of sterile distilled water, and reacted at 16 ° C. for 3 hours. Incubation was performed at 70 ° C. for 10 minutes to inactivate T4 DNA Ligase.

Using 5 μL of this reaction solution, Escherichia coli XL-1 was used.
The method using the method described in Inoue et al. (Inoue etal, Gene, 96 (199)
0) Transform according to 23-28), X-gal, IPTG
Was plated on an L-amp plate containing, and a white colony was selected to obtain a colony having a plasmid resistant to ampicillin and lacking β-galactosidase. This white colony was cultured overnight in 3 mL of an LB medium supplemented with 50 mg / mL of ampicillin.
ABO: PI-100) to recover plasmid DNA. This plasmid DNA was digested with the restriction enzymes ApaI and Ps
Cleavage at tI, a plasmid DNA containing a fragment of the desired length was selected.

The obtained plasmid DNA was prepared using WizerdTM Min.
Using ipreps DNA Purification System (Promega),
Purified according to the manufacturer's protocol. Purified DN
A for PRISM ^TM Ready Reactiondye DeoxyTM terminator c
y7 Sequencing Kit (Applied Biosystems) using T7 and SP6 primers (Applied Biosystems)
ytems) according to the method recommended by the manufacturer. This reaction product was converted to 370 DNA sequence.
r (Applied Biosystems) to determine the nucleotide sequence. The clone containing the PCR fragment of about 190 bp was named pGEM-TAs, and the clone containing the PCR fragment of about 30 bp was named pGEM-TAL. Since the sequences of the two clones overlap, SEQ ID NO: 1
1 shows a single sequence.

Embodiment 3 FIG. Non-B non-C chronic hepatitis patient serum 4
Isolation of GBV-C Gene from Sample The GBV-C gene was isolated from four sera of non-B non-C chronic hepatitis patients in the same manner as the operation method shown in Example 2. However, the following four primers were used, and a PCR product of about 300 bp was obtained. GBC-11: 5'-GCT CGC CTA TGA CTC AGC ATC CAT CCA
-3 '(SEQ ID NO: 18) GBC-14: 5'-GTC ACC TCA ACG ACC TCC TCC ACC ACC
A-3 '(SEQ ID NO: 19) GBC-12: 5'-TGA CTC AGC ATC CAT CCA TAA TTG AGA
-3 '(SEQ ID NO: 20) GBC-13: 5'-CGA CCT CCT CCA CCA CCA ACC CAC AGT
-3 '(SEQ ID NO: 21) The sequence of the gene obtained in this example is SEQ ID NO: 2 to 5
Shown up to.

Embodiment 4 FIG. Gene isolation by RACE method From the sample of No. 26, Rapid Amplification of cDNA Ends
The 5 'gene was isolated by the (RACE) method. RNA was extracted from serum of a non-B non-C hepatitis patient in the same manner as in Example 2 (1), and dissolved in 10 μL of diethylpyrocarbonate-treated distilled water. From 2 μL of this RNA, Mara
Using the thon ^™ cDNA amplification Kit (Clontech), the manufacturer's protocol was slightly modified to obtain PCR products. The difference was that random hexamer was used as a primer for cDNA synthesis, and the first PCR was performed using TA. as-4
(5'-GCA ATG GCA TTA ACC CCC CGA GAG GAG AAT TG
G-3 ′) (SEQ ID NO: 22) and AP-1 primer were used to perform the second PCR on TA. as-5 (5'-TCA CAC
TCC GCC TTG GAA TGG CAG AAT-3 ') (SEQ ID NO: 2)
3) and AP-2 primer.

This PCR product was digested with the restriction enzyme Sau3AI, and the gene was amplified using LA PCR in vitro Cloning Kit (TAKARA). The primer used in this PCR was 26. as-1 (5'-TCA CCC ACG TCC AGT TTG
GTCTCAATTA-3 ') (SEQ ID NO: 24) and C1-2
Kind of primer. By this operation, about 300 b of gene was amplified. This PCR product was cloned into a pGEM-T vector in the same manner as in Example 2 (3), and the sequence was determined. The obtained clone was extended about 200 b to the 5 'side from the clone obtained in Example 3. Since this sequence overlaps with the sequence determined in Example 3, it is described in SEQ ID NO: 5.

Embodiment 5 FIG. GBV-C by RT-PCR
Detection of Gene (1) Design of Oligonucleotide Primers Primers were synthesized from the sequence of the GBV-C gene isolated in Examples 2 and 3. The synthesized primers are as follows. ns3D-1: GT (AG) TTC TGC CA (TC) TC (CA) AAG GCG (SEQ ID NO: 6) ns3D-4: CTG TC (CT) TT (TGAC) CCC C (GT) (AG) TAA TAG
(SEQ ID NO: 7) ns3D-2: AGG CGG AGT G (TC) G AG (CA) G (CGA) CT (GT) GC
(SEQ ID NO: 8) ns3D-3: ATA ATA GGC (AG) AT GGC ATT (AGTC) AC C (SEQ ID NO: 9) ns3DI-1: GT (AG) TTC TGC CAI TCI AAG GCG (SEQ ID NO: 10) ns3DI-2: AGG CGG AGT G (TC) G AGI GIC TIG C (SEQ ID NO: 11) ns3DI-3: ATA ATA GGC IAT GGC ATT IAC C (SEQ ID NO: 12) ns3DI-4: GA (AG) CTG TCI TTI CCC CII TAA TAG (SEQ ID NO: 13) The synthesis method followed the method described in Example 1.

(2) Extraction of RNA from 30 Serums of Non-B Non-C Hepatitis Patients From 28 sera of non-B non-C hepatitis patients, (1) of Example 2
RNA was extracted and adjusted to 10 μL of DEPC-treated distilled water according to the method described in (1). (3) Detection of GBV-C by RT-PCR RT-PCR was performed from the RNA prepared in (2). 5 μL of the extracted RNA was placed in a silicon-treated tube (0.5 mL), heated at 70 ° C. for 3 minutes, and then rapidly cooled on ice. Add 5x RT buffer (250 mM Tris-
HCl (pH 8.3), 375 mM KCl, 15 mM Mg
Cl ₂ ) 2 μL, 100 mM dithiothreitol 1 μL, 2 μL
0.5 μL of 0 mM dNTPs, 0.125 μL of RNase inhibitor (Takara Shuzo, 160 units / μL), 1st
Antisense primer (100 pmol / μL) 0.2μ
L, MMLV RTase (BRL, 200 units / μ
L) Add 0.5 μL of 0.675 μL of distilled water treated with diethylpyrocarbonate, and keep at 42 ° C. for 1 hour.

The nested PCR was performed as follows. 10 μL of synthesized cDNA, 5 μL of 10 × PCR buffer, first sense primer (100 pmol / μL)
Add 0.2 μL, 0.5 μL of Taq DNA polymerase (Boehringer Mannheim, 5 units / μL), and make up to 50 μL with sterile distilled water. One drop of mineral oil (Sigma) is layered on this, and after stirring, lightly centrifuged. This is DNA thermal cycler PJ2000 from PerkinElmer Cetus.
And a PCR reaction was performed. The reaction profile was DNA denaturation at 94 ° C for 0.5 minutes, annealing at 50 ° C,
This reaction was repeated 35 cycles at 72 ° C. for 1 minute and extension reaction for 1 minute. After the completion of the 35 cycles, the temperature was kept at 72 ° C. for 7 minutes and cooled to 4 ° C.

[0040] 2.5 µL of this PCR product was added to 10 × PCR.
5 μL of buffer, 5 μL of 10 mM dNTPs, 0.2 μL of second sense primer (100 pmol / μL), 0.2 μL of second antisense primer (100 pmol / μL),
Add 0.5 μL of Taq DNA polymerase (Boehringer Mannheim, 5 units / μL) and make up to 50 μL with sterile distilled water. This was reacted with the same profile as the first PCR. 10 μL of the second PCR product was subjected to electrophoresis in the same manner as in Example 2 to confirm the PCR product.

The following three combinations of primers were used for detection by RT-PCR. Combination First sense primer: GBC.NS3-1: 5'-TTA TGG GC
A TGG (T, C) AT (C, A) CC (C, T) CT (C, G) GA-3 '(SEQ ID NO: 14) First antisense primer: GBC.NS3-4: 5'- (G,
C) GA (A, G) GA (G, A, T) AGCGC (G, A) TC (T, A) GT (C, G, A) G
C (A, G) CA-3 '(SEQ ID NO: 15) Second sense primer: GBC.NS3-2: 5'-TGC CAT TC
C AAG GCG GAG TGC GA-3 '(SEQ ID NO: 16) Second antisense primer: GBC.NS3-3: 5'-TCC
(GA) TC (TC) TT GAT GATGGA ACT GTC-3 '(SEQ ID NO: 17)

Combination First sense primer: ns3D-1: GT (AG) TTC TGC CA (T
C) TC (CA) AAG GCG (SEQ ID NO: 6) First antisense primer: ns3D-4: CTG TC (CT) TT
(TGAC) CCC C (GT) (AG) TAA TAG (SEQ ID NO: 7) Second sense primer: ns3D-2: AGG CGG AGT G (TC) GA
G (CA) G (CGA) CT (GT) GC (SEQ ID NO: 8) Second antisense primer: ns3D-3: ATA ATA GGC (A
G) AT GGC ATT (AGTC) ACC (SEQ ID NO: 9)

Combination First sense primer: ns3DI-1: GT (AG) TTC TGC CAI
TCI AAG GCG (SEQ ID NO: 10) First antisense primer: ns3DI-4: CTG TCI TTI
CCC CII TAA TAG (SEQ ID NO: 13) Second sense primer: ns3DI-2: AGG CGG AGT G (TC) G
AGI GIC TIG C (SEQ ID NO: 11) Second antisense primer: ns3DI-3: ATA ATA GGC
IAT GGC ATT IAC C (SEQ ID NO: 12)

The combination primers give a 108 bp PCR product, and the combination primers give a 65 bp PCR product. Table 1 shows the results of PCR. Among the 28 samples, 4 of the combined primers were 5
The sample became PCR positive. Many of these samples are listed in Table 1.
As shown in the figure, the antibody is negative for HCV antibodies and negative for HBV antigens and has liver disease.

[0045]

[Table 1]

From the results in Table 1, GBV announced by Simons et al.
The combination of primers synthesized from the sequence of -C
By using Degenerat primer, G with mutation
The purpose is to detect BV-C.
It is possible to detect the BV-C gene. However, there is a GBV-C in Japan that cannot be detected by this combination for the sample No. 13 in Table 1, and such a sample is a combination or combination of the combination synthesized from the GBV-C gene sequence isolated by the applicant. Such oligonucleotides can be used for detection. Thus, the oligonucleotide of the present invention is considered to be useful for detecting the GBV-C gene of Japanese.

[0047]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 258 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA sequence CAAAATTGGA CGTGGGTGAG ATCCCCTTTT ATGGGCATGG TATCCCCCTG GAGCGAATGC 60 GGACTGGTAG GCACCTCGTA TTCTGCCATT CCAAGGCGGAGTGTGAGCTCGCGGCTGG CCTATTATCG GGGAAAGGAT AGCTCTATTA 180 TCAAAGATGG AGATCTCGTG GTCTGTGCTA CTGATGCGCT TTCCACGGGG TATACCGGCA 240 ACTTCGACTC TGTGACTG 258

SEQ ID NO: 2 Sequence length: 258 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence CCAAATTGGA CGTGGGTGAG ATCCCCTTTT ACGGGCATGG CATCCCCTTA GAGAGGATGC 60 GGACCGGAAG GCATCTCGTA TTCTGCCGGCAGC AGTTCTCTTC CCGGGGAGTT AACGCTATCG CCTATTATCG GGGAAAAGAC AGTTCTATCA 180 TCAAAGATGG AGACCTTGTG GTGTGCGCCA CTGATGCGCT CTCCACGGGG TATACCGGAA 240 ACTTCGACTC CGTGACTG 258

SEQ ID NO: 3 Sequence length: 258 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence CAAAGCTTGA TGTAGGAGAG ATTCCCTTTT ATGGGCACGG CATACCCTTG GAGCGGATGC 60 GAACCGGCAG GCACCTCGTA TTCTGCCATT CAAAAGCGGAGCGCGCGCAGGCGCGCAG AGTTCTCTTC CAGGGGAGTA AATGCTATTG CCTATTACCG GGGAAAGGAC AGTTCAATCA 180 TTAAGGATGG GGACCTCGTG GTGTGCGCGA CCGACGCGCT CTCCACGGGG TACACCGGCA 240 ACTTCGATTC CGTGACCG 258

SEQ ID NO: 4 Sequence length: 258 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA sequence CCAAACTGGA TGTGGGCGAG ATCCCCTTCT ATGGGCATGG CATCCCTTTG GAGAGGATGC 60 GGACCGGAAG GCACCTCGTA TTCTGCCGGCTGAA 120 AGTTCTCTTC TCGGGGGGTG AACGCCATTG CCTATTACAG GGGAAAGGAC AGTTCTATCA 180 TCAAAGATGG AGACCTCGTG GTGTGCGCAA CAGACGCGCT GTCCACGGGG TACACCGGAA 240 ACTTCGATTC CGTGACTG 258

SEQ ID NO: 5 Sequence length: 497 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence GATCACAGAC TCCCCCTTAA CGTACTCGAC CTACGGGAGG TTCTTAGCCA ACCCTAGGCA 60 GATGCTGCGA GGGGTGTCTG TGGTCATATG TGATGAGTCACAGT GTTGCTGGGC ATTGGACGGG TCAGGGAGTT GGCGCGTGGC TGTGGGGTCC AATTGGTCCT 180 CTATGCCACT GCCACACCTC CCGGGTCCCC CATGGTTCAG CATCCTTCAA TAATTGAGAC 240 CAAACTGGAC GTGGGTGAGA TCCCCTTTTA TGGGCATGGC ATACCCCTGG AGCGGATGCG 300 GACCGGCAGG CATCTCGTAT TTTGCCATTC AAAGGCGGAG TGCGAGCGTC TGGCGGGCCA 360 GTTCTCCTCC AGGGGTGTAA ACGCCATTGC CTATTATAGG GGCAAGGACA GTTCTATCAT 420 CAAGGATGGA GACCTCGTGG TGTGCGCTAC AGATGCGCTA TCCACGGGGT ACACTGGCAA 480 CTTCGATTCC GTGACCG 497

SEQ ID NO: 6 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GTRTTCTGCC AYTCMAAGGC G 21

SEQ ID NO: 7 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CTGTCYTTNC CCCKRTAATA G 21

SEQ ID NO: 8 Sequence length: 22 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence AGGCGGAGTG YGAGMGVCTK GC 22

SEQ ID NO: 9 Sequence length: 22 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: synthetic DNA sequence ATAATAGGCR ATGGCATTNA CC 22

SEQ ID NO: 10 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTRTTCTGCC AITCIAAGGC G 21

SEQ ID NO: 11 Sequence length: 22 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence AGGCGGAGTG YGAGIGICTI GC 22

SEQ ID NO: 12 Sequence length: 22 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATAATAGGCI ATGGCATTIA CC 22

SEQ ID NO: 13 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GARCTGTCIT TICCCCIITA ATAG 24

SEQ ID NO: 14 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TTATGGGCAT GGYATMCCYC TSGA 24

SEQ ID NO: 15 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence SGARGADAGC GCRTCWGTVG CRCA 24

SEQ ID NO: 16 Sequence length: 23 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TGCCATTCCA AGGCGGAGTG CGA 23

SEQ ID NO: 17 Sequence length: 24 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TCCRTCYTTG ATGATGGAAC TGTC 24

SEQ ID NO: 18 Sequence length: 27 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GCTCGCCTAT GACTCAGCAT CCATCCA 27

SEQ ID NO: 19 Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTCACCTCAA CGACCTCCTC CACCACCA 28

SEQ ID NO: 20 Sequence length: 27 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: synthetic DNA sequence TGACTCAGCA TCCATCCATA ATTGAGA 27

SEQ ID NO: 21 Sequence length: 27 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: synthetic DNA sequence CGACCTCCTC CACCACCAAC CCACAGT 27

SEQ ID NO: 22 Sequence length: 33 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCAATGGCAT TAACCCCCCG AGAGGAGAAT TGG 33

SEQ ID NO: 23 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence TCACACTCCG CCTTGGAATG GCAGAAT 27

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Shintaro Yagi, Inventor 1-33-1 Nishitsurugaoka, Oi-machi, Iruma-gun, Saitama Prefecture Tonen Co., Ltd. (72) Inventor Akira Hasegawa Nishi-Tsuruga, Oi-machi, Iruma-gun, Saitama 1-3-1 Oka Inside Tonen Research Institute

Claims (10)

[Claims] 1. An oligonucleotide capable of hybridizing to any of the nucleic acids of SEQ ID NOs: 1 to 5 or a nucleic acid complementary thereto. 2. The oligonucleotide according to claim 1, wherein any of the nucleotide bases is replaced by inosine. 3. The oligonucleotide primer according to claim 1 or 2, comprising the following sequence: ns3D-1: 5'-GT (AG) TTC TGC CA (TC) TC (CA) AAG GCG
-3 '(SEQ ID NO: 6) ns3D-4: 5'-CTG TC (CT) TT (TGAC) CCC C (GT) (AG) TA
A TAG-3 '(SEQ ID NO: 7) ns3D-2: 5'-AGG CGG AGT G (TC) G AG (CA) G (CGA) CT
(GT) GC-3 '(SEQ ID NO: 8) ns3D-3: 5'-ATA ATA GGC (AG) AT GGC ATT (AGTC) AC
C-3 '(SEQ ID NO: 9) ns3DI-1: 5'-GT (AG) TTC TGC CAI TCI AAG GCG-
3 '(SEQ ID NO: 10) ns3DI-2: 5'-AGG CGG AGT G (TC) G AGI GIC TIG C-
3 '(SEQ ID NO: 11) ns3DI-3: 5'-ATA ATA GGC IAT GGC ATT IAC C-
3 '(SEQ ID NO: 12) ns3DI-4: 5'-GA (AG) CTG TCI TTI CCC CII TAA TAG
3 '(SEQ ID NO: 13). A primer for amplifying a GBV-C gene, comprising the oligonucleotide according to any one of claims 1 to 3. A probe for detecting a GBV-C gene, comprising the oligonucleotide according to any one of claims 1 to 3. 6. A method for amplifying a GBV-C gene, comprising using the primer according to claim 4. 7. The method according to claim 5, which is a PCR method. 8. A method for detecting a GBV-C gene, comprising using the probe according to claim 6. A kit for amplifying a gene for GBV-C, comprising the primer according to claim 4. 10. The kit according to claim 9, further comprising the probe according to claim 5.