CN106282408B

CN106282408B - Method for detecting clade2.1.3 branch H5N1 subtype avian influenza virus by pyrosequencing

Info

Publication number: CN106282408B
Application number: CN201610685766.7A
Authority: CN
Inventors: 孙洪磊; 王晨曦; 张谞霄; 蒲娟; 孙怡朋; 刘金华
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2016-08-18
Filing date: 2016-08-18
Publication date: 2019-12-24
Anticipated expiration: 2036-08-18
Also published as: CN106282408A

Abstract

The invention discloses a method for detecting clade2.1.3 branch H5N1 subtype avian influenza virus by pyrosequencing. The invention provides a complete set of primers for detecting clade2.1.3 branch H5N1 avian influenza virus, which comprises the following primer pairs: the primer pair consists of a single-stranded DNA molecule shown in a sequence 1in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table. The primer set also comprises a sequencing primer of which the nucleotide sequence is a sequence 3 in a sequence table. The method has strong specificity and high sensitivity, and compared with the identification results of conventional experimental methods such as virus separation, hemagglutination inhibition experiment and the like, the coincidence rate reaches 100%, and the method can timely and quickly detect and diagnose the avian infectious disease condition and the virus pollution condition of the region and the environment, and has important significance for timely monitoring the clade2.1.3 branch H5N1 avian influenza virus entry transmission.

Description

Method for detecting clade2.1.3 branch H5N1 subtype avian influenza virus by pyrosequencing

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting clade2.1.3 branch H5N1 subtype avian influenza virus by pyrosequencing.

Background

The traditional avian influenza virus detection method is to inoculate chick embryos for virus isolation and culture (2-3 days), and then to identify influenza virus subtypes by adopting a hemagglutination inhibition test (1 day). The traditional detection method has the defects of time and labor waste, is difficult to diagnose quickly and timely, and can not distinguish the viruses with higher homology accurately.

Highly pathogenic avian influenza virus subtype H5N1 has been prevalent in poultry in asia, europe, africa 15 countries and causes infections in humans. In these countries, the incidence and mortality of Indonesia is the first worldwide. By 9 months of 2015, 199 people had been diagnosed with avian influenza virus H5N1in Indonesia, 167 people died, and the number of diseases and the mortality rate were the first worldwide. The H5N1 strain, which is predominantly prevalent in indonesia, is the clade2.1 branch, including 2.1.1, 2.1.2, 2.1.3. Since 2005, the proportion of calde 2.1.1 and clade2.1.2 has been significantly reduced, while clade2.1.3 has gradually become the main epidemic strain in Indonesia, and almost all strains infecting humans belong to the clade2.1.3 branch. The clade2.1.3 branch virus is shown to be more infectious and pathogenic to human than other branch influenza viruses of subtype H5. At present, no report that people or birds infect clade2.1.3 branched H5N1 avian influenza virus exists in China, but both trade communication of avian products and migratory bird migration can transfer the clade2.1.3 branched H5N1 avian influenza virus into China. Therefore, a method for quickly and effectively diagnosing the clade virus is established, and the method has important significance for monitoring the immigration and transmission of the clade2.1.3 clade H5N1 avian influenza virus.

Conventional detection methods, such as virus isolation, hemagglutination inhibition assay, neuraminidase assay, etc., are time consuming, laborious, and do not allow for rapid and timely diagnosis. For related molecular biology detection methods, such as a single RT-PCR method and a multiple RT-PCR method, viruses with high homology cannot be accurately distinguished only through PCR electrophoresis results, and Sanger sequencing and gene evolution analysis are required. Therefore, it is very important to establish a novel detection method for the clade2.1.3 branch H5N1 avian influenza virus.

The pyrosequencing technology is a novel sequence analysis technology invented in recent years, the pyrosequencing technology triggers enzyme cascade reaction through pyrophosphoric acid released after nucleotide is combined with a template, so that fluorescein is promoted to emit light and be detected, gene sequence analysis can be performed on a target sequence in a short time aiming at a known short sequence, and the pyrosequencing technology has the advantages of rapidness, accuracy, real time and the like, and the repeatability and the accuracy of the pyrosequencing technology can be comparable to those of a sanger DNA sequencing method. In addition, the Sanger method has advantages in that the sequence of unknown DNA can be analyzed, and the read sequence of the one-way reaction is long. In practical work, sequence verification is required for DNA fragments with known sequences in many cases, and the analysis can be carried out by detecting dozens of bases. In this case, Sanger's method is not necessarily the most suitable DNA sequence analysis technique.

Disclosure of Invention

The invention aims to provide a set of primers for detecting clade2.1.3 branch H5N1 avian influenza virus, which comprises the following primer pairs:

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table.

In the above set of primers, one primer in the primer pair is labeled with biotin at its end.

The primer set also comprises a sequencing primer of which the nucleotide sequence is a sequence 3 in a sequence table.

A PCR kit comprising the above primer set or a kit comprising the primer set or the PCR kit is also within the scope of the present invention.

In the above-mentioned PCR reagent set, the PCR reagent set consists of PCR reagent 1 and PCR reagent 2;

the PCR reagent 1 is a reagent containing the primer pair in the primer set;

or, the concentration of each primer in the primer pair in the PCR reagent 1 is 20 μ M;

the PCR reagent 2 is a reagent containing the sequencing primer in the primer set;

alternatively, the concentration of the sequencing primer in the PCR reagent 2 is 0.3. mu.M.

The primer set or the PCR reagent set or the kit is applied to the detection of clade2.1.3 branched H5N1 avian influenza virus;

or the primer set or the PCR reagent set or the kit is applied to the preparation of the products for detecting the clade2.1.3 branched H5N1 avian influenza virus;

or, the primer set or the PCR reagent set or the kit is applied to detecting whether the sample to be detected contains the clade2.1.3 branched H5N1 avian influenza virus;

or, the primer set or the PCR reagent set or the kit is applied to the preparation of the product for detecting whether the sample to be detected contains the clade2.1.3 branched H5N1 avian influenza virus;

or the primer set or the PCR reagent set or the kit is applied to detecting the amino acid residue at the P6 position of the HA protein cleavage site of the H5N1 subtype avian influenza virus;

or the primer set or the PCR reagent set or the kit is applied to the preparation of products for detecting the amino acid residue at the P6 position of the HA protein cleavage site of the H5N1 subtype avian influenza virus;

the HA protein cleavage site P6 amino acid sequence is 325 th amino acid of sequence 4 in the sequence table.

The invention also aims to provide a method for detecting or assisting in detecting whether the sample to be detected contains the clade2.1.3 branched H5N1 avian influenza virus.

The method comprises the following steps:

1) carrying out PCR amplification on a sample to be detected by using a primer pair consisting of a single-stranded DNA molecule shown in a sequence 1in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table to obtain a PCR amplification product;

2) carrying out pyrosequencing on the PCR amplification product by using a sequencing primer with a nucleotide sequence of sequence 3 in the sequence table to obtain a sequencing result of the PCR amplification product;

3) carrying out reverse complementation on the sequencing result of the PCR amplification product to obtain a reverse complementary strand; translating the reverse complementary strand to obtain the amino acid sequence of the PCR amplification product; if the 8 th position from the C end in the amino acid sequence of the PCR amplification product is S, the sample to be detected contains or is candidate to contain clade2.1.3 branch H5N1 avian influenza virus; if the 8 th position from the C end in the amino acid sequence of the PCR amplification product is not S, the sample to be detected does not contain or does not contain the clade2.1.3 branch H5N1 avian influenza virus.

The third purpose of the invention is to provide a method for detecting the amino acid residue at the P6 position of the HA protein cleavage site in a sample to be detected.

The method provided by the invention comprises the following steps:

3) carrying out reverse complementation on the sequencing result of the PCR amplification product to obtain a reverse complementary strand; and translating the reverse complementary strand to obtain an amino acid sequence of the PCR amplification product, and obtaining an amino acid residue at the position P6 of the HA protein cleavage site in the sample to be detected.

In the method, the annealing temperature of the PCR amplification is 55 ℃.

In the method, the template amplified by PCR is the cDNA of the sample to be detected.

Experiments prove that the method is faster than virus separation, RT-PCR and serological typing methods, and can detect a large number of samples. The method has strong specificity and high sensitivity, and compared with the identification results of conventional experimental methods such as virus separation, hemagglutination inhibition experiment and the like, the coincidence rate reaches 100%, and the method can timely and quickly detect and diagnose the avian infectious disease condition and the virus pollution condition of the region and the environment, and has important significance for timely monitoring the clade2.1.3 branch H5N1 avian influenza virus entry transmission.

Drawings

FIG. 1 shows the results of rg325GPCR product sequencing, rg325IPCR product sequencing, rg325RPCR product sequencing, and rg325 PCR product sequencing, where A-E are pyrosequencing peak diagrams, and F is pyrosequencing sequence results.

Figure 2 is a translation of rg325GPCR product sequencing results, rg325IPCR product sequencing results, rg325RPCR product sequencing results, rg325 PCR product sequencing results, and rg325SPCR product sequencing results into amino acid alignment using MAGA6.0, wherein 1 represents H5N1 standard sequence a/Anhui/1/2005 (sequence 4), 2 is rg325, 3 is rg325I, 4 is rg325R, 5 is rg 325S; rg325G for 6; it can be seen that, in column 8 from the C-terminal (i.e. right), positions P6 of HA protein cleavage sites in the test sample contain corresponding G, I, R,. star and S, respectively, indicating that the method of the present invention is correct.

FIG. 3 shows the results of sensitive sequencing, in which A, B, C, D represents the virus content of 1X 10³、1×10²、10、1TCID₅₀Rg325S virus HA cleavage site pyrosequencing peak plot per mL; panel E shows the pyrosequencing sequence resulting from the sensitivity detection. WellA1, B1, C1, D1 represent rg325S virus detection sequences with virus contents of 1 × 103, 1 × 102, 10, 1TCID50/mL, respectively.

FIG. 4 shows the results of amino acid sequence alignment, in which 1 represents H5N1 standard sequence A/Anhui/1/2005, 2, 3, 4 represent virus content of 1X 10³、1×10²、10、1TCID₅₀Rg325S virus/mL; as can be seen, the HA protein cleavage site P6 in column 8 from the C-terminus (i.e., right) of the protein sequence is S at position 2, 3, 4, indicating 10TCID₅₀The samples with/mL and above concentrations can detect that the HA protein cleavage site P6 contains S.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The clade2.1.3 branch H5N1 avian influenza virus in the following examples is described in the following references: zhang, y., Sun, H., Pu, j., Bi, y., Shi, y., Lu, x., Li, j., Zhu, q., Gao, g.f.2012.a single amino acid to the pharmacological diagnosis and neuro diagnosis of H5N1 underfluor virus in micro.j.virol.86, 6924-31.

Example 1 design of primers for pyrosequencing and establishment of method

Design of pyrophosphate sequencing primer

The HA protein HAs an amino acid sequence of sequence 4 and a nucleotide sequence of sequence 5.

According to the sequence of H5N1 avian influenza virus uploaded in GenBank, a specific PCR primer (the target sequence is the sequence 5 at the 920 th position 1092) and a sequencing primer (the target sequence is the sequence 5 at the 997 th position 1044) are designed aiming at the nucleotide of the HA protein cleavage site region of the H5N1 subtype avian influenza virus as a target sequence, which are concretely as follows:

specific upstream primer H5-F: 5'-TCC ACAACATAC ACC CTC TCAC-3' (SEQ ID NO: 1) (5 ' Biotin Label)

Specific downstream primer H5-R: 5'-TAC CAT TCC CTg CCATCC TC-3' (sequence 2)

Pyrosequencing primer: 5'-CCT gCTATA gCT CCAAA-3' (sequence 3)

Second, method for detecting clade2.1.3 branch H5N1 avian influenza virus by pyrosequencing primer

1. Extraction and reverse transcription of sample RNA

Extracting RNA of a sample and carrying out reverse transcription to obtain cDNA, wherein the method comprises the following steps:

1) collection of samples

Tissue samples may be as follows: killing or extinguishing diseased fowl, and collecting liver, brain, lung, etc.; cotton swab: nasal swab, cloaca swab. Storing at 2-8 deg.C, and testing in laboratory. The tested disease material is required to be fresh.

2) And sample treatment: each sample was treated separately

(1) Tissue sample treatment: 100mg of a disease material to be detected is weighed and placed in a grinder, 0.8ml of lysis solution (guanidine thiocyanate, 0.8M, ammonium thiocyanate, 0.4M, sodium acetate buffer, 0.1M, 5% of glycerol and 38% of phenol are mixed uniformly) is added for grinding, the ground disease material is moved into a 1.5ml centrifuge tube, the centrifugation is carried out for 5min at 8000rpm of 4 ℃, 250 mul of supernatant is taken and placed in a 1.5ml sterilized centrifuge tube, 750 mul of lysis solution is added, the mixture is mixed uniformly by violent oscillation, and the mixture is placed for 5min at room temperature.

(2) Cotton swab treatment: twisting cotton swab in the extractive solution thoroughly, wringing, discarding swab, centrifuging at 4 deg.C and 8000rpm for 5min, collecting supernatant 250 μ l, placing in 1.5ml sterilized centrifuge tube, adding 750 μ l lysate (guanidine thiocyanate, 0.8M, ammonium thiocyanate, 0.4M, sodium acetate buffer, 0.1M, 5% glycerol and 38% phenol), mixing, shaking vigorously, and standing at room temperature for 5 min.

(3) Negative control (RNase free water): placing 250 μ l of sterilized double distilled water in 1.5ml sterilized centrifuge tube, adding 750 μ l of lysis solution (guanidine thiocyanate, 0.8M, ammonium thiocyanate, 0.4M, sodium acetate buffer, 0.1M, 5% glycerol and 38% phenol), mixing under vigorous shaking, and standing at room temperature for 5 min.

3) Extraction and reverse transcription of viral RNA

(1) Take 300. mu.l of the treated sample solution, add 900. mu.l TRIZOL, mix by gently inverting several times, and ice-wash for 10 min.

(2) Add 200. mu.l chloroform, mix by inversion for 15s, ice-cool for 5min, centrifuge at 4 ℃, 13000r/m, 15 min.

(3) Transferring 700 μ l of the supernatant into a new centrifuge tube, adding equal amount of isopropanol, slightly inverting, mixing, centrifuging at 4 deg.C, 13000r/m, and 15 min.

(4) The supernatant was discarded, 1ml of 75% DEPC treated alcohol was slowly added to the adherent wall, and the vessel was gently turned for two cycles after the addition.

(5)4℃，13000r/m，5min。

(6) And pouring the supernatant, and placing on ice for air drying for 5-10 min.

(7) The precipitate was dissolved in 50. mu.l of RNase free water and 1. mu.1 RNase inhibitor (30U/. mu.l, manufactured by Genview Co.) and used immediately or stored at-80 ℃ until use.

The RT system is: the reaction system of each tube is 20 mul, and the reaction system is as follows:

the reaction procedure was as follows:

after the addition of RNase-free water and oligo, the mixture was instantaneously separated and frozen at 65 ℃ for 5 min. After the addition of the latter four samples, the samples were immediately separated and stored at 37 ℃ for 1h, then at 70 ℃ for 5min and 4 ℃.

cDNA was obtained for each sample.

2. Pyrosequencing assay identification

1) PCR amplification reaction

PCR amplification was carried out using the cDNA obtained in the above 1 as a template and the specific forward primer and the specific reverse primer synthesized in the above design.

25 mul of PCR amplification system is used in each tube, and the reaction system is as follows:

add double distilled water to a final volume of 25. mu.l, mix well and mark well.

The PCR amplification reaction procedure was as follows:

94℃5min；

94℃30s；

55℃30s；

72℃30s

30 cycles from the second step

72℃10min；

4℃10h。

The PCR amplification product with the molecular weight of 170 bpwas obtained.

2) Preparation of pyrophosphate Single chain template

(1) Add 3. mu.l of Sepharose probes (Streptavidin Sepharose high Performance, GE, 71500440AD) and 47. mu.l of binding buffer (Pyromark binding buffer, QIAGEN, 979006) to the PCR plate to make an average volume of 50. mu.l per sample, mix the mixture well and shake;

(2) adding 10. mu.l of the PCR amplification product obtained in the above 1) to the above mixture;

(3) horizontally shaking the mixture treated in the step (2) at normal temperature for 15 minutes to combine the beads and the biotin;

(4) in the Vacuum prep works, high-purity water, 70% ethanol, washing Buffer (PyroMark Wash Buffer, QIAGEN, 979008) and destination Buffer (PyroMark destination Sol, QIAGEN, 979007) are sequentially added into four sample plates, and each sample plate is at least 100 ml;

(5) add 45. mu.l of Annealing Buffer (Pyromark Annealing Buffer, QIAGEN, 979009) containing pyrophosphate sequencing primers (10. mu.M) to PSQ 96 plate in advance, with the final concentration of sequencing primers in the final well being 0.3. mu.M);

(6) opening a pump of the vacuum prep workstation, and cleaning the vacuum prep tool in high-purity water for 30 seconds;

(7) then moving the vacuum prep tool to the PCR plate processed in the step (3) and grabbing sepharose beads; picking up the PCR plate and checking whether most of the beads are adsorbed on the vacuum prep tool;

(8) putting vacuumprep tool into 70% ethanol for 5-10 seconds;

(9) moving to the concentration buffer for 5-10 seconds;

(10) moving to washing buffer for washing for 5-10 seconds;

(11) the pump is turned off;

(12) aligning the vacuum prep tool to a PSQ 96 plate, immediately putting the plate containing the sequencing primer, and slightly shaking the probe to release sepharose beads;

(13) washing the vacuum prep tool by using high-purity water; obtaining a pyrophosphate single-chain template;

3) pyrosequencing

(1) Placing the PSQ 96 Plate with the single-chain template of pyrophosphate on a Thermo Plate, heating to 80 ℃ for 2 minutes, and cooling to room temperature;

(2) the enzyme mixture, substrate mixture and four types of dNTPs (dATP. alpha.S, dTTP, dCTP, dGTP) were added separately to the reagent compartment (Pyromark Q96 Cartridge (3), QIAGEN, 979004) in fixed positions. The program was set up and the reagent compartments and 96-well sequencing plate were placed in the cabinet for sequencing reactions.

Obtaining the sequencing result of the PCR product of the sample to be tested (sequence 5, 997-th position 1044).

Performing Reverse compensation on the measured sequence by EditSeq software to obtain a Reverse complementary sequence, translating the Reverse complementary sequence into a PCR product coding amino acid sequence (333 rd to 348 th site of the sequence 4) of the sample to be detected by MAGE 6.0 software, wherein if the 8 th site of the PCR product coding amino acid sequence of the sample to be detected from the C end, the HA protein cleavage site P6 of the sample to be detected is S, the sample to be detected contains or is candidate to contain the clade2.1.3 branched H5N1 avian influenza virus, and if the 8 th site of the PCR product coding amino acid sequence of the sample to be detected is not S from the C end, the HA protein cleavage site P6 of the sample to be detected is not S, and the sample to be detected does not contain or is candidate to not contain the clade2.1.3 branched H5N1 avian influenza virus.

Example 2 specific detection of pyrophosphate sequencing primers

1. Extraction and reverse transcription of sample RNA

H5N1 avian influenza viruses rg325G (6), rg325I (3), rg325R (4), rg325 × 2, and rg325S (5) are described in the following documents: zhang, y, Sun, H, Pu, j, Bi, y, Shi, y, Lu, x, Li, j, Zhu, q, Gao, g.f.2012.a single amino acid at the magic viral infection at the clinical diagnosis of virus, 6924-31.rg325G, rg325I, rg325R, rg325, and rg325S represent different clade H5N1 avian influenza viruses, respectively, wherein rg325S cleavage site P6 (position 325 of amino acid residue) is serine, consistent with the parent H2.1 branch H1, as published in other publications.

The method of step 1 of the second step of example 1 is adopted to extract total RNA of H5N1 avian influenza virus rg325G, rg325I, rg325R, rg325 and rg325S virus allantoic fluid and negative control samples respectively, and the cDNA of each virus sample and the cDNA of the negative control sample are obtained by reverse transcription and synthesis of cDNA.

2. Identification of specificity of pyrosequencing primers by pyrosequencing

1) PCR amplification reaction

PCR was carried out using the cDNA obtained in the above 1 as a template and H5-F and H5-R synthesized in the above first design in the same manner as in the second embodiment of example 1 to obtain a PCR product.

2) Preparation of pyrophosphate Single chain template

The PCR product obtained in the above 1) was amplified with the pyrosequencing primer by the method of the second example 1 to obtain a pyrophosphate single-stranded template.

3) Pyrosequencing

Pyrosequencing was performed using the method of example 1 two, yielding rg325GPCR product sequencing results, rg325IPCR product sequencing results, rg325RPCR product sequencing results, rg325 PCR product sequencing results, and rg325SPCR product sequencing results.

The rg325GPCR product sequencing result, the rg325IPCR product sequencing result, the rg325RPCR product sequencing result, the rg325 PCR product sequencing result and the rg325SPCR product sequencing result are subjected to Reverse Complement by EditSeq software to obtain a Reverse complementary sequence, and the Reverse complementary sequence is aligned with a protein sequence 4 coded by a standard sequence A/Anhui/1/2005(H5N1) HA gene by using MAGA6.0 software.

Simultaneously using a control primer F': 5'-CAA ACA AAT TAG TCC TTG CGA CTG-3', R ': 5' -CGG CCT CAAACT GAG TAT TCA-3, the PCR amplification reaction of the cDNA template obtained in the step 1) is carried out by adopting the method of the step 1), no amplified band exists, and the effect of the specific primer of the invention is good.

Example 3 detection of sensitivity of pyrosequencing primer

1. Preparation of each dilution and Total RNA extraction and reverse transcription

The samples for sensitive detection are that the rg325S strain has the virus content of 1 multiplied by 10 respectively³、1×10²、10、1TCID₅₀Viral dilutions at/mL. Total RNA was extracted and cDNA was synthesized by reverse transcription (the total RNA extraction and reverse transcription were performed in the same manner as in step 1 of example 1).

2. Sensitivity of pyrosequencing method for identifying pyrosequencing primer

1) PCR amplification reaction

PCR was carried out using the cDNA obtained in the above 1 as a template and H5-F and H5-R synthesized in the above first design in the same manner as in the second embodiment of example 1 to obtain PCR products.

2) Preparation of pyrophosphate Single chain template

3) Pyrosequencing

Pyrosequencing was performed by the method of example 1 to obtain virus contents of 1X 10³、1×10²、10、1TCID₅₀Results of sequencing of rg325S viral PCR products per mL.

The virus content is 1 × 10 respectively³、1×10²、10、1TCID₅₀Rg325S Virus PCR product sequencing results in mL Reverse Complement obtained by Reverse completion of the determined sequence using EditSeq software, which was aligned with the HA-encoded protein (SEQ ID NO: 4) of the standard sequence A/Anhui/1/2005(H5N1) using MAGA6.0 software.

FIG. 3 shows the sequencing results, in which A, B, C, D represents the virus content of 1X 10³、1×10²、10、1TCID₅₀Rg325S virus HA cleavage site pyrosequencing peak plot per mL; panel E shows the pyrosequencing sequence resulting from the sensitivity detection. Well A1, B1, C1 and D1 represent virus contents of 1X 10³、1×10²10, 1TCID50/mL rg325S virus detection sequences.

FIG. 4 shows the results of amino acid sequence alignment, in which 1 represents H5N1 standard sequence A/Anhui/1/2005, 2, 3, 4 represent virus content of 1X 10³、1×10²、10、1TCID₅₀Rg325S virus/mL; as can be seen, the HA protein cleavage site P6 (HA protein 325) in the third column from the left of the protein sequence, 2, 3, 4 is S, indicating 10TCID₅₀The samples with the concentration of/mL and above can detect that the HA protein cleavage site P6 (HA protein 325) contains S. The pyrosequencing detection method is very goodHigh sensitivity.

Claims

1. A set of primers for detecting clade2.1.3 branch H5N1 avian influenza virus comprises the following primer pairs:

the primer pair consists of a single-stranded DNA molecule shown in a sequence 1in a sequence table and a single-stranded DNA molecule shown in a sequence 2 in the sequence table; the primer set also comprises a sequencing primer of which the nucleotide sequence is a sequence 3 in a sequence table.

2. The set of primers according to claim 1, wherein: one primer in the primer pair is labeled with biotin at the tail end.

3. A PCR kit comprising a primer set according to claim 1 or 2 or a kit comprising said primer set or said PCR kit.

4. The kit PCR reagents according to claim 3, wherein:

the PCR reagent set consists of a PCR reagent 1 and a PCR reagent 2;

the PCR reagent 1 is a reagent containing the primer set of any one of claims 1 to 3;

the concentration of each primer in the primer pair in the PCR reagent 1 is 20 mu M;

the PCR reagent 2 is a reagent containing the sequencing primer of the primer set of any one of claims 1 to 3;

the concentration of the sequencing primer in the PCR reagent 2 was 0.3. mu.M.