JPH06121700A - Method for detecting hepatitis c virus gene - Google Patents

Abstract

PURPOSE:To well detect a hepatitis C virus at a low concentration by converting a hepatitis C virus gene RNA into a DNA, amplifying the resultant DNA and hybridizing the amplified DNA with a specific labeling DNA probe. CONSTITUTION:A hepatitis C virus gene is converted into a DNA, which is subsequently amplified. The resultant amplified DNA is then subjected to the hybridization reaction with a probe of the formula 5'- GAGACTGCTAGCCGAGT#AGTGTTGGG-3' or the formula 5'- CGCTCAATGCCT#GGAGATTTGGG-3' (acridinium N-hydroxysuccininide ester is bound through a linker to the site indicated by #). The label is subsequently detected by a hybridization.protection.assay.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a hepatitis C virus gene. Specifically, in the base sequence of the hepatitis C virus gene, a DNA fragment consisting of a specific base sequence of the 5'untranslated region is labeled with acridinium N-hydroxysuccinimide ester (appropriate labeling substance) D
After the NA probe and the test substance hepatitis C virus gene are subjected to a hybridization reaction, a hybridization protection assay (Hybrid
The present invention relates to a method for detecting a hepatitis C virus gene, which is characterized in that a label is detected by an aging protection assay (HPA) method.

[0002]

[Problems to be Solved by the Invention] Hepatitis C virus (H
As a method for detecting CV), H in a sample is analyzed by a method based on an antigen-antibody reaction using an antigen against the virus.
Methods for detecting CV antibodies have become used. However, the detection rate of HCV by this method is not always satisfactory. In addition to the above detection method based on the antigen-antibody reaction, a DNA probe method based on a hybridization technique is also under study.

In the serum of hepatitis C patients, 1 × 10 ² to
It is said that there may be only 1 × 10 ³ cells / ml of HCV. Therefore, H by DNA probe method
When the CV gene is to be detected, the HCV gene present in the sample is subjected to a PCR method using appropriate primers (Michael A Inn et al. PCR Protocols. Academic Pre
ss Inc. (1989)) and 3SR method (John C Guatelli. et a
l. Proc. Natl. Acad. Sci. USA 87,2451-2455 (199
After amplification by a known amplification method such as 1)), the amplified test gene and labeled DNA probe are subjected to a hybridization reaction, and then the label is detected.

However, the HCV gene has an extremely long nucleotide sequence with a total length of about 9 kilobases, and in the case of detecting HCV by the DNA probe method, there is a problem of what nucleotide sequence to use for the DNA probe. Also, there is a problem of how to detect a DNA probe, and establishment of a practical DNA probe method is desired.

[0005]

In the detection of the HCV gene by the DNA probe method, the present inventors often target a region in which a specific nucleotide sequence of the HCV gene is well conserved as a test subject. It was considered important in obtaining the detection rate. Therefore, with the aim of establishing an HCV detection method using the HCV gene (fragment) encoding the 5'untranslated region, which is said to be well conserved for specific nucleotide sequences in the HCV gene, as the test subject. Repeated research.

For this purpose, first, a primer based on the PCR method is designed as a means for amplifying the HCV gene fragment in the sample, and a part of the untranslated region on the 5'side of the HCV gene (regions NC1, NC2) is designed. ) And for comparison, a primer comprising the following DNA sequence for amplifying the gene of the non-structural region (NS5) was prepared and subjected to the experiment. The non-translated region and the non-structured region were selected in order to examine which of these two can be used as a test object to obtain a high detection rate. The synthesis of the primer will be described in detail in Example 1 below. Primer synthesis is automated DNA
An oligomer synthesizer (Applied Biosystems) was used.

Primer nucleotide sequence (1) 5'non-translated region NC1 region name Primer nucleotide sequence 1st-PCR P-(-) 246 5'-ATG AGT GTC GTG CAG CCT CCA G-3 '(SEQ ID NO: : 3) P-(-) 48R 5′-GGT ACT CGC AAG CAC CCT ATC A-3 ′ (SEQ ID NO: 4) 2nd-PCR P-(-) 208 5 ′ -AGA GCC ATA GTG GTC TGC GGA A- 3 '(SEQ ID NO: 5) P-(-) 71R 5'-GCA GTA CCA CAA GGC CTT TCG C-3' (SEQ ID NO: 6)

NC2 region name Primer nucleotide sequence 1st-PCR P-(-) 208 5'-AGA GCC ATA GTG GTC TGC GGA A-3 '(SEQ ID NO: 7) P-(-) 24R 5'-TGC ACG GTC TAC GAG ACC TCC C-3 ′ (SEQ ID NO: 8) 2nd-PCR P-(-) 138 5 ′ -CAA CCC GCT CAA TGC CTG GAG A-3 ′ (SEQ ID NO: 9) P-(-) 51R 5′-ACT CGC AGA CAC CCT ATC AGG C-3 ′ (SEQ ID NO: 10)

(2) Nonstructural region NS5 region name Primer nucleotide sequence 1st-PCR P-6187 5'-AAC GCA TAC ACC ACA GGC CCC T-3 '(SEQ ID NO: 11) P-6493R 5'-GTG AGC ACC GCC ACG TCC GGT TC-3 ′ (SEQ ID NO: 12) 2nd-PCR P-6253 5′-GTG GCT GCG GAG GAG TAC GTG GA-3 ′ (SEQ ID NO: 13) P-6421R 5′-GAA CGT GAC CTC ATC CCG CAG-3 '(SEQ ID NO: 14)

In the DNA probe method, the most important point for obtaining a high detection rate is labeled DN.
The present inventors considered that the problems of what type of base sequence probe is used as the A probe and the problem of what detection method is suitable for detecting the label of the labeled probe are considered by the present inventors.

First, in designing a DNA probe, when the nucleotide sequences of nucleic acids are compared between isolated HCV clones, there are few mutations between corresponding nucleotide sequences, and the location where the sequence is well conserved among clones is DNA. The study was advanced based on the idea that it is preferable as a probe. And HC
The 5'untranslated region, which is generally recognized as a region in which the V gene specificity is well conserved, was selected. As elements for determining the base sequence of the probe, the content (GC%) of guanine (G) and cytosine (C), which are bases contained in the sequence within the region, and H, which is the test object, are included.
The degree of mutation of the CV gene must be taken into consideration.

For example, between the −120 bases and −100 bases upstream from the translation start point of the 5 ′ untranslated region of HCV, −22.
Where the GC% is extremely high, such as between 2 bases and -209 bases, and the adenine (A) and thymine (T) content (AT
It is empirically understood that a position with a high (%) is not suitable as the position of the probe. Therefore, the normal GC% is 5
It was considered most appropriate to select about 0% as the position of the labeled DNA probe. The method for producing the labeled probe will be described in detail in Example 2 below. Further labeled DNA
The present inventors have selected the HPA method as the most suitable method for detecting a label used in combination with a probe.

In designing a labeled DNA probe applicable to the HPA method, it is important that the probe has a Tm value (Melting Point) suitable for the HPA format. For this purpose, the probe must have an appropriate GC% and at the same time be composed of a DNA strand of an appropriate length. It is clear from the study by the present inventors that the Tm of the probe used in the HPA method is appropriately around 65 to 70 ° C., and consideration must be given to such a design.

Further, even if the Tm value is appropriate, the probe length becomes a problem, and if it is too short, hybridization will not be successful due to mismatch with the gene DNA as the test substance and detection can be performed. It may disappear. For example, when a test gene DNA containing a mismatch of 4 bases is detected by the HPA method, it may not be detected by a probe of 18mer, but it may be detected by a probe of 26mer.

In order to detect a test gene sequence containing a mismatch, it is desirable to increase the length of the probe. However, if the probe is too long, the reaction rate of the probe becomes slow, or the Tm value is suitable for the HPA method. Problems such as disappearance occur, and it is difficult to establish a DNA probe method with a high detection rate unless there is a labeled probe having a length suitable for the HPA method.

As a result of repeated studies in consideration of the above situation, the inventors of the present invention have a base sequence shown in (1) or (2) below and bind a label at the position indicated by #. A linker for binding is attached, and a labeled DNA probe produced by binding it to acridinium N-hydroxysuccinimide ester as a labeling substance is used to carry out a hybridization reaction with the HCV gene to be tested, and then labeled. It was found that HCV can be detected at an extremely high detection rate by detecting HPV by the HPA method, and the present invention has been completed based on this finding. (1) 5'- GAG ACT GCT AGC CGA GT # AGT GTT GGG -3 '(SEQ ID NO: 1) (2) 5'- CGC TCA ATG CCT #GG AGA TTT GGG -3' (SEQ ID NO: 2)

That is, according to the present invention, after converting the hepatitis C virus gene RNA, which is the test object, into DNA using reverse transcriptase, the DNA is amplified by a known DNA amplification method,
Next, a method comprising subjecting the amplified DNA and a labeled DNA probe to a hybridization reaction, and then measuring a labeling substance, which comprises the nucleotide sequence shown in (1) or (2) above as a labeled DNA probe, and A hybridization protection assay (HP) was performed using any of the DNA fragments obtained by binding acridinium N-hydroxysuccinimide ester as a labeling substance to the site indicated by # via a linker.
A method for detecting a hepatitis C virus gene, which comprises detecting a label by the method A). The base sequences of the DNA probes (1) and (2) are Q.-L. Choo et al.
Proc. Natl. Acad. Sci. USA 88 2451-2455 1991 Translation initiation codon AT of hepatitis C virus gene
A of G is 1st place, -99 to -74, and-, respectively.
It roughly corresponds to the positions 133 to -111.

In carrying out the method of the present invention, first, H extracted and purified from the serum or liver tissue to be tested.
D obtained by reverse transcriptase treatment of CV-derived RNA gene
NA is amplified by, for example, a method known per se such as the PCR method or the 3SR method, and then a labeled DNA probe is added and subjected to a hybridization reaction. A primer is used for the amplification of DNA, and this primer is, for example, the above-mentioned primer “P-(−) 24 produced by the present inventors.
6 "," P-(-) 48R "," P-(-) 208 ",
"P-(-) 71R", "P-(-) 138", "P-
(−) 24R ”,“ P − (−) 51R ”and the like can be used.

However, the primers that can be used in the present invention are not limited to those listed here,
A pair of primers corresponding to arbitrary positions sandwiching the site on the hepatitis C virus gene corresponding to the probe (1) or (2) can be used. As the gene amplification method, known methods can be used as described above, and the method is not limited to these methods. The value following P in the above primer is Q.-L.Choo et al Proc.Nat
l.Acad.Sci.USA 88 2451-2455 1991 shows the position of the primer counted from A of translation initiation codon ATG of the hepatitis C virus gene as A-position, for example P-
(−) 246 means a primer having a base sequence extending from −246 to the downstream side, and P-(−) 48R means a primer having a sequence complementary to the base sequence extending from −48 to the downstream side. means. In the NS5 region primer, for example, P-6187 is 6187.
Position means a primer having a base sequence extending downstream from the position, and P-6493R means a primer having a sequence complementary to the base sequence extending downstream from the position 6493.

The feature of the present invention is to provide a labeled DNA probe having the base sequences shown in (1) and (2) above and labeled with acridinium N-hydroxysuccinimide ester via a linker at a specific position indicated by #. In addition, the HPA method is combined as a method for detecting the label of the labeled DNA probe.

The probe used in the present invention can be synthesized using an automatic DNA oligonucleotide synthesizer. Also, a linker for labeling a labeling substance can be simultaneously bound to the position # when the probe is synthesized using the same device. Examples of usable linkers include RXL linker (see Attachment-1) and commercially available Amino.
modifier II (Glen-Research) can be used. After purifying the probe with a linker, the linker portion of the probe can be labeled with acridinium N-hydroxysuccinimide ester.

The labeled probe can be used as a labeled DNA probe after purified by HPLC using a reverse phase column. The probe is manufactured in Japanese Patent Application No. 63-
See 508490. For the "HPA" method for detecting labels, see Arnold et al.
inical Chemistry (1989) vol.35; 1588-1594 "," Japanese Patent Application No. 63-508490 ").

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0024]

EXAMPLES Example 1 Of the base sequence of the DNA fragment as a primer
Determination and preparation In the serum of hepatitis C patients (hereinafter referred to as patients), 1 × 10 ² ~
About 1 × 10 ³ / ml of HCV (Hepatitis C
It is said that there may be only the virus (Japanese Patent Application No. 1-500565). Therefore, amplification of the HCV gene is essential to find more infected people. HCV is an RNA virus, and RT-nested PCR (Michael A In
PCR Protocols, Academic Press Inc. (1989)).

In other words, HCV RNA was prepared using reverse transcriptase.
PCR is performed twice with a nested primer after converting DNA to DNA
(Polymerase Chain Reactio
n), the HCV gene was amplified. First, when making the primers for PCR amplification, the total length is about 9000 ba
5'-noncod among HCV genes across ses
ing and NS5 region genes (QL. Choo et al., Proc.
Natl. Acad. Sci. USA 88 , 2451-2455 (1991)) was prepared. The nucleotide sequence of the primer was determined in consideration of the following points.

1-a. When the nucleotide sequences of nucleic acids were compared between known HCV clones, the location where the nucleotide sequence was well conserved and there were few mutations between the corresponding nucleotide sequences was selected as the position of the primer. At this time, the base sequence of the primer is most preferably perfectly conserved among the target HCV clones, but amplification is possible even if the homology between the primer and the corresponding HCV gene is about 90%. The homology is 70% to 80
Amplification is possible even by adjusting the sequence and length of the primer even if it is about%.

1-b. The length of the primer is 15-30
About bases, and GC% in the sequence is 40-70%
And The length of the primer depends on the ratio of mismatch expected between the primer and the HCV gene corresponding thereto and the GC% of the primer. In the case of perfect complementarity, a length of around 20 bases was considered appropriate. In addition, the Tm (Melting Point) of the primer is different depending on the case where a mutation in the target nucleotide sequence is expected at the position of the primer or the GC% of the primer, and thus an appropriate length was examined.

Under these conditions, a nested primer set was selected from the 5'-noncoding region and NS5 region of the HCV gene. Two sets were made in the 5'-noncoding region and one set was made in the NS5 region. The nucleotide sequences of the nested primer sets are shown below.

I. 5'-noncoding region-NC-1 name Primer nucleotide sequence 1st-PCR P-(-) 246 5'-ATG AGT GTC GTG CAG CCT CCA G-3 '(SEQ ID NO: 3) P-(-) 48R 5 ′ -GGT ACT CGC AAG CAC CCT ATC A-3 ′ (SEQ ID NO: 4) 2nd-PCR P-(-) 208 5′-AGA GCC ATA GTG GTC TGC GGA A-3 ′ (SEQ ID NO: 5) P- (-) 71R 5'-GCA GTA CCA CAA GGC CTT TCG C-3 '(SEQ ID NO: 6)

* NC-2 name Base sequence of primer 1st-PCR P-(-) 208 5'-AGA GCC ATA GTG GTC TGC GGA A-3 '(SEQ ID NO: 7) P-(-) 24R 5'- TGC ACG GTC TAC GAG ACC TCC C-3 '(SEQ ID NO: 8) 2nd-PCR P-(-) 138 5'-CAA CCC GCT CAA TGC CTG GAG A-3' (SEQ ID NO: 9) P-(- ) 51R 5′-ACT CGC AGA CAC CCT ATC AGG C-3 ′ (SEQ ID NO: 10)

II. NS-5 Region Name Primer nucleotide sequence 1st-PCR P-6187 5′-AAC GCA TAC ACC ACA GGC CCC T-3 ′ (SEQ ID NO: 11) P-6493R 5′-GTG AGC ACC GCC ACG TCC GGT TC-3 ′ (SEQ ID NO: 12) 2nd-PCR P-6253 5′-GTG GCT GCG GAG GAG TAC GTG GA-3 ′ (SEQ ID NO: 13) P-6421R 5′-GAA CGT GAC CTC ATC CCG CAG-3 '(SEQ ID NO: 14)

Each primer was synthesized by an automatic DNA oligonucleotide synthesizer (Applied Biosystems 380B). OPC column (Applie
d Biosystems # 400771) according to the instruction manual. The purified primer is TE
(10 mM Tris-HCl pH 8.0, 1 mM EDT
It was dissolved in A) and used as a primer for PCR.

Example 2 DNA fragment as a probe
Determination of base sequence and preparation of labeled probe The following formula (I):

[Chemical 1] According to the chemiluminescent substance acridinium ester (AE)
The base sequence of the probe labeled with was selected according to the following criteria.

2-a. When the nucleotide sequences of nucleic acids were compared between the isolated HCV clones, the location where the sequence was well conserved among the clones with few mutations between the corresponding nucleotide sequences was selected as the probe position. Most preferably, the nucleotide sequence of the probe is completely conserved among HCV clones, but the probe and the corresponding HCV clone
Even if the homology with a gene is about 90%, it can be detected, and even if the homology is about 70% to 80%, detection can be sufficiently performed by adjusting the sequence and length of the probe.

2-b. The length of the probe is 15-40base
s, and the GC% in the nucleotide sequence is within the range of 40-70%. The length of the probe depends on the GC% and the expected mutation of the gene, but is 20 to 36 bases.
I thought the degree was appropriate. Moreover, when a mutation was expected between the base sequence of the probe and the corresponding base sequence of the HCV gene, the probe length was adjusted while taking into consideration the GC% of the probe.

The base sequence of the probe was selected in consideration of the above conditions. The base sequence of the probe is shown below. In order to label the probe with AE, the following formula (II):

[Chemical 2] RXL linker (Japanese Patent Application No. 63-507).
941) was inserted near the center of each probe. The position of the RXL linker is indicated by #.

I. 5'-noncoding region Amplification position Name Base sequence of probe NC-1 CP-(-) 133 5'- CGC TCA ATG CCT #GG AGA TTT GGG -3 '(SEQ ID NO: 2) NC-2 CP-(-) 99 5'-GAG ACT GCT AGC CGA GT # AGT GTT GGG -3 '(SEQ ID NO: 1) II. NS5 region Amplification position Name Base sequence of probe NS CP-10 5'-GGT GGG GGA CTT CC # ACT ACG TGA CGG GTA TGA TGA C-3 ′ #: RXL linker ⁵⁾ (SEQ ID NO: 15)

The probe was synthesized using an automatic DNA oligonucleotide synthesizer. The RXL linker for labeling the probe was attached during the synthesis of the probe using an automated DNA oligonucleotide synthesizer. In addition to the RXL linker used this time, it is also possible to use the commercially available linker Aminomodifier II (Glen-Reseach # 10-1950-90). The probe with a linker is an OPC column (Applie
d Biosystems # 400771), and then the chemiluminescent substance AE was bound to the linker portion of the probe (Weeks et al., Clin. Chem. 29 , 1474-1479 (1983)).
. The outline will be described below.

1 nmol of probe with RXL linker (dry state) was added with 3 μl of Dimethyl sulfo.
xide (Nacalai Tesque # 130-45 100
G), 2 μl of 1.6 mg / ml acridinium N-hydroxysuccinimide ester (synthesis requested to Dojindo Laboratories), 1 μl of 1M Hepes Buffer (Dojindo Laboratories # 346-01373), 4 μl of H ₂
O was added and incubated at 37 ° C for 20 minutes to label the probe with AE. After the incubation, 5 μl of 125 μM glycine was added for the purpose of blocking unreacted acridinium N-hydroxysuccinimide ester, the reaction was carried out at room temperature for 5 minutes, and the probe was recovered by ethanol precipitation.

The probe labeled with AE was purified by HPLC using an ODS column (Tosoh TSKgel OligoDNA PR column # 13352). For purification, solution A: 0.1M triethylammonium acetic acid pH
7.0 (Applied Biosystems # 400613), B
Liquid: Acetonitrile (Nacalai Tesque # 004-30S
P) was used to separate the solution B: 0 → 20 min., 10 → 30% (linear gradient). The AE label probe obtained under the above conditions was used for detection of the HCV gene.

Example 3 Detection of test substance by PCR amplification
NC-2 region primer sets shown in out Example 1, i.e. P - (-) 208, P - (-) 24R, P-
Using (-) 138 and P-(-) 51R, the 5'-noncoding region of HCV from -208 to -3.
Amplification of the gene sequence up to the th position (the first base of the translation region of the HCV gene was number 1) was performed. As the test substance, about 800 bases of synthetic RNA (hereinafter abbreviated as H-M800 RNA) spanning the 5'-noncoding region, core region, and envelope region of HCV was used.

In the amplification reaction, RNA is d
After converting to NA, P using a nested primer set
CR (Polymerase Chain React)
Ion) twice (RT nested PC
R). An example of the amplification reaction is shown below. The amplification reagent I represented by the following composition was placed in a reaction tube, and 70, 17.5 or 4 copies of H-M800 RNA was added thereto to make the total volume 90 μl.

Composition of Amplification Reagent I 10 mM Tris-HCl pH 8.3 0.5 μg Random primer (TaKaRa Shuzo, Catalog No. 3801) 2.5 mM MgCl ₂ 50 mM KCl 0.2 mM dATP (Pharmacia, Catalog No. 3801)
27-5500) 0.2 mM dTTP (Pharmacia, Catalog No.
27-5800) 0.2 mM dGTP (Pharmacia, Catalog No.
27-5700) 0.2 mM dCTP (Pharmacia, Catalog No.
27-5600)

This reaction solution was incubated at 60 ° C. for 5 minutes. After cooling the reaction solution to room temperature, 1 µl of M-MLV reverse transcriptase (200 units / µl manufactured by BRL, Catalog No. 8025SB) was added thereto and the mixture was kept at 37 ° C for 3 hours.
CDNA was synthesized by reacting for 0 minutes. Then, 100 pmol of each of P-(-) 208 and P-(-) 2 was added to this reaction solution.
4R Primer and 2.5 Unit of Taq DNA Polymerase (AmpliTaq ^™ , TaKaRa Sake, Catalog No. 2
531) was added to make the total volume 100 μl, and the first P
CR was performed.

The PCR reaction is IWAKI Thermal Sequence.
r After denaturing with TSR300 type at 95 ℃ for 2 minutes,
The reaction was performed by carrying out 40 cycles of reaction at 1.5 ° C. for 1.5 minutes, at 55 ° C. for 1.5 minutes, at 72 ° C. for 2 minutes, and then at 72 ° C. for 9 minutes. Starting from 10 μl of this first PCR solution, P-(−) 138,
Second P using P-(-) 51R Primer
CR was performed. The second PCR was performed as follows. That is, 10 μl of the first amplification reaction solution was added to the amplification reagent II shown in the following composition to make 100 μl in total.
It was set to l.

Composition of Amplification Reagent II 10 mM Tris-HCl pH 8.3 100 pmol P-(-) 138 Primer 100 pmol P-(-) 51R Primer 2.5 mM MgCl ₂ 50 mM KCl 0.2 mM dATP 0.2 mM dTTP 0.2 mM dGTP 0.2 mM dCTP 2.5 Unit Taq. DNA polymerase (AmpliTa
q ^TM )

Using this reaction solution, PCR was performed under the same conditions as the first PCR to obtain a nucleic acid amplification solution. The amplified nucleic acid was detected by a method called the hybridization protection assay (HPA) method shown below. That is, 10 of the reaction solution after the above amplification reaction was completed.
μl was heat denatured at 95 ° C. for 3 minutes. The following probe solution 9 containing a chemiluminescent-labeled CP-(−) 99 probe (CP-(−) 99 probe shown in Example 2 chemiluminescently labeled with an acridinium ester)
0 μl was added and the reaction was carried out at 60 ° C. for 20 minutes.

Then, a reagent (0.6 M Bolic) which selectively inactivates the chemiluminescence of the unreacted probe is added here.
Acid-182 mM NaOH-1% Triton
X100) was added in an amount of 300 μl and reacted at 60 ° C. for 6 minutes, and the remaining chemiluminescence was measured. For the method of quantifying the amplified nucleic acid shown above, see Arnold.
Report (Japanese Patent Application No. 63-508490 and Clinical Che
Hybridization protection assay (HPA) described in mistry (1989) vol.35; 1588-1594).
Please refer to the method called.

Probe solution 111 mM Lithium succinate buffer ^* 10% Lithium lauryl sulfate (Sigma # L-
4632) 1.1 mM EDTA (2Na) (Nacalai Tesque #
151-11GR) 1.1 mM EGTA (Sigma # E-4378) 1 × 10 ⁶ RLUs ^** AE-label CP-133 probe * 1M Succinic acid (Nacalai Tesque # 324-02GR)
1M Lithium hydroixide (Nacalai Tesque # 206-
(34EP) was added to adjust the pH to 5.2 ** Relative Light Units

The results obtained by the above amplification reaction are shown in Table 1.
It was shown to.

[Table 1]

As a result, 17.5 copies of H-M800
In the reaction to which RNA was added, chemiluminescence of about 100 times that in the case where H-M800 RNA was not added was detected. This indicates that a very small amount of RNA equivalent to 17.5 copies can be detected using the nucleic acid amplification method shown here.

Example 4 Hybridization pro
HCV gene by traction assay (HPA) method
In order to examine that the HCV gene can be detected by the HPA method, the amplification method (RT-nested PCR) described in Example 3 from hepatitis C patient serum (hereinafter referred to as patient serum).
Method) was used to amplify the HCV gene, and then the HPA method was used to detect the HCV gene. The patient serum was obtained by treating blood collected from a patient clinically diagnosed with non-A non-B hepatitis according to a conventional method. Further, as a control, serum was collected from a healthy person by the same method, and this was also examined.

Extraction of HCV RNA from patient sera was performed as follows. That is, RN was added to 100 μl of serum.
Azol ^™ (Biotecx Lab., Inc. # CS-101) 90
After adding 0 μl and 100 μl of chloroform, the mixture was stirred for 1 minute and left on ice for 15 minutes. 14,000 × g, 4
After centrifugation at 10 ° C for 10 minutes, about 500 µl of the supernatant was collected, and 2 µl of glycogen (20 µg / µl B
oehringer Mannheim # 90139
3), 2-propanol (Nacalai Tesque # 291-
(13GR) (500 μl) was added, and the mixture was stirred and then left at −20 ° C. for 1 hour. Then, the mixture was centrifuged at 14,000 xg at 4 ° C for 15 minutes, the supernatant was removed, and the pellet was washed with 1
It was washed twice with ml of 80% ethanol (ethanol: Nacalai Tesque # 147-13GR). The obtained pellet was dissolved in 10 μl of H ₂ O to prepare an RNA sample.

The obtained RNA sample was RT-nested P
It was amplified by the CR method. That is, using the RNA sample obtained by the above RNA extraction method, P-(−) 20
8, P-(-) 24R, P-(-) 138 and P-
RT-neste using (-) 51R Primer
5'-noncoding of HCV by d PCR
The sequence of the region was amplified. The details of the amplification method were according to the method shown in Example 3.

The amplified gene was detected by the HPA method shown below. That is, 10 μl of the reaction solution after completion of the amplification reaction was heat denatured at 95 ° C. for 3 minutes. here,
90 μl of a probe solution containing a chemiluminescence-labeled CP-(−) 99 probe (CP-(−) 99 probe shown in Example 2 chemiluminescence-labeled with an acridinium ester) was added, and the mixture was added at 60 ° C. The reaction was carried out for 20 minutes (see Example 3 for the composition of the probe solution). Next, 300 μl (0.6 M Boric Acid, 18 M) of a reagent that selectively inactivates the chemiluminescence of the unreacted probe was added to this.
After adding 2 mM NaOH and 1% Triton X100) and reacting at 60 ° C. for 6 minutes, the remaining chemiluminescence was measured.

The method for detecting the amplified gene shown here is reported by Arnold et al. (Japanese Patent Application No. 63-50).
8490 and Clinical Chemistry (1989) vol.35; 158.
8-1594), referred to as a hybridization protection assay (HPA). Table 2 shows an example of the detection result of the HCV gene from the patient serum by the above method.

[0057]

[Table 2]

HCV infection was determined to be negative by obtaining chemiluminescence of 265 RLUs (Relative Light Units indicating chemiluminescence amount) in healthy human serum, while 30,000-250,000 RLUs was detected in hepatitis C patient serum. It was determined to be positive for HCV infection by obtaining chemiluminescence. This is H
It has been shown that the PA method can specifically detect the HCV gene of hepatitis C patients.

Comparative Tests Primers and Pros of the Invention
The DNA sequence of the probe is superior to those of other DNA sequences.
HCV RNA was extracted from the clinical specimen of Comparative Test Example 42 showing that
Ested PCR, HPA (Hybridization Protecti
on Assay) and 5'-noncoding region and NS
The difference in detection rate between the 5 regions was compared. 100 μl of C
900 μl of RNAzol ^™ (Biotec
x Lab., Inc. # CS-101), after adding 100 μl of chloroform (Nacalai Tesque # 084-02GR),
The mixture was stirred for 1 minute and left on ice for 15 minutes. 14,000 ×
After centrifugation at 4 ° C. for 10 minutes, about 500 μl of the supernatant was collected.

2 μl of glycogen (20 μg /
μl Boehringer Mannheim # 9
01393), 500 μl of 2-propanol (Nacalai Tesque # 291-13GR) and then stirred, -20
It was left at ℃ for 1 hour. Next, 14,000 × g, 4 ° C,
After centrifuging for 15 minutes and removing the supernatant, the pellet was mixed with 1 ml of 80% ethanol (ethanol: Nacalai Tesque # 1).
47-13 GR) and washed twice. 10 of these pellets
It was dissolved in μl of H ₂ O to obtain an RNA solution. This 10 μl
RNA is first converted into DNA by reverse transcriptase and then PCR (Polymerase) is performed using nested primers.
Amplification was carried out twice by Chain Reaction (RT-nested PCR). The amplified DNA fragment was detected by HPA. The outline is shown below. To the reaction tube, add the amplification reagent I shown in the following composition, and add 10 μl of the RNA solution to make the total amount 9
The volume was 0 μl.

Composition of Amplification Reagent I 10 mM Tris-HCl pH 8.3 0.5 μg Random primer (TaKaRa Shuzo, Catalog No. 3801) 2.5 mM MgCl ₂ 50 mM KCl 0.2 mM dATP (Pharmacia, Catalog No. 3801)
27-5500) 0.2 mM dTTP (Pharmacia, Catalog No.
27-5800) 0.2 mM dGTP (Pharmacia, Catalog No.
27-5700) 0.2 mM dCTP (Pharmacia, Catalog No.
27-5600)

This reaction solution was incubated at 60 ° C. for 5 minutes. After cooling the reaction solution to room temperature, 1 μl of M-MLV reverse transcriptase (200 units / μl manufactured by BRL, Catalog No. 8025SB) was added thereto, and the mixture was incubated at 37 ° C. for 3 days.
CDNA was synthesized by reacting for 0 minutes. Then, 100 pmol of each of the two primers (see Table 1 for the combination of primers) and 2.5 Units of Taq D were added to the reaction solution.
NA polymerase (AmpliTaq ^™ , TaKaRa Shuzo, Catalog No. 2531) was added to make the total volume 100 μl, and the first PCR was performed. The PCR conditions were set as follows. IWAKIThermal Sequencer TSR300 type was used for heat denaturation at 95 ° C for 2 minutes, then at 95 ° C for 1.5 minutes, 55
The reaction was carried out for 40 cycles of 2 minutes at 72 ° C. for 40 minutes at 72 ° C. for 1.5 minutes, and then an extension reaction at 9 minutes at 72 ° C.

A second PCR was carried out using 10 μl of the first PCR solution as a starting material and two types of primers (see Table 3 for primer combinations). The second PCR was performed as follows. That is, 10 μl of the first amplification reaction solution was added to the amplification reagent II having the following composition to make the total amount 100 μl.

Composition of Amplification Reagent II 10 mM Tris-HCl pH 8.3 100 pmol Primer A (see Table 3 for primer combinations) 100 pmol Primer B (see Table 3 for primer combinations) 2.5 mM MgCl ₂ 50 mM KCl 0.2 mM dATP 0.2 mM dTTP 0.2 mM dGTP 0.2 mM dCTP 2.5 Unit Taq. DNA polymerase (AmpliTa
q ^TM )

Using this reaction solution, PCR was performed under the same conditions as in the first PCR. The amplified gene was detected by a method called the hybridization protection assay (HPA) method shown below. That is, 10 μl of the reaction solution after completion of the above amplification reaction was heated to 95 ° C.
Heat denatured for 3 minutes. 90μ of the following probe solution
1 was added and incubated at 60 ° C. for 20 minutes. Then, a reagent (0.6M Boric Acid-1) that selectively inactivates the chemiluminescence of the unreacted probe is present here.
After adding 300 μl of 82 mM NaOH-1% Triton X100) and incubating at 60 ° C. for 6 minutes, the remaining chemiluminescence was measured. The above-mentioned amplification of the HCV gene and detection of the amplified gene were carried out according to "Example 3". The primers and probes used for PCR are summarized in Table 3.

[Probe Solution] 111 mM Lithium succinate buffer ^* 10% Lithium lauryl sulfate (Sigma # L-
4632) 1.1 mM EDTA (2Na) (Nacalai Tesque #
151-11GR) 1.1 mM EGTA (Sigma # E-4378) 1 × 10 ⁶ RLUs ^** AE-label probe (see Table 1) * 1M Succinic acid (Nacalai Tesque # 324-02GR)
1M Lithium hydroixide (Nacalai Tesque # 206-
(34EP) was added to adjust the pH to 5.2 ** Relative Light Units

[0067]

[Table 3]

Serum of a healthy person was used for positive determination by HPA. That is, healthy human serum was treated in the same manner as patient serum, PCR was applied twice, and then HPA was performed. 5 times or 10 times the measured value (RLU) at that time (Cutoff value)
Those exceeding the range were determined to be positive (Tables 4 and 5).

[0069]

[Table 4]

[0070]

[Table 5]

The Cutoff value is the RLU derived from the serum of a healthy person.
Even in the case of 10 times, the detection rate of the 5'untranslated region was 100% in the first PCR. On the other hand, in the non-structural region, the maximum detection rate was about 70% after the second PCR. As is clear from the above, the 5'untranslated region is optimal for amplifying and detecting the HCV gene.

[0072]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Features: X indicates a linker. Sequence GAGACTGCTA GCCGAGTXAGT GTTGGG 26

SEQ ID NO: 2 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Feature: X represents a linker. Sequence CGCTCAATGC CTXGGAGATTT GGG 23

SEQ ID NO: 3 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence ATGAGTGTCG TGCAGCCTCC AG 22

SEQ ID NO: 4 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GGTACTCGCA AGCACCCTAT CA 22

SEQ ID NO: 5 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AGAGCCATAG TGGTCTGCGG AA 22

SEQ ID NO: 6 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Synthetic DNA Sequence GCAGTACCAC AAGGCCTTTC GC 22

SEQ ID NO: 7 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AGAGCCATAG TGGTCTGCGG AA 22

SEQ ID NO: 8 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence TGCACGGTCT ACGAGACCTC CC 22

SEQ ID NO: 9 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CAACCCGCTC AATGCCTGGA GA 22

SEQ ID NO: 10 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence ACTCGCAGACA CCCTATCAG GC 22

SEQ ID NO: 11 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AACGCATACA CCACAGGCCC CT 22

SEQ ID NO: 12 Sequence Length: 23 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence GTGAGCACCG CCACGTCCGG TTC 23

SEQ ID NO: 13 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GTGGCTGCGG AGGAGTACGT GGA 23

SEQ ID NO: 14 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence GAACGTGACC TCATCCCGCA G 21

SEQ ID NO: 15 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Features: X is a linker GGTGGGGGAC TTCCXACTACG TGACGGGTAT GATGAC 36

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Claims (1)

[Claims] 1. A hepatitis C virus gene RNA, which is an object to be tested, is converted into DNA, the DNA is amplified, and then the amplified DNA and a labeled DNA probe are subjected to a hybridization reaction, followed by labeling with a labeling substance. In the method consisting of measuring, the labeled DNA probe is composed of the base sequence shown in (1) or (2) below, and an acridinium N-hydroxysuccinimide ester is bound as a labeling substance via a linker to the position indicated by #. A method for detecting a hepatitis C virus gene, characterized in that the label is detected by a hybridization protection assay method using any of the resulting DNA fragments. (1) 5'-GAG ACT GCT AGC CGA GT # AGT GTT GGG -3 '(SEQ ID NO: 1) (2) 5'- CGC TCA ATG CCT #GG AGA TTT GGG -3' (SEQ ID NO: 2) ( In the above sequence, # indicates the binding position of the linker for label binding.)