JPH07203996A - Oligonucleotide for detecting human herpes simplex virus and use thereof - Google Patents

Abstract

PURPOSE:To obtain an oligonucleotide having high homology to a specific base sequence or its complementary sequence, capable of easily, quickly, specifically and sensitively detecting human herpes simplex virus and useful for labeled probe, primer, etc. CONSTITUTION:This new oligonucleotide has >=70% homology to a nucleic acid sequence expressed by formula I to VIII [A is adenine; C is cytosine; G is guanine; T is thymine; T at arbitrary position may be replaced with uracil (U)] or >=70% homology to a sequence complementary to the above sequence and is capable of easily, quickly, specifically and sensitively detecting human herpes simplex virus. The oligonucleotide can be chemically synthesized by a phosphoamidite method using a DNA synthesizer. Human herpes simplex virus can be detected by labeling the oligonucleotide with <32>P-phosphoric acid group, etc., to form a probe or primer and amplifying by PCR method.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to human herpes simplex virus (HSV).
And a use thereof.

[0002]

2. Description of the Related Art HSV is often subclinical in the initial infection, but it is often severe when it is overt. H
It is known that after the initial infection, SV is latent in the ganglia and is reactivated due to fever, overwork, administration of immunosuppressive agents, and the like. HSV is mainly type 1 (HSV-1), which infects the upper body, and type 2 (HSV-), which infects the lower body.
It is classified into 2). HSV is stomatitis, pharyngitis, keratitis,
The diagnostic significance is high because it shows an extremely diverse clinical picture, ranging from diseases that are frequently treated in daily medical care such as genital herpes to serious diseases that are fatal such as herpes encephalitis and meningitis. In addition, due to the recent development of effective therapeutic agents such as acyclovir,
It is desired to establish a rapid early diagnosis method.

Culture methods and immunization methods are known as methods for detecting HSV. However, in the culture method, it takes 5 to 7 days from separation culture to identification, and rapid diagnosis cannot be performed. On the other hand, in the case of the immunization method, in the method of testing the antigen, the diversity of antigenicity becomes a problem in HSV in which the variation among strains is severe. In addition, the method of testing an antibody generally only indicates that the virus has been affected in the past, and does not reflect the state at the time of testing.

[0004]

Therefore, an object of the present invention is to provide an oligonucleotide capable of detecting human herpes simplex virus conveniently, rapidly, specifically and with high sensitivity.

[0005]

Means for Solving the Problems As a result of various investigations concerning the gene of HSV, the present inventors have obtained an oligonucleotide having a sequence capable of detecting HSV simply, rapidly, specifically and with high sensitivity. Established the detection method used,
The present invention has been completed.

The present invention is directed to HSV-specific oligonucleotides, that is, SEQ ID NOS: 1 to 8 (provided that A
Represents adenine, C represents cytosine, G represents guanine, and T represents thymine. Also, T at any position has 70% or more homology with the nucleic acid sequence shown in uracil (U), or has 70% or more homology with the sequence complementary thereto. An oligonucleotide for detecting human herpes simplex virus.

Further, the present invention is characterized in that a labeled nucleic acid probe labeled with the above-mentioned human herpes simplex virus detection oligonucleotide is hybridized with DNA or RNA in the sample, and the label of the hybridized conjugate is measured. It is a method for detecting human herpes simplex virus.

Further, the present invention is obtained by hybridizing a nucleic acid primer, which is the above-mentioned oligonucleotide for detecting human herpes simplex virus, or a labeled nucleic acid primer labeled with the oligonucleotide, with DNA or RNA in a sample and extending the primer. A method for detecting human herpes simplex virus in a sample, which comprises measuring the primer extension product.

The present invention also provides a reagent kit for detecting human herpes simplex virus in a sample, which comprises a labeled nucleic acid probe labeled with the above-mentioned oligonucleotide for detecting human herpes simplex virus.

Further, the present invention comprises a nucleic acid primer which is the above-mentioned oligonucleotide for detecting human herpes simplex virus, or a labeled nucleic acid primer obtained by labeling the oligonucleotide, and a reagent for detecting human herpes simplex virus in a sample. It's a kit.

The oligonucleotides for detecting human herpes simplex virus of the present invention are shown in Sequence Listing, SEQ ID NOs: 1 to 8 (provided that A
Represents adenine, C represents cytosine, G represents guanine, and T represents thymine. Also, T at any position has 70% or more homology with the nucleic acid sequence shown in uracil (U), or has 70% or more homology with its complementary sequence. And reacts specifically with the human herpes simplex virus. In general, since oligonucleotides can be prepared by chemical synthesis, compared to cloned oligonucleotides or polynucleotides, it is possible to obtain oligonucleotides of constant quality in a large amount and at low cost.

The oligonucleotide of the present invention may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In the case of ribonucleic acid, it goes without saying that the thymidine residue (T) is read as the uridine residue (U). Alternatively, a DNA containing a uridine residue may be used by synthesizing by changing T at any position to U at the time of synthesis. Similarly, any U
It may be RNA containing a thymidine residue in which is changed to T. The oligonucleotide may have point mutations such as deletion, insertion or substitution, and modified nucleotides.

The labeled nucleic acid probe of the present invention is one in which a label is bound to the above-mentioned oligonucleotide for detecting human herpes simplex virus, and the labeled substance is a radioactive substance, an enzyme, a fluorescent substance, a luminescent substance, an antigen, a hapten, an enzyme substrate. A known label such as an insoluble carrier can be used. The labeling method may be end labeling or labeling in the middle of the sequence. It may also be a label on the sugar, phosphate group, or base moiety.

As the nucleic acid primer of the present invention, the above-mentioned human herpes simplex virus detection oligonucleotide is used as it is, or the oligonucleotide is labeled.

When HSV is detected using the human herpes simplex virus detection oligonucleotide of the present invention, (1)
There is a method of detecting the oligonucleotide of the present invention as a probe, and (2) a method of detecting the oligonucleotide of the present invention by using the oligonucleotide of the present invention as a primer for extension reaction with a DNA polymerase or the like. In the case of the above (2), detection can be easily performed by introducing a label into the above oligonucleotide. In the case of the above (1), the labeled nucleic acid probe labeled with the oligonucleotide of the present invention is used as DNA or R in a sample.
It is achieved by hybridizing with NA and detecting the label of the hybridized conjugate by an appropriate detection method. On the other hand, in the case of the above (2), the oligonucleotide of the present invention is used as a nucleic acid primer as it is, or a labeled nucleic acid primer obtained by labeling the oligonucleotide is hybridized with DNA or RNA in a sample, and the primer is treated with DNA polymerase or the like. Or measure the resulting primer extension directly, or
It measures using a labeled probe.

When the oligonucleotide of the present invention is used as a primer, it is possible to specifically amplify only the HSV gene by carrying out gene amplification (PCR etc.) using DNA polymerase or the like. In the amplification reaction, HSV can be easily detected by a method of incorporating a labeled nucleotide such as a radioactive substance during the reaction or by fractionating the amplification product by electrophoresis to detect a specific band. Furthermore, by using the type-specific oligonucleotide of the present invention, the type can be easily determined by the size of the amplification product after electrophoresis. Further, the amplification product can be directly detected by using the above-mentioned labeled oligonucleotide as a primer.

The human herpes simplex detection reagent kit of the present invention comprises the human herpes simplex virus detection oligonucleotide of the present invention or a labeled oligonucleotide obtained by labeling the oligonucleotide with an appropriate labeling agent. The reagent kit containing the nucleic acid primer may contain a labeled probe. Further, other components include a labeled detection substance, a buffer solution and the like. Furthermore, in the reagent kit of the present invention containing a nucleic acid primer or a labeled nucleic acid primer, as other components, a nucleic acid synthase (DNA polymerase, RNA polymerase, reverse transcriptase, etc.), a substrate corresponding to the enzyme (dNTP, rNTP, etc.) , Labeled detection substances and buffers.

Further, the oligonucleotide of the present invention can be bound to a solid phase carrier and used as a capture probe. In this case, the sandwich assay may be performed with a combination of a capture probe and a labeled (nucleic acid) probe, or there is a method of labeling and capturing a target nucleic acid. There is also a method in which an oligonucleotide is labeled with biotin, and after hybridization, it is captured with an avidin-binding carrier.
In the sandwich assay, if the oligonucleotide of the present invention is used for either one of them, specific measurement becomes possible due to the specificity of the oligonucleotide, and there is no problem even if the specificity of the other oligonucleotide is slightly low.

[0019]

EXAMPLES The effects of the present invention will be further clarified by exemplifying the examples of the present invention.

Example 1 (Synthesis of Various Oligonucleotides) A variety of oligonucleotides having the sequences shown in the sequence listing and SEQ ID NOs: 1 to 8 were synthesized by the phosphoamidite method using ABI DNA Synthesizer Model 391. Less than,
The various oligonucleotides shown in Sequence Listing and SEQ ID NOS: 1 to 8 are referred to as oligonucleotides (1) to (8), respectively. The method was carried out on a 0.2 μM scale according to the ABI manual. Deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Purification was carried out by FPLC manufactured by Pharmacia on a reverse phase column. If necessary, the synthesized oligonucleotide had a ³² P-phosphate group bonded to the 5'end by the following method.

Reaction solution composition Oligonucleotide 5-20 pmoles 10 × T4 polynucleotide kinase buffer 10 μl 1 mM [γ- ³² P ATP (10 mCi / ml) 1 μl T4 polynucleotide kinase (manufactured by Toyobo) 10 units The reaction mixture prepared as described above was reacted at 37 ° C. for 1 hour. Here, the 10 × T4 polynucleotide kinase buffer means 0.5M Tris-HCl (pH8.0), 0.1M MgCl ₂ , 0.1M 2
-Indicates mercaptoethanol.

Example 2 (Common Amplification of Human Herpes Simplex Virus) (1) Kit A: Kit for Amplifying Human Herpes Simplex Virus Nucleic Acid (a) Oligonucleotide of Example 1 (1) (b) Example Oligonucleotide 1 (2) (c) Tth DNA polymerase (manufactured by Toyobo), dAT
P, dCTP, dGTP, dTTP

(2) Preparation of Viral Nucleic Acid The Seivert strain was used as a standard strain of human herpes simplex virus type 1 (HSV-1), and the UW268 strain was used as a standard strain of human type 2 (HSV-2). In addition, varicella-zoster virus (VZV) estuary strain and EB virus (EBV; Epstein-Barr virus) were used to examine specificity.
s) B95-8 strain, human cytomegalovirus (CM
V; cytomegalovirus) AD169 strain,
Human herpesvirus type 6 A (HHV-6A) U11
The strain 02, the same type B (HHV-6B) Hashimoto strain, the human herpesvirus type 7 (HHV-7) RK strain, and human placental DNA (manufactured by Sigma) were used. Viral nucleic acid was prepared by a conventional method.

5 μl of the nucleic acid solution and 1 μl each of the oligonucleotides (1) and (2) of Example 1 were added as primers to 93 μl of the PCR reaction solution to amplify human herpes simplex virus-derived nucleic acid. The composition of the reaction solution is as follows. Reaction solution composition KCl 50 mM Tris-HCl (pH 8.3) 10 mM MgCl ₂ 1.5 mM Gelatin 0.01% dATP, dCTP, dGTP, dTTP 0.2 mM Tth DNA polymerase 40 units / ml

The reaction conditions are as follows. Heat denaturation: 94 ° C., 1 minute, annealing: 55 ° C., 2 minutes Polymerization reaction: 75 ° C., 1.5 minutes 30 times. These operations are Perkin-Elmer /
It was performed using a DNA thermal cycler manufactured by Cetus (Perkin-Elmer / Cetus).

Detection 10 μl of the reaction solution was electrophoresed on a 2% agarose gel and stained with ethidium bromide, and then fluorescence from ultraviolet rays was detected (FIG. 1). In FIG. 1, lanes 2 to 10 are each H
SV-1, HSV-2, VZV, EBV, CMV, HH
V-6 (A), HHV-6 (B), HHV-7 and human placental DNA are shown. Lanes 1 and 11 show molecular weight markers. The electrical conditions for electrophoresis are a constant voltage of 100 V,
The time was 30 minutes. Operating method and other conditions are Maniat
The method described by Is et al., Molecular Cloning (1982) was followed. In addition to the reaction solution, a molecular weight marker was also electrophoresed at the same time, and the length of the detected nucleotide fragment was calculated by comparing the relative mobilities.

Results In the PCR product, a band was observed only when the human herpes simplex virus was used (FIG. 1: lanes 2 and 3). Its size was identified by molecular weight markers to be about 320 base pairs.
This was in good agreement with the nucleotide length calculated from the nucleic acid sequence.

Example 3 (Specific Amplification of Human Herpes Simplex Virus) (1) Kit B: Kit for Amplifying Human Herpes Simplex Virus-Derived Nucleic Acid (a) Oligonucleotide of Example 1 (1) (b) Example Oligonucleotide 1 (3) (c) Oligonucleotide 1 of Example 1 (4) (d) Tth DNA polymerase (manufactured by Toyobo), dAT
P, dCTP, dGTP, dTTP

5 μl of the nucleic acid solution and 1 μl each of the oligonucleotides (1), (3) and (4) of Example 1 were added as primers to 93 μl of the PCR reaction solution to perform type-specific amplification of human herpes simplex virus-derived nucleic acid. It was The composition of the reaction solution and the detection method were the same as in Example 2. In FIG. 2, lanes 2 to 10 are HSV-1, HSV-2, VZV, EBV, and CM, respectively.
V, HHV-6 (A), HHV-6 (B), HHV-7
And human placental DNA. Lanes 1 and 11 show molecular weight markers.

Results In the PCR product, a band was observed only when human herpes simplex virus was used (FIG. 2: lanes 2 and 3). From the molecular weight marker, the size was identified as about 210 base pairs for HSV-1 and about 280 base pairs for HSV-2. This was in good agreement with the nucleotide length calculated from the nucleic acid sequence.

Example 4 (Specific detection of human herpes simplex virus) Dot blot hybridization was carried out to identify which human herpes simplex virus the amplification product of Example 2 was derived from. (1) Kit C: Kit for detecting nucleic acid derived from human herpes simplex virus Oligonucleotides (5) and (6) of Example 1, and
It contains (7) or (8) and has a ³² P-phosphate group at the 5'end. (2) Dot Blot Hybridization 100 μl of 0.3 N NaOH was added to 1 μl of the PCR product of Example 2, denatured at room temperature for 15 minutes, and wetted with 5 × SSC using a dot blotter (manufactured by BRL). It was blotted on a nylon membrane (High Bond N Plus, manufactured by Amersham). SSC is a 15 mM trisodium citrate and 15 mM sodium chloride solution, and 5 × SSC is a 5-fold concentrated solution of SSC. This membrane was heated at 80 ° C. for 30 minutes to immobilize the nucleic acid, and then hybridized. First, the dried membrane (8 × 12 cm) was immersed in 5 × SSC for 5 minutes. Next, transfer the membrane to a hybridization bag (manufactured by BRL), add 5 ml of hybridization buffer (5 × SSC, 0.5% bovine serum albumin, 0.5% polyvinylpyrrolidone, 1% sodium dodecyl sulfate) to the policy. Seal with a stirrer and pre-hybridize at 50 ° C. for 15 minutes. Next, the oligonucleotide probe of kit B
Hybridization was carried out at 55 ° C. for 15 minutes with 5 ml of a hybridization buffer containing 5 μl of any one of (5) to (8). The membrane was taken out from Polyvac and washed with Washing Solution-1 (1 × SSC, 1% sodium dodecyl sulfate) at 55 ° C. for 5 minutes with shaking twice. The membrane was thoroughly dried after it was washed with Shake Solution-2 (1 × SSC) twice at room temperature for 5 minutes with shaking.

(3) Detection An X-ray film (New AIF RX made by Fuji Photo Industry Co., Ltd.) was adhered to the nylon film and exposed at -80 ° C for a whole day and night. Human herpes simplex virus was detected from the photosensitivity of the film.

Results The oligonucleotide probes specific to the respective viruses specifically reacted only with the amplification products derived from the respective viruses, and no reaction with the amplification products derived from other viruses or human DNA was observed. (Table 1).

[0034]

[Table 1]

Example 5 (High Sensitivity of Human Herpes Simplex Virus)
Degree-specific detection) Amplification products derived from human herpes simplex virus are more sensitive
Southern blot hybridization to detect
I went to (1) Kit D: Detects human herpes simplex virus-derived nucleic acid
Kit for the preparation of the oligonucleotide of Example 1 (5),
(7) or (6), (8) at the 5'end, ³²P-phosphate group
have. (2) Southern Blot Hybridization The HSV-1-derived PCR product of Example 3 and HSV-2
Each of the derived PCR products is diluted 5 times in 5 steps to obtain the original product.
5 μl of each of the 6 concentrations of PCR product solution
Electrophoresis was performed using a 2% agarose gel. Etisi
After staining with umbromide solution and taking a photograph, Maniat
By the method described in Molecular Cloning (1982) of is et al.
For nylon membrane (High Bond N Plus, Amersham)
Blotted. Hybridization as a probe
Oligonucleotide (6) of Example 1 (for HSV-1:
Example using (3) and (8) (for HSV-2: FIG. 4)
The same procedure as in 4 was performed.

(3) Detection The same procedure as in Example 4 was performed (FIG. 3: HSV-1, FIG. 4: H).
SV-2). In FIG. 3, lanes 2 to 7 are PCRs, respectively.
The stock solution of the product, the same 5-fold diluted solution, the 25-fold diluted solution, the 125-fold diluted solution, the 625-fold diluted solution, and the 3125-fold diluted solution are shown. Lanes 1 and 11 show molecular weight markers. In FIG.
Lanes 2 to 7 show the stock solution of PCR product, the same 5-fold diluted solution, the 25-fold diluted solution, the 125-fold diluted solution, the 625-fold diluted solution, and the 3125-fold diluted solution, respectively. Lanes 1 and 11 show molecular weight markers. Results Both HSV-1 and HSV-2 could be detected up to a 125-fold diluted solution by electrophoresis (Fig. 3-
A, FIG. 4-A), and by performing Southern blot hybridization, it was possible to detect up to a 3125-fold diluted solution (FIG. 3-B, FIG. 4-B).

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to measure human herpes simplex virus, which has conventionally been time-consuming to culture, quickly, simply, and specifically and highly sensitively by type. The oligonucleotide of the present invention can be used both as a primer for amplification reaction and as a probe for direct detection.
Also. By detecting the amplification product amplified by the primer with a probe, it is possible to detect with higher sensitivity and specificity, which is of great clinical significance.

[0038]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method: S Other features: Sequence common to human herpes simplex virus. Array GTTTGTGTGT TGTTCGGTCA TAAGC 25

SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..22 Features Method determined: S Other features: Sequence common to human herpes simplex virus. Array GCGTAGTACA CCGTGATCGG GA 22

SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other features: human herpes simplex virus type 1 (HSV-1) specific sequence. Sequence TCGGCCATCT TGAGAGAGGC ATCC 24

SEQ ID NO: 4 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..24 Method determined: S Other characteristics: human herpes simplex virus type 2 (HSV-2) specific sequence. Sequence GGCTGAATGT GGTAAACACG CTTC 24

SEQ ID NO: 5 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: human herpes simplex virus type 1 (HSV-1) specific sequence. Array AGCGCGAACG ACCAACTACC CCGAT 25

SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other features: human herpes simplex virus type 1 (HSV-1) specific sequence. Sequence TCAGTTATCC TTAAGGTCTC TTTTG 25

SEQ ID NO: 7 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other characteristics: human herpes simplex virus type 2 (HSV-2) specific sequence. Sequence TCCCAATCGA TTTCGCGGGA AGAAC 25

SEQ ID NO: 8 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Both forms Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Location: 1..25 Features Method determined: S Other characteristics: human herpes simplex virus type 2 (HSV-2) specific sequence. Sequence TTCCGGTTTT GGACCAGCTG ACCGA 25

[Brief description of drawings]

FIG. 1 shows the results of electrophoresis of Example 2.

FIG. 2 shows the results of electrophoresis of Example 3.

FIG. 3 shows the results of electrophoresis (A) and Southern hybridization (B) when using the HSV-1-derived PCR product of Example 5.

FIG. 4 shows the results of electrophoresis (A) and Southern hybridization (B) when using the HSV-2-derived PCR product of Example 5.

Claims (11)

[Claims] 1. Sequence Listing: SEQ ID NOS: 1 to 8 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). The nucleic acid sequence shown in (1) may have a homology of 70% or more, or a complementary sequence thereof with a homology of 70% or more, for detecting human herpes simplex virus. 2. SEQ ID NO: 1 to 8 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). An oligonucleotide for detecting human herpes simplex virus, which has a homology of 70% or more with the nucleic acid sequence shown in FIG. 3. Sequence Listing, SEQ ID NOS: 1 to 8 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). Oligonucleotide for detecting human herpes simplex virus. 4. SEQ ID NO: 1 to 8 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). An oligonucleotide for detecting human herpes simplex virus, which has a homology of 70% or more with the complementary sequence of the nucleic acid sequence shown in FIG. 5. SEQ ID NO: 1 to 8 (where A is adenine, C is cytosine, G is guanine, T is thymine, and T at any position is replaced with uracil (U). An oligonucleotide for detecting human herpes simplex virus, which is a complementary sequence of the nucleic acid sequence shown in 6. A human herpes simplex virus detection oligonucleotide according to claim 1 is labeled, the obtained labeled nucleic acid probe is hybridized with DNA or RNA in a sample, and the label of the hybridized conjugate is measured. A method for detecting human herpes simplex virus in a sample, comprising: 7. A nucleic acid primer, which is the oligonucleotide for detecting human herpes simplex virus according to claim 1, or a labeled nucleic acid primer labeled with the oligonucleotide is hybridized with DNA or RNA in a sample to extend the primer, A method for detecting human herpes simplex virus in a sample, which comprises measuring the obtained primer extension product. 8. A reagent kit for detecting human herpes simplex virus in a sample, which comprises the labeled nucleic acid probe labeled with the oligonucleotide for detecting human herpes simplex virus according to claim 1. 9. A human herpes simplex virus in a sample, which comprises a nucleic acid primer that is the oligonucleotide for detecting human herpes simplex virus according to claim 1 or a labeled nucleic acid primer labeled with the oligonucleotide. Reagent kit for detection. 10. A labeled nucleic acid probe labeled with the oligonucleotide for detecting human herpes simplex virus according to claim 1, and a nucleic acid primer or the oligo that is the oligonucleotide for detecting human herpes simplex virus according to claim 1. A reagent kit for detecting human herpes simplex virus in a sample, which comprises a labeled nucleic acid primer labeled with a nucleotide. 11. A reagent kit for detecting human herpes simplex virus in a sample according to claim 8, which further comprises a nucleic acid synthase, dNTP and / or rNTP, and a buffer solution.