JPH05260999A - Specific detection of herpes simplex virus 2 - Google Patents

Abstract

PURPOSE:To enable quick type-specific discrimination of HSV2-type infectious diseases in high accuracy by using a base sequence specifically hybridizing with HSV2-type DNA as a primer for amplification. CONSTITUTION:A liquid mixture containing (A) two DNA primers containing oligonucleotide fragments having at least 15 bases of GCGCGAAGGC GGGCGGCGGC and at least 15 bases of GCCGCGGAAG GCAGCCCGCG, respectively, (B) a DNA polymerase and (C) an aqueous liquid specimen to be analyzed is subjected to amplification cycle by PCR method. The presence of HSV2-type DNA in the obtained reaction liquid is examined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for specifically detecting human herpes simplex virus type 2 (hereinafter sometimes referred to as HSV2).

[0002]

2. Description of the Related Art Human herpes simplex virus (hereinafter referred to as HS
(Sometimes called V) is latent in human sensory ganglia,
Once infected, it will recur. HSV is classified into type 1 and type 2, and the recurrence rate and drug sensitivity differ depending on the type, so HSV type discrimination is an important issue. HSV
The most reliable method for diagnosing infectious diseases is to isolate and determine the virus, but this method requires the preparation of cultured cells and requires several days before the determination, so it is appropriate for treatment. It is difficult to reflect. There is also a method of making a determination by the fluorescent antibody method, but in this case, affected cells are required. There is also a method of inspecting a patient sample by a dot blot method using a DNA fragment that identifies HSV as a type-specific probe, but in the conventional method, a large amount of sample (for example, 2
In addition to requiring 100 to 500 μl), it may not be possible to determine the type even if it is possible to determine that it is HSV. As described above, in the conventional method,
It was difficult to fully meet the demands of the medical field. Therefore, it has been desired to develop a method and an in-vitro diagnostic agent capable of accurately discriminating between HSV type 1 and type 2 in a short time.

On the other hand, although it has been said that HSV does not have a nucleotide sequence that enables type-specific discrimination, the present inventors have already developed a HSV type-specific DNA probe. .. That is, HSV1 type or HSV2 type DNA
DNA fragment obtained by cutting with a specific restriction enzyme [or by inserting the DNA fragment into an appropriate vector (plasmid) and cloning the resulting vector (plasmid) DNA with a specific restriction enzyme D
NA fragment] can be labeled to obtain a type-specific DNA probe. These labeled DNA fragments have a size of several hundreds to several kilobp (base pairs) and are excellent in specificity by type (Japanese Patent Laid-Open No. 142242/1990 and Japanese Patent Application No. 2242/1990).
-90198).

[0004]

DISCLOSURE OF THE INVENTION In addition to the above method, the present inventors newly searched for a base sequence specific to the above HSV2 type in order to detect HSV2 type quickly and reliably. , Newly found that a nucleotide sequence that specifically hybridizes with HSV2 type DNA can be used as a primer. The present invention is based on such findings.

[0005]

Accordingly, the present invention provides a first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 1 in the sequence listing, and the sequence listing Sequence number 2
A combination of a second DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence of SEQ ID NO: 2, a DNA polymerase, and an aqueous liquid test sample are subjected to a DNA amplification step, and thus obtained. The present invention relates to a method for detecting human herpes simplex virus type 2, which comprises subjecting the reaction solution to a DNA inspection step. In the nucleotide sequences of the present specification, A means an adenine residue, C means a cytosine residue, G means a guanine residue, and T means a thymine residue.

The test sample used in the method of the present invention is human HS.
There is no particular limitation as long as it is a sample suspected of containing V2 type. For example, a solution in a patient's blisters, a pharyngeal swab, a cerebrospinal fluid and the like can be used.

The HSV type 2 detection method according to the present invention mainly comprises (1) a DNA amplification step and (2) a DNA inspection step. In this DNA amplification step (1), PC
R (Polymerase Chain Reacti)
on) can be used. Using the PCR method,
From a very small amount of DNA, only the target DNA region can be automatically amplified up to about 1,000,000 times (Scie
nce, 239, 487-491, 1988). PCR
In the method, two kinds of primers, a primer (1) for the + strand and a primer (2) for the − strand, are used with the region to be amplified sandwiched therebetween.

The sequence consisting of 20 bases of the sequence of SEQ ID NO: 1 [DNA primer (1)] and the sequence consisting of 20 bases of the sequence of SEQ ID NO: 2 [DNA primer (2)] are a'of HSV2 type DNA. Sequence (J. Gen. Virol., 55, 315-331,
1981). The 20 base sequence of SEQ ID NO: 1 is a '
It corresponds to 20 bases from 62 bases downstream to 81 bases downstream from the 5'end of the sequence, and the 20 base sequence of SEQ ID NO: 2 is 20 bases from the complementary strand from 54 bases upstream to 73 bases upstream from the 3'end of the a'sequence. Equivalent to a base. When HSV2 type DNA is present in the test sample, the combination of primer (1) and primer (2)
137 bp corresponding to a part of a'sequence gene
Since a large amount of the portion is specifically amplified in a short time, the presence of HSV2 type DNA can be detected very specifically.

The above-mentioned primer (1) and primer (2) can each be 15 mer to 30 mer, but generally 20 mer to 25 mer is preferable. When it is less than 15 mer, specificity upon annealing is decreased and non-specific binding is increased. Also,
When it exceeds 30 mer, a secondary structure between the primer molecules or in the molecule is easily obtained, which is not preferable. Each base constituting each of the first primer and the second primer used in the method of the present invention may be modified (for example, biotinylated or labeled with a luminescent substance) in any known manner. Each of the first primer and the second primer according to the present invention may be prepared by a known DNA synthesis method (for example, phosphoamidite method) using an ordinary automatic DNA synthesizer (for example, Applied Biosystems). it can.

In the DNA amplification step (1) of the present invention, the first step
The amplification cycle is repeated using a DNA polymerase, especially a thermostable DNA polymerase, together with the primer and the second primer. As a thermostable polymerase, a DNA capable of maintaining its activity especially at a temperature of up to 95 ° C
Polymerase For example, commercially available Taq polymerase can be used.

In the DNA amplification step of the present invention, a mixed solution containing the first primer, the second primer, the DNA polymerase and the liquid test sample is used. The amounts of the first primer, the second primer, and the DNA polymerase used vary depending on the type of the liquid test sample.
It can be easily determined within a range in which the amplification step can be performed. This mixture may optionally be a buffer (eg Tris-HCl buffer), a stabilizer (eg gelatin),
Alternatively, it may contain salts such as sodium chloride.

In the method of the present invention, P
A CR method amplification cycle is performed. The amplification cycle includes (i) a denaturation step of DNA (about 90 ° C to 98 ° C, about 10
(2 seconds) to (2 minutes) (ii) an aryling step of the single-stranded DNA with the first primer and the second primer (at about 37 ° C to 70 ° C, about 30
Seconds to about 3 minutes), and (iii) a DNA synthesis step using a DNA polymerase (at about 65 ° C to 80 ° C for about 30 seconds to about 5 minutes). The amount of DNA is amplified up to 2 times per cycle,
After the cycle is amplified to 2 ⁿ times. In the present invention, the amplification cycle is performed 10 to 60 times, preferably 2 times.
Repeat 0-40 times. In the last cycle, the heating time in step (iii) was extended to about 5-10 minutes
It is preferred that the synthesis be complete.

When HSV2 is present in the test sample, after the amplification cycle is completed, the DN of about 140 bp is reached.
A is synthesized in a large amount. This DNA is detected by the following DNA inspection step. As the DNA inspection step, a method using gel electrophoresis and ethidium bromide staining,
The base sequence determination method by the Southern hybrid method or the dideoxy method, the radiolabeling method and the like can be used. When performing gel electrophoresis, for example, submarine electrophoresis using agarose gel as a carrier or slab electrophoresis using acrylamide can be used.

When the dot blot method, the Southern blot hybrid method or the in situ hybrid method is performed, radioactive probes, non-radioactive probes (for example, enzyme-labeled probes, biotinylated probes, digoqueshigenylated probes or chemiluminescent substances, fluorescent substances are labeled. Probe) can be used. Furthermore, when utilizing the nucleotide sequence determination method by the dideoxy method, a DNA autosequencer (Applied Biosystems) using a fluorescent label can be used.

The present invention further provides a DNA probe which can be used for specific detection of HSV2 type DNA. Therefore, in the present invention, a probe containing a oligonucleotide containing at least 10 bases of the base sequence represented by SEQ ID NO: 3 in the sequence listing and carrying a label is contacted with a test sample, and a signal from the label is transmitted. It also relates to a method for detecting herpes simplex type 2, which is characterized by detecting.

The sequence consisting of 40 bases of SEQ ID NO: 3 in the sequence listing corresponds to 40 bases between 100 bases and 139 bases downstream from the 5'end of the a'sequence of the L region. Therefore, the probe of the present invention containing at least 10 bases of the 40 bases is specific to the a'sequence of the L region of HSV2 type DNA. The length of the probe depends on the type of pretreatment for the test sample. PC to be tested
In the case of having undergone the DNA amplification step by the R method,
The size can be 10 bp to the size of the DNA fragment amplified by the PCR method (excluding the primer portion). When the test sample is not subjected to the amplification step by the PCR method and the probe is used for the dot blot or in situ hybrid method, the length of the probe can be up to 10 bp to 137 bp. DNA probe containing 40 bases of SEQ ID NO: 3 in the sequence listing (40 me
r) is preferable because it can be used in any case whether the test sample has undergone the DNA amplification step by the PCR method or in the case of the in situ hybrid method. As the method for preparing the probe of the present invention, a method using succinoimide (for example, disuccimidyl suberate), an active halogen method, a photoreaction using azide, or a carbodiimide method can be used.

Any conventionally known substance can be used as the labeling substance of the probe. Preferably, a non-radioactive substance (enzyme, fluorescent dye, luminescent substance, biotin, etc.) is used. The method for synthesizing the labeled DNA is roughly classified into (1) a method of directly preparing the labeled DNA in the process of synthesizing the DNA, and (2) synthesizing the DNA to which the linker is bound and then isolating it, followed by labeling reagent In particular, the method (2) is preferable because the type of the label can be easily changed according to the purpose and is excellent in application. As the linker used in the method (2), 5'-dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2-cyanoethyl) is used. Examples include-(N, N-diisopropyl)] phosphoramidite and the like. As a method of generating a signal from the label bound to the probe and further measuring the signal, any conventionally known method can be used.

[0018]

When the combination of the primer (1) and the primer (2) is used in the method of the present invention, HS will be present in the test sample.
Only when V2-type DNA is present, a 137 bp base portion corresponding to a part of the L region a'sequence gene is specifically amplified and synthesized in a large amount within a short time. Furthermore, even if the amount of HSV2-type DNA in the test sample is very small,
Since it is amplified and synthesized, it has high sensitivity.

In the method of the present invention, labeled DNA specific to (optionally amplified) HSV2 type DNA
Since a probe is used, HSV2 type DNA can be extremely accurately
Can be detected. Furthermore, even when the amplification step using the first and second primers and the inspection step using the probe are used in combination, the time required for the inspection is about 4 to 5 hours in the case of ethidium staining. Results can be obtained quickly, even within 48 hours.

[0020]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these do not limit the scope of the present invention. Example 1: Synthesis of primer A cytosine CPG column was attached to a 381 type automatic DNA synthesizer (Applied Biosystems) to synthesize a primer (1) consisting of 20 bases of the sequence of SEQ ID NO: 1 in the sequence listing. Further, a guanine CPG column was further attached, and a primer (2) consisting of 20 bases of the sequence of SEQ ID NO: 2 in the sequence listing was synthesized. Ammonia water (about 3
0%) Disposable syringe containing 2.5 ml (about 2.5 m
l) was connected to a CPG column where the synthesis process was completed, and aqueous ammonia was pushed out into the column to elute the synthesized DNA fragment. The vial bottle containing the recovered DNA ammonia solution was sealed, heated at 65 ° C. for 6 hours, cooled to room temperature, and then concentrated. The concentrate was freeze-dried and 10 mM triethylammonium acetate (hereinafter,
It is dissolved in TEA-A) (pH 7.4) and the precipitate is removed, and then a separation column (YMC-Pack ODS) is applied to a L-6200 high performance liquid chromatography apparatus (Hitachi, Ltd.) (hereinafter referred to as HPLC). -AM313: YMC
Co., Ltd.) and 95 mM-T containing 5% acetonitrile
Purification was performed using a concentration gradient of EA-A and acetonitrile, and the main peak was collected. 80% acetic acid (prepared with acetonitrile) was added to the obtained residue to suspend it, and the suspension was kept at room temperature for 30 minutes and then dried under reduced pressure. The dried product is 10 mM-T
It was dissolved in EA-A, extracted with diethyl ether, and dried under reduced pressure. The dried DNA sample was dissolved in TEA-A to remove the precipitate, and then purified by the second HPLC to collect the main peak. Purified D thus obtained
The NA primer was dried under reduced pressure and stored.
Used in.

Example 2 Preparation of Enzyme-Labeled Probe A DNA probe was synthesized under the same conditions as in Example 1, except that the cytosine CPG column was used in the synthesis of this probe. The thymine at the 20th position from the end is 5'-dimethoxytriethyl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2-cyanoethyl) (N , N-diisopropyl)] phosphoamidite (hereinafter referred to as a linker). In addition,
The position of the linker is not limited to this site and can be replaced with another thymine site. Purification of the probe with linker was also performed under the conditions described in Example 1. 3 nmol of the purified probe with linker was dissolved in 8 μl of sterilized water. To this solution 0.2M-NaHCO ₃ / 4mM-EDT
After adding 8 μl of the solution A, 50 μl of a dimethylsulfoxide solution (10 mg / ml) of disuccinimylsverate (hereinafter referred to as DSS) was added, and the mixture was reacted at room temperature in the dark for 15 minutes. After the reaction was completed, 30 μl of sterilized water was added and the mixture was centrifuged, and the supernatant was subjected to HPLC (Toso-G30 as a column).
00PW and water as the moving bed)
The S-bonded DNA (hereinafter referred to as modified linker DNA) was collected and freeze-dried. Dried modified linker D
To NA, 40 μl of alkaline phosphatase EIA (Boehringer Mannheim Yamanouchi) (20 mg / ml) (hereinafter referred to as AP) was added and reacted at room temperature in the dark for 16 hours. After completion of the reaction, 0.1 M Tris-HCl buffer (pH 8.
4) Add 2.5 ml and HPLC (column: Toso-DEA
E-5PW). First, unreacted AP with 0.1 M Tris buffer (pH 8.4) containing 0.1 M NaCl
And the target AP-labeled DNA was eluted with 0.1 M Tris buffer (pH 8.4) containing 0.33 M NaCl.
About 6 ml of the eluate of the collected purified AP-labeled DNA was mixed with 3M-N.
After dialysis against 0.1 M Tris buffer (pH 8.4) containing aCl, the solution was concentrated to 1 ml. This eluate was used as an AP-labeled probe in Example 6 described later and the like.

Example 3: Extraction of HSV2 type DNA UW268 was used as a standard strain of HSV2 type, and
Six clinical isolates identified as SV2 were used. Each virus was added to CV-1 cells derived from African green monkey 1
The cells were infected at a concentration of pfu / cell. Virus-infected cells were cultured in the gas phase of 5% carbon dioxide-95% air. Sucrose method (V
Viral DNA was extracted by irology, 93, 260-264, 1979).

Example 4: Extraction of DNA from clinical sample 10 μl of clinical sample was added to 0.8% NaCl, 0.02% KC
1, 0.115% Na ₂ HPO ₄ , 0.02% KH ₂ P
Specimen 500μ diluted with 490μl PBS with O ₄ composition
After adding 6M guanidine isothiocyanate solution dissolved in the same amount of 50 mM Tris-HCl buffer (pH 7.4) to l, 10 μl of glass powder suspension [PrepDNA: DIAYATRON] was added and stirred. , At room temperature 1
It was left for 0 minutes. Next, the precipitate was collected by centrifugation at 15,000 rpm for 2 minutes. The obtained precipitate was suspended in 1 ml of 10 mM Tris-HCl buffer (pH 7.4) containing 50% ethyl alcohol and 50 mM-NaCl, and washed by centrifugation at 15,000 rpm for 2 minutes. The precipitate was washed by centrifugation twice more. Washed glass powder with distilled water 50μ
suspended in 1 l. Nucleic acid adsorbed on glass powder is 5
The mixture was left standing at 5 ° C for 15 minutes to elute, then centrifuged at 15,000 rpm for 2 minutes, and the supernatant was collected. This supernatant was used as a sample for PCR.

Example 5: HSV2-DN by PCR method
Amplification of A 10 ng each of the DNA of the HSV type 2 standard strain extracted in the above Example 3 and the DNA of the HSV wild type strain of a known type, or 10 μl of the DNA extract from the clinical specimen extracted in the above Example 4
125 μM-dATP, 125 μM-dCTP, 1
25 μM-dTTP, 31.25 μM-dGTP, 9
3.75 μM-deaza c ⁷ GTP (hereinafter dC ⁷ dGTP
, 0.01% gelatin, 20 mM Tris-HCl buffer (pH 8.8), 1.5 mM-MgCl ₂ , 50 mM-K.
Cl, 10% dimethyl sulfoxide, 2 μM primer and 2.5 units of Taq polymerase (AmpliTa
qDNA polymerase: Cetus) was added to 100 μl of the PCR reaction solution. In the PCR method, 30 cycles of (i) 93 ° C. for 1 minute, (ii) 65 ° C. for 1 minute, and (iii) 75 ° C. for 2 minutes were performed for 30 cycles, and finally, the program was performed at 75 ° C. for 5 minutes. Was amplified.

Example 6: Generation of PCR reaction by electrophoresis
Confirmation and diagnosis of the gel: The gel for electrophoresis is agarose gel in 1 liter of buffer (pH 8.0) containing 4.84 g of Tris base, 1.142 g of glacial acetic acid and 2 ml of 0.5M-EDTA (hereinafter referred to as 1xTAE). It was dissolved at 4% and poured on a gel plate of Mupid (Advance) to prepare. next,
Ficoll type 400 in 10 μl of solution after PCR reaction
2 μl of 15% aqueous solution (Pharmacia) was mixed, and the whole amount of the mixture was put into a sample well. 1xTAE
Was placed in an electrode tank and subjected to electrophoresis at 100 V for 2 hours at room temperature. After the electrophoresis was completed, the agarose gel was stained with ethidium bromide, and the results of the electrophoresis were confirmed under UV illuminator irradiation (Fig. 1). According to the ethidium bromide staining of FIG. 1, a plurality of bands are observed in the PCR method using the HSV type 2 primer.
Southern hybridization was performed using a P-labeled probe. That is, DN from the agarose gel after electrophoresis
A Hybridization Membrane
(GeneScreenPlusNEF976: DuP
on) and the AP-labeled probe were hybridized. As is clear from the results of FIG. 2, by amplifying the DNA of the patient sample with the primer of the present invention and confirming the amplified DNA with the probe of the present invention, the virus causing HSV infection can be identified as HSV2.
A type-determining diagnosis of type can be performed. 1 and 2, "M" is a DNA marker, "1" is HS
V2 type standard strains UW268 and "2" to "7" are clinical isolates, and "137b" indicates the position of the DNA fragment amplified by the probes 1 and 2 of the present invention. In addition, a DNA marker (φ × 174-HaeIII dige
st) is 72 bp, 118 bp, 194 from the bottom.
bp, 234bp, 271bp, 281bp, 310b
p, 603 bp, 872 bp, 1078 bp and 135
The one appearing at 7 bp was used.

Example 7: For dot blotting of clinical specimens
Type identification of HSV type 2 by 5 μl of a blistering fluid solution collected from a patient suspected of having a herpes infection was suspended in 2 ml of physiological saline and diluted. DNA was extracted from 10 μl of the diluted sample under the conditions described in Example 4, and PCR was performed under the conditions described in Example 5.
The same amount of 0.5N-Na was added to the reaction solution in which the DNA was amplified.
OH was added and left at room temperature for 10 minutes. 10 μl of the treated sample, which had been denatured of DNA, was added to GeneScreen.
The liquid was removed by suction after placing it on a blotting apparatus equipped with nPlusNEF976 (DuPont) and allowing it to stand at room temperature for 30 minutes. The DNA-blotted filter was placed in a plastic bag, followed by 0.75M-
NaCl, 75 mM trisodium citrate, 0.1% N-
5 ml of a hybridization buffer having a composition of lauryl sarcosine, 0.2% SDS, 30% formamide, 5% nucleic acid hybridization blocking agent (Boehringer Mannheim) was added and sealed. After leaving at 37 ° C. for 60 minutes, the hybridization buffer was removed. 1.50 ml of a hybridization solution prepared by dissolving 3 picomoles of the AP-labeled HSV type 2 discrimination probe prepared in Example 2 was added, and the mixture was left overnight at 37 ° C. The filter in which the hybridization of the probe with the viral DNA in the sample has been completed is taken out from the plastic bag, and first, 2xSSC for 5 minutes and twice, and then 2xSSC containing 0.1% sodium dodecyl sulfate for 15 minutes, 3
Washed 3 times with 2 × SSC for 10 minutes. Washed filter with 0.1 M NaCl and 10 mM
0.1 M Tris-HCl buffer containing MgCl ₂ (pH 9.
5) (Hereinafter, referred to as a color-developing liquid) was immersed for 30 seconds to 1 minute.

75 mg of nitroblue tetrazolium (Boehringer Mannheim) was dissolved in 1 ml of a 30% sterilized water-70% dimethylformamide mixed solution (hereinafter, this solution is referred to as an NBT solution). In addition, 5-bromo-4-chloro-3-indolyl phosphoric acid (Boehringer Mannheim)
50 mg was dissolved in 1 ml of dimethylformamide (hereinafter,
This solution is called BCIP solution). NBT solution 40μ
1 and 40 μl of BCIP solution were added,
It was put in a plastic bag containing a filter soaked in the color developing solution and kept at room temperature for 5 minutes to 16 hours for color development.
The reaction with AP was stopped by immersing the filter in 50 ml of Tris-HCl buffer (pH 7.5) containing 10 mM-EDTA.

The results are shown in Table 1. Among 40 clinical specimens tested, 16 specimens of HSV1 type, 14 specimens of HSV2 type, 2 specimens that could not be determined (species of type 1 or 2 that could not be determined) and 8 specimens were negative in virus isolation. In the direct dot blot method, 14 samples of HSV1 type and 17 samples of HSV2 type
The specimens were 1 specimen that could not be determined and 8 specimens that were negative. On the other hand, the result of the HSV2 type discrimination probe of the present invention is
HSV type 2 was 20 specimens, and there were no specimens that could not be determined or were negative. In addition, from another test result, it was confirmed that the remaining 20 specimens were HSV1 type. In addition, Table 1
FIG. 3 shows the test results by the PCR method of the present invention, which is the basis of the above. In FIG. 3, lanes A to C are dot-blotted HSV type 1-specific PCR products, and lanes D
-F are dot blots of HSV type 2 specific PCR products. In addition, C9 and F9 are HSV1 type standard strains (KOS), C10 and F10 are HSV2 type standard strains (UW268), and 1 to 8 of each lane A to F are obtained by blotting clinical samples after PCR treatment. Yes, the samples 1 to 8 in lanes A to C and the samples 1 to 8 in lanes D to F are the same samples blotted after PCR.

[0029]

[Table 1]

[0030]

INDUSTRIAL APPLICABILITY The method of the present invention has a size such that it can be chemically synthesized as a pair of primers that can be used for amplification of a DNA fragment specific to HSV2 type DNA and a probe specific to HSV2 type DNA. Moreover, the present invention provides an oligonucleotide having a base sequence that does not reduce the specificity of type discrimination. Therefore, the infection of HSV type 2
Highly accurate, rapid, and type-specific identification is possible, and the result can be used for diagnosis and treatment.

[0031]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GCGCGAAGGC GGGCGGCGGC

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GCCGCGGAAG GCAGCCCGGG

SEQ ID NO: 3 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GCAGCCCCGC GCGCCCCCTT CCCCGTCCCT CCCCCGGAGC

[Brief description of drawings]

FIG. 1 is a photograph replacing a drawing showing a result of electrophoresis performed by an inspection method of the present invention.

FIG. 2 is a photograph instead of a drawing, which shows the results of Southern hybridization in FIG.

FIG. 3 is a photograph instead of a drawing, which shows the result of dot blot of HSB type PCR products.

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[Procedure amendment]

[Submission date] February 4, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

The sequence consisting of 20 bases of the sequence of SEQ ID NO: 1 [DNA primer (1)] and the sequence consisting of 20 bases of the sequence of SEQ ID NO: 2 [DNA primer (2)] are a'of HSV2 type DNA. Sequence (J. Gen. Virol., 55, 315-331,
1981). The 20 base sequence of SEQ ID NO: 1 is a '
Corresponding to 20 bases from 61 bases downstream to 80 bases downstream from the 5'end of the sequence, the 20 base sequence of SEQ ID NO: 2 has 20 complementary sequences from 178 bases downstream to 197 bases downstream from the 5'end of the a'sequence. Equivalent to a base.
When HSV2-type DNA is present in the test sample, the combination of primer (1) and primer (2) corresponds to a part of the gene of a'sequence 137.
Since a large amount of the bp portion is specifically amplified in a short time, the presence of HSV2 type DNA can be detected very specifically.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

The sequence consisting of 40 bases of SEQ ID NO: 3 in the sequence listing corresponds to 40 bases between 98 bases and 137 bases downstream from the 5'end of the a'sequence of the L region. Therefore, the probe of the present invention containing at least 10 bases of the 40 bases is specific to the a'sequence of the L region of HSV2 type DNA. The length of the probe depends on the type of pretreatment for the test sample. PC to be tested
In the case of having undergone the DNA amplification step by the R method,
The size can be 10 bp to the size of the DNA fragment amplified by the PCR method (excluding the primer portion). When the test sample is not subjected to the amplification step by the PCR method and the probe is used for the dot blot or in situ hybrid method, the length of the probe can be up to 10 bp to 137 bp. DNA probe containing 40 bases of SEQ ID NO: 3 in the sequence listing (40 me
r) is preferable because it can be used in any case whether the test sample has undergone the DNA amplification step by the PCR method or in the case of the in situ hybrid method. As the method for preparing the probe of the present invention, a method using succinoimide (for example, disuccimidyl suberate), an active halogen method, a photoreaction using azide, or a carbodiimide method can be used.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Sequence: GCCGCGGAAG GCAGCCCGCG

Claims (2)

[Claims] 1. A first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence number 1 in the sequence listing, and the base represented by the sequence number 2 in the sequence listing. A second containing an oligonucleotide portion of at least 15 bases of sequence
Of a human herpes simplex virus type 2, characterized in that a mixed solution containing a combination of DNA primers, DNA polymerase, and an aqueous liquid test sample is subjected to a DNA amplification step, and the resulting reaction solution is subjected to a DNA inspection step. Detection method. 2. A signal containing an oligonucleotide portion having at least 10 bases of the base sequence represented by SEQ ID NO: 3 in the sequence listing and carrying a label is contacted with a test sample to detect a signal from the label. A method for detecting herpes simplex type 2, characterized by: