JP3414766B2 - Specific method for detecting herpes simplex virus type 2 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to a human herpes simplex virus.
Russ type 2 (hereinafter sometimes referred to as HSV2)
The method relates to a statistical detection method. [0002] 2. Description of the Related Art Human herpes simplex virus (hereinafter referred to as HS)
V), which hides in the human sensory ganglia,
Once infected, recurrent episodes repeat. HSV has type 1
There are two types, and the regression rate and drug sensitivity differ depending on the type.
Therefore, HSV type discrimination is an important issue. HSV
The most reliable diagnosis of infectious disease is to isolate the virus and determine
This method requires the preparation of cultured cells.
And it takes several days to make a decision,
Difficult to reflect properly. By fluorescent antibody method
There is a method to determine, but in this case the affected cells are needed
I do. In addition, a DNA fragment that specifically identifies HSV is
Testing patient samples by dot blot as a probe
Although there are methods, the conventional method requires a large amount of sample (for example, 2
Not only requires 100-500 μl), but also with HSV
Even if it can be determined that there is, until the type is determined
There were times when I could not. As described above, in the conventional method,
It was difficult to sufficiently meet the needs of the medical field. Obedience
To quickly and accurately distinguish between HSV1 and HSV2
And the development of in vitro diagnostics
Was. On the other hand, HSV enables type-specific identification.
It has been said that there is no base sequence
We have already opened a HSV type-specific DNA probe.
Has emitted. That is, HSV1 type or HSV2 type DNA
Fragment obtained by cleaving DNA with a specific restriction enzyme
[Alternatively, the DNA fragment is ligated to an appropriate vector (plasmid
Vector and cloned vector
Smid) D obtained by cutting DNA with a specific restriction enzyme
NA fragment] to obtain a type-specific DNA probe.
Can be These labeled DNA fragments are several hundred to several
The size is kilobp (base pairs), and the specificity of each type is excellent.
(Japanese Unexamined Patent Application Publication No. 2-142499 and Japanese Patent Application No.
-90198). [0004] DISCLOSURE OF THE INVENTION The present inventors have found that
Separately and quickly detect HSV type 2
For this purpose, a new nucleotide sequence specific to the above HSV type 2 was newly added.
Search and hybridize specifically with HSV type 2 DNA
Base sequenceprobeCan be used as
Was newly found. The present invention is based on this finding.
It is. [0005] SUMMARY OF THE INVENTION Accordingly, the present invention providesArray
Oligonucleotide consisting of the sequence of SEQ ID NO: 1 in the table
20- to 25-mer first DN containing a thiol moiety
A primer and the sequence of SEQ ID NO: 2 in the sequence listing
20 mer- containing an oligonucleotide moiety consisting of
A combination of a 25mer second DNA primer;
Mixing containing DNA polymerase and aqueous liquid test sample
The solution was subjected to a DNA amplification step, and the obtained reaction solution was compared with the sequence listing.
Consisting of the nucleotide sequence of SEQ ID NO: 3
Contact with a probe that contains a peptide moiety and carries a label.
Detecting a signal from the sign,
The present invention relates to a method for detecting herpes simplex type 2.Base in the present specification
In the sequence, A is an adenine residue, C is a cytosine residue,
G is a guanine residue, and T is a thymine residue.
You. The test sample used in the method of the present invention is human HS.
Especially restricted if it is suspected of containing V2 type
Not done. For example, patient intravesical solution, pharyngeal swab, cerebrospinal fluid
Etc. can be used. [0007] The above HSV type 2 detection method according to the present invention.
Mainly consisted of (1) DNA amplification step and (2) DNA test
Process. In this DNA amplification step (1), PC
R (Polymerase Chain Reacti)
on) can be used. Using the PCR method,
Automatically detects only the target DNA region from a small amount of DNA
Can be amplified up to about one million times (Scie
nce, 239, 487-491, 1988). PCR
In the method, the primer for the + strand is
Primer (2) for the primer (2)
Use primers. It consists of 20 bases of the sequence of SEQ ID NO: 1.
Sequence [DNA primer (1)] and SEQ ID NO: 2
A sequence consisting of 20 bases of the sequence [DNA primer
(2)] is a ′ sequence of HSV type 2 DNA
(J. Gen. Virol., 55, 315-331,
1981). The 20 nucleotide sequence of SEQ ID NO: 1 is a '
80 bases 61 bases downstream from the 5 'end of the sequence
It corresponds to 20 bases to the downstream, and the 20 base sequence of SEQ ID NO: 2
Is the column 178 bases downstream from the 5 'end of the a' sequence?
This corresponds to 20 bases in the complementary strand from 197 bases downstream.
If HSV type 2 DNA is present in the test sample,
By combination of primer (1) and primer (2)
137 corresponding to a part of the gene of the a 'sequence
The bp part is amplified in a large amount specifically in a short time
Therefore, the presence of HSV type 2 DNA can be detected very specifically.
Can be The above-mentioned primer (1) and primer
(2)20 mer to 25 mer.Book
Of the first and second primers used in the method of the invention
Each base constituting each is modified in any known manner
(Eg, biotinylated or labeled with a luminescent material)
It may be. Each of the first steps according to the invention;
The primer and the second primer are used for normal DNA automatic synthesis.
Machine (for example, manufactured by Applied Biosystems)
By a known DNA synthesis method (for example, a phosphoramidite method)
Can be prepared. In the DNA amplification step (1) of the present invention, the first
DNA polymerase with primer and second primer
Amplification, especially with thermostable DNA polymerase.
Repeat the cycle. As a thermostable polymerase,
DNA capable of maintaining activity at temperatures up to 95 ° C
Polymerase For example, using a commercially available Taq polymerase
Can be In the DNA amplification step of the present invention, the first primer
And second primer, DNA polymerase and liquid coating
Use a mixture containing the test sample. First primer, second primer
The amount of primer and DNA polymerase used should be
It depends on the type of sample, but DNA by PCR
Easy determination as long as the amplification process can be performed
be able to. This mixture may optionally contain a buffer (eg,
For example, Tris-HCl buffer), a stabilizer (eg, gelatin),
Or can contain salts (eg sodium chloride)
Wear. In the method of the present invention, P
The amplification cycle of the CR method is performed. The amplification cycle is (I) DNA denaturation step (about 90 to 98 ° C. for about 10
2 seconds to 2 minutes) (Ii) Single-stranded DNA and first and second primers
(About 37 ° C to 70 ° C, about 30 ° C)
Seconds to about 3 minutes), and (Iii) DNA synthesis step using DNA polymerase (about
(65 ° C to 80 ° C for about 30 seconds to about 5 minutes)
You. The amount of DNA is amplified up to two times at each cycle, and n
2 after the cycleⁿIt is amplified twice. In the present invention
Means that the above amplification cycle is performed 10 to 60 times, preferably 2 to 60 times.
Repeat 0-40 times. In the last cycle, the process
The heating time of (iii) is extended to about 5 to 10 minutes to increase the DNA
Preferably, the synthesis takes place completely. When HSV2 is present in the test sample
Is approximately 140 bp DN after the amplification cycle
A is synthesized in large quantities. This DNA is used for the next DNA test
Detect according to the process. In the DNA testing process, gel
Method using electrophoresis and ethidium bromide staining,
Base arrangement by Southern hybrid method or dideoxy method
Sequence determination, radiolabeling, and the like can be used. Get
When performing electrophoresis, for example, agarose gel
Submarine electrophoresis or acrylamide as carrier
Can be used. Dot blot method, Southern blot hive
When performing the lid method or the in situ hybrid method
Include radioactive and non-radioactive probes (eg, enzymes
Labeled probe, biotinylated probe, digokeshigenination
Probes or chemiluminescent substances, probes labeled with fluorescent substances
B) can be used. Furthermore, by the dideoxy method
When using sequencing, use fluorescent labels.
DNA Autosequencer (Applied Biosys
Tem Company) can be used. The present invention further relates to the specificity of HSV type 2 DNA.
To provide a DNA probe that can be used for dynamic detection
You. BookinventionsoIs represented by SEQ ID NO: 3 in the sequence listing.
Base sequenceConsisting of 40 basesThe oligonucleotide part of
The probe containing the label and the test sample
And detect the signal from the sign.You. From the 40 bases of SEQ ID NO: 3 in the sequence listing
From the 5 'end of the a' sequence of the L region
Equivalent to 40 bases between 100 bases and 139 bases downstream
You. Therefore,At least 40 bases ofIncluding the present invention
The probe was a 'sequence of the L region of HSV type 2 DNA.
Is specific to The length of the probe is
It depends on the type of preprocessing. The test sample is
If the DNA has undergone a DNA amplification step,40bp
Or the size of the DNA fragment to be amplified by the PCR
Excluding the immer portion). Test sample is PCR
Do not use an amplification process
Used for blot or in situ hybrid method
The length of the probe40bp or 137b
It is possible to have a size of p. SEQ ID NO: 3 in Sequence Listing
A DNA probe (40mer) containing 40 bases of
The test sample has undergone a DNA amplification step by PCR.
Or in the case of in situ hybrid method
Is preferred because it can be used for any
No. As a method for preparing the probe of the present invention,
(For example, disuccimidyl suberate)
Reaction, active halogen method, photoreaction using azide, or
A carbodiimide method or the like can be used. As the labeling substance for the probe, a conventionally known substance can be used.
Any substance can be used. Preferably, non-release
Radioactive substances (enzymes, fluorescent dyes, luminescent substances, biotin, etc.)
Used. The method of synthesizing labeled DNA can be roughly classified (1) Preparation of labeled DNA directly during DNA synthesis
How to (2) Isolation after synthesizing DNA with linker
And how to apply a labeling reagent to this In particular, method (2) uses a type of label depending on the purpose.
It is preferable because it can be easily changed and is excellent in application. Method
As the linker used in (2), 5'-dimethoxy
Lityl-5- [N- (trifluoroacetylaminohexyl
Sil) -3-acrylimide] -2'-deoxyuridine
-3 '-[(2-cyanoethyl)-(N, N-diiso
Propyl)] phosphoramidite
You. A signal is generated from the label attached to the probe and updated.
In addition, the method of measuring the signal is also any conventionally known method.
A method can be used. [0018] In the method of the present invention, the primer (1) and the primer (1) are used.
When the combination of the primers (2) is used, HS is contained in the test sample.
L region a'sequence only when V2 type DNA is present
137 bp corresponding to a part of the gene
It is amplified and synthesized in large quantities specifically within a certain time. In addition,
Even if the amount of HSV type 2 DNA in the test sample is very small,
Is amplified and synthesized, so that the sensitivity is high. In the method of the present invention,
Labeled DNA specific to HSV type 2 DNA
Since the probe is used, HSV type 2 DNA
Can be detected. Further, the first and second pumps
Amplification step using primer and test using the probe
Even if the process is used together, the time required for inspection is
About 4 to 5 hours in the case of
Quick within 48 hours, even when performing up to the hybrid method
Results can be obtained. [0020] The present invention will be described in more detail with reference to the following examples.
However, these do not limit the scope of the present invention.
No.Example 1: Synthesis of primer Model 381 Automatic DNA Synthesizer (Applied Biosystem
And a cytosine CPG column attached to the
Consisting of 20 bases of the sequence of SEQ ID NO: 1
-Synthesize (1) and attach a guanine CPG column
From the 20 bases of the sequence of SEQ ID NO: 2 in the sequence listing
Primer (2) was synthesized. Ammonia water (about 3
0%) disposable syringe containing 2.5 ml (about 2.5 m
l) is connected to the CPG column where the synthesis process is completed, and
Methanol water is pushed into the column and the synthesized DNA
The fragment eluted. Recovered DNA ammonia solution
Was sealed and heated at 65 ° C. for 6 hours.
Thereafter, the mixture was cooled to room temperature and concentrated. Lyophilize concentrate
And 10 mM triethylammonium acetate (hereinafter, referred to as
TEA-A) (pH 7.4)
L-6200 high performance liquid chromatography
(Hitachi) (hereinafter referred to as HPLC)
Ram (YMC-Pack ODS-AM313: YMC
95mM-T containing 5% acetonitrile
Purification using a concentration gradient with EA-A and acetonitrile
And collected the main peak. 80% vinegar in the residue obtained
Add acid (prepared with acetonitrile) and suspend.
It was dried for 30 minutes and then dried under reduced pressure. Dry product is 10mM-T
Dissolve in EA-A, extract with diethyl ether and reduce
Pressure dried. Dissolve dried DNA sample in TEA-A
After removing the precipitate, the second purification by HPLC was performed.
And collected the main peak. Purified D thus obtained
The NA primer was dried under reduced pressure and stored.5
Used in [0021]Example 2: Preparation of enzyme-labeled probe For the synthesis of the DNA probe, the same apparatus as in Example 1 was used.
The procedure was performed under the same conditions.
Using a Tocin CPG column, the 20th from the 5 'end
Thymine was converted to 5'-dimethoxytriethyl-5- [N- (g
(Trifluoroacetylaminohexyl) -3-acrylic acid
Mido] -2'-deoxyuridine-3 '-[(2-sia
Noethyl) (N, N-diisopropyl)] phosphoramida
(Hereinafter referred to as a linker). In addition,
The position of the linker is not limited to this site, but may be
It is also possible to replace it. Of the probe with linker
Purification was also performed under the conditions described in Example 1. Purified
Dissolve 3 nmoles of probe with anchor in 8 μl of sterile water
Was. 0.2 M NaHCO was added to this solution._Three/ 4mM-EDT
After adding 8 μl of solution A, disuccinimil verate
(Hereinafter referred to as DSS) in dimethyl sulfoxide
(10 mg / ml) for 15 minutes in the dark at room temperature
Reaction. After the reaction is completed, add 30 μl of sterilized water and centrifuge.
The supernatant was subjected to HPLC (Toso-G30 as a column).
00PW using water for the mobile bed)
S-linked DNA (hereinafter referred to as modified linker DNA)
Was collected and freeze-dried. Dried modified linker D
NA for alkaline phosphatase EIA (Boehringer
Mannheim Yamanouchi) (20mg / ml) (hereinafter referred to as AP)
40 μl) and react at room temperature in the dark for 16 hours.
Was. After completion of the reaction, 0.1 M Tris-HCl buffer (pH 8.
4) Add 2.5 ml, HPLC (column: Toso-DEA)
E-5PW). First, 0.1 M NaCl
Not reacted with 0.1 M Tris buffer (pH 8.4)
Except for 0.1M Tris buffer containing 0.33M-NaCl
The target AP-labeled DNA was eluted with a buffer (pH 8.4).
Approximately 6 ml of the eluted fractionated purified AP-labeled DNA was added to 3M-N
against 0.1M Tris buffer (pH 8.4) containing aCl
After dialysis, the mixture was concentrated to 1 ml. This eluate is labeled with AP
The probe was used in Example 6 described later. [0022]Example 3: Extraction of HSV type 2 DNA UW268 was used as a standard strain of HSV2 type.
Six clinical isolates identified as SV2 were used. each
Virus was transferred to African green monkey-derived CV-1 cells for 1
Infected at a concentration of pfu / cell. Infected with a virus
Cells are cultured in the gas phase of 5% carbon dioxide-95% air
Was. Saccharose method (V
irology, 93, 260-264, 1979)
Virus DNA was extracted therefrom. [0023]Example 4: DNA extraction of clinical specimen 10 μl of a clinical sample is 0.8% NaCl, 0.02% KC
1, 0.115% Na_TwoHPO_Four, 0.02% KH_TwoP
O_Four500 μl of sample diluted with 490 μl of PBS
Dissolve in the same volume of 50 mM Tris-HCl buffer (pH 7.4).
6M guanidine isothiocyanate solution was added.
Then, a glass powder suspension [PrepDNA:
Iayatron], add 10 μl, stir, and bring to room temperature.
Left for 0 minutes. Then centrifuge at 15,000 rpm for 2 minutes
And the sediment was collected. The resulting sediment is washed with 50% ethyl alcohol
10 mM Tris-HCl buffer containing urea and 50 mM NaCl
(PH 7.4) Suspend in 1 ml and rotate at 15,000 rpm for 2 minutes
Was washed by centrifugation. Wash the sediment by centrifugation.
Was performed twice. Wash the washed glass powder with distilled water 50μ
l. 5 nucleic acids adsorbed on glass powder
Leave at 5 ° C for 15 minutes to elute, then 15,000 times
After centrifugation for 2 minutes, the supernatant was collected. This supernatant is used for PCR
Was used as a sample. [0024]Example 5: HSV2-DN by PCR method
A amplification The DNA of the HSV type 2 standard strain extracted in Example 3 and
10 ng of each DNA of a known HSV wild type strain, or
10 μl of DNA extract from clinical specimen extracted in Example 4
With 125 μM-dATP, 125 μM-dCTP, 1
25 μM-dTTP, 31.25 μM-dGTP, 9
3.75 μM-deazaC ⁷ dGTP (hereinafter dC⁷dGT
P), 0.01% gelatin, 20 mM Tris-HCl
Buffer solution (pH 8.8), 1.5 mM-MgCl_Two, 50 mM-
KCl, 10% dimethyl sulfoxide, 2 μM primer
And 2.5 units of Taq polymerase (AmpliT
aq DNA polymerase (Cetus)
The solution was added to 100 μl. The PCR method is (i)
Minutes, (ii) 1 minute at 65 ° C, (iii) 2 minutes at 75 ° C
30 cycles, and finally at 75 ° C
Amplification of DNA was performed by a program for 5 minutes. [0025]Example 6: PCR reaction generation by electrophoresis
Confirmation and diagnosis of objects The gel for electrophoresis was 4.84 g of Tris base and glacial acetic acid.
Buffer containing 1.142 g and 2 ml of 0.5 M EDTA
(PH 8.0) (hereinafter referred to as 1xTAE) 1 liter
Dissolve agarose gel to 4% and add mupido (A
(Dovance Co., Ltd.). next,
Add 10 μl of the solution after PCR to Ficoll type 400
(Pharmacia) and 2 μl of a 15% aqueous solution.
Of the mixture was placed in a sample well. 1xTAE
Into an electrode tank and electrophoresed at 100 V for 2 hours at room temperature
Was done. After completion of electrophoresis, agarose gel is
Umbromide stained and charged under UV illuminator irradiation.
The result of the electrophoresis was confirmed (FIG. 1). Ethidium bu in FIG.
According to romide staining, using HSV type 2 primer
In the PCR method, multiple bands were observed.
Southern hybridization using P-labeled probe
Was done. That is, the agarose gel after electrophoresis
A Hybridization Membrane
(GeneScreenPlusNEF976: DuP
ontSouthern blot and AP-labeled pro
The probe was hybridized. It is clear from the result of FIG.
As can be seen, the DNA of a patient sample can be
And confirm the amplified DNA with the probe of the present invention.
By doing so, the virus causing HSV infection is
Type 2 definitive diagnosis can be performed. Figure 1 and
In FIG. 2, “M” is a DNA marker, “1” is H
SV2 standard strain UW268, "2" to "7"
"137b" is a probe 1 of the present invention.
2 and 3 show the positions of the DNA fragments amplified by the method. Ma
DNA marker (φ174-HaeIII dige)
As st), from the bottom, 72 bp, 118 bp, 194
bp, 234 bp, 271 bp, 281 bp, 310 b
p, 603 bp, 872 bp, 1078 bp and 135
Those appearing at 7 bp were used. [0026]Example 7: For dot blotting of clinical samples
Classification of HSV2 Type Blister content from a patient suspected of having a herpes infection
The solution (5 μl) was suspended in 2 ml of physiological saline and diluted. Dilution
DNA was obtained from 10 μl of the sample under the conditions described in Example 4.
The DNA was extracted and subjected to PCR 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. Denatured DNA
10 μl of the processed sample thus obtained was placed in GeneScreen
nPlusNEF976 (DuPont) was installed.
Placed on blotting device and left at room temperature for 30 minutes
Thereafter, the liquid was removed by suction. DNA blotted DNA
Luter in a plastic bag, followed by 0.75M-
NaCl, 75 mM trisodium citrate, 0.1% N-
Lauryl sarcosine, 0.2% SDS, 30% formua
Mid, blocking for 5% nucleic acid hybridization
Of the composition of the agent (Boehringer Mannheim)
5 ml of a buffer for lysis was added and sealed. At 37 ° C
After standing for 60 minutes, hybridization buffer
Was removed. AP-labeled HSV type 2 prepared in Example 2
Hybridization with 3 picomoles of the discriminating probe
1.50 ml of the reaction solution was added, and the mixture was left at 37 ° C. overnight. Inspection
Hybridization of probe with viral DNA in the body
Remove the completed filter from the plastic bag
First, 2x SSC twice for 5 minutes, then 0.1%
15 minutes in 2 × SSC containing sodium decyl sulfate
Twice, and finally 3 times with 2 × SSC for 10 minutes. Washing
The filtered filter was washed with 0.1 M NaCl and 10 mM.
-MgCl_Two0.1M Tris-HCl buffer (pH 9.
5) Immerse for 30 seconds to 1 minute (hereinafter referred to as coloring solution)
Was. Nitro blue tetrazolium (boehringer
-Mannheim) 75 mg in 30% sterile water-70% dimethi
Dissolved in 1 ml of mixed formamide (hereinafter, this solution
Is called NBT solution). Also, 5-bromo-4-chloro
B-3-Indolyl phosphoric acid (Boehringer Mannheim)
50 mg was dissolved in 1 ml of dimethylformamide (hereinafter referred to as “
This solution is called BCIP solution). NBT solution 40μ
and 10 ml of a color developing solution to which 40 μl of BCIP solution was added.
Plastic bag containing filter soaked in the coloring solution
And kept at room temperature for 5 minutes to 16 hours to develop color.
The reaction with AP was performed using Tris-HCl buffer containing 10 mM EDTA.
To immerse the filter in 50 ml of buffer (pH 7.5)
Stopped more. The results are shown in Table 1. Clinical trial
Among 40 samples, HSV1 type 16 test was used for virus isolation.
Body, HSV type 2 14 samples, undeterminable (determined as type 1 or type 2)
2) and 8 negative samples. Direct dot
In the blot method, 14 samples of HSV1 type, 17 samples of HSV2 type
Samples, 1 undeterminable sample, 8 negative samples. Against this
However, the result of the HSV2 type discriminating probe of the present invention is as follows:
HSV type 2 became 20 samples, undetermined and negative samples
There was no. Also, according to another test result, the remaining 20 samples
Has been confirmed to be of HSV1 type. Table 1
Fig. 3 shows the test results based on the PCR method of the present invention based on
Show. In FIG. 3, lanes AC are HSV type 1 specific.
Dot blot of PCR product, lane D
~ F shows dot blots of HSV type 2 specific PCR products.
It is a thing. C9 and F9 are HSV type 1 standard strains.
(KOS), C10 and F10 are HSV2 type standard
(UW268), and lanes A to F 1 to 8
The floor sample was blotted after PCR treatment.
Samples 1 to 8 in lanes A to C and samples 1 to 8 in lanes DF are the same.
The same sample was blotted after PCR treatment. [0029] [Table 1] [0030] The method of the present invention is specific for HSV type 2 DNA.
A pair of plasmids that can be used for
As a probe specific to immersion and HSV type 2 DNA
Large enough for chemical synthesis, with a unique type discrimination
Oligonucleotides consisting of a base sequence that does not decrease
To provide. Therefore, HSV type 2 infections
Accurate, rapid and type-specific identification, resulting in
The results can be used for diagnosis and treatment. [0031] [Sequence list] SEQ ID NO: 1 Sequence length: 20 Sequence type: nucleic acid Number of chains: single strand Array: GCGCGAAGGC GGGCGGCGGC SEQ ID NO: 2 Sequence length: 20 Sequence type: nucleic acid Number of chains: single strand Array: GCCGCGGAAG GCAGCCCGCG SEQ ID NO: 3 Sequence length: 40 Sequence type: nucleic acid Number of chains: single strand Array: GCAGCCCCGC GCGCCCCCTT CCCCGTCCCT CCCCCGGAGC

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph instead of a drawing showing the result of electrophoresis performed by the inspection method of the present invention. FIG. 2 is a photograph instead of a drawing showing the result of the Southern hybridization of FIG. FIG. 3 is a photograph instead of a drawing, showing the results of dot blots of HSB-type-specific PCR products.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-3-290189 (JP, A) JP-A-2-142499 (JP, A) Gen. Virol. 55, Pt. 2 (1981), p. 315-331 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-1/70 C12N 15/00-15/90 BIOSIS / WPI (DIALOG) CA / REGISTRY (STN) PubMed

Claims (1)

(57) [Claim 1] 20mer to 25 containing an oligonucleotide portion consisting of the sequence of SEQ ID NO: 1 in the sequence listing
a combination of a first DNA primer of mer and a second DNA primer of 20 mer to 25 mer containing an oligonucleotide portion consisting of the sequence of SEQ ID NO: 2 in the sequence listing, a DNA polymerase, and an aqueous liquid test sample The mixed solution is subjected to a DNA amplification step, and the resulting reaction solution is brought into contact with a probe containing an oligonucleotide portion consisting of the nucleotide sequence of SEQ ID NO: 3 in the sequence listing and carrying a label to detect a signal from the label. A method for detecting herpes simplex type 2.