CN101153324A - Reagent kit for detecting susceptibility of cancer of the lungs with SNPs of gene XRCC1 etc. - Google Patents

Abstract

The invention discloses a regent box for detecting the susceptibility to lung cancer, comprising a specific primer and a specific fluorescent probe which are capable of synchronously detecting an rs1136410 SNP locus on an PARP1 gene, an rs25487 SNP locus on a XRCC1 gene and an rs13181 SNP locus and an rs1799793 SNP locus on an ERCC2 gene, and common components for fluorescent quantitative detection of PCR. The reagent box of invention predicts the susceptibility to lung cancer of an individual by detecting the genotype of genes on the SNP loci of the PARP1 gene, the XRCC1 gene and the ERCC2 gene of the individual.

Description

Test kit by the isogenic SNPs detection of lung cancer of XRCC1 susceptibility

Technical field

The present invention relates to the test kit that a kind of disease susceptibility detects usefulness, especially a kind of test kit of detection of lung cancer susceptibility, by detecting poly ADP ribose transferring enzyme family member 1 (poly (ADP-ribose) polymerase family simultaneously, member 1, PARP1, be abbreviated as ADPRT again), staggered complementary gene 1 (the X-ray repair cross--complementing gene 1 that repairs of X ray, XRCC1) and excision repairing composite 2 (Excision Repair Cross-complementing Rodent Repair Deficiency, Complementation Group 2, mononucleotide polymorphism site ERCC2) (SNP) predict individual susceptibility to lung cancer.

Background technology

Primary bronchogenic carcinoma of lung is called for short lung cancer, is one of modal malignant tumour in our times various places, is the disease of a kind of serious threat human health and life.Tumour cell comes from tunica mucosa bronchiorum or body of gland, and regional lymphoglandula and hematogenous metastasis are often arranged, and respiratory symptoms such as irritable cough, sputum mixed with blood are often arranged in early days, and disease progression speed is relevant with the biological nature of cell.

Since half a century, the sickness rate of countries in the world lung cancer and case fatality rate all have and obviously increase trend.The World Health Organization's year 2000 report: totally 706.5 ten thousand people of malignant tumour are died from the whole world in 1997, account for 12.6% of death toll, and wherein lung cancer accounts for 19% of malignant tumour death, occupies first of the malignant tumour cause of the death.China sample survey in 1990～1992 years shows, in the tumor mortality of urban population, lung cancer rises to the 1st by original the 4th, and rising the fastest on the rural area also is lung cancer.The lung cancer morbidity rate of Yunnan tin ore is to be to rise to 219.10/10 ten thousand in the period of 197.87/10 ten thousand, 1970～1979 in the period of 28.02/10 ten thousand, 1960～1969 between 1954～nineteen fifty-nine, and this also is rare in the whole world.The famous oncologist R.Peto prophesy of Britain: if untimely control smoking of China and atmospheric pollution will become the first in the world lung cancer big country above 1,000,000 to the annual lung cancer of China in 2025.

The cause of disease and pathogenesis are not clear and definite as yet so far.Consistently think that the morbidity of lung cancer is relevant with following factors: 1. smoking (comprising initiatively smoking and passive smoking); 2. professional carcinogen, the occupational factor that causes Human Lung Cancer that has been identified comprise the polycyclic aromatic hydrocarbons in asbestos, inorganic arsenic chemicals, dichlormethyl ether, chromium and compound thereof, nickel, radon, yperite, vinylchlorid, coal smoke, tar and the oil, the heating product of tobacco etc.; 3. atmospheric pollution (comprising the pollution of indoor subenvironment and outdoor overall situation); 4. ionizing rays; 5. the presentation of results that the perspective crowd that diet and nutrition, USA New York and Chicago are carried out observes, in the food intake of natural vitamin A class, beta carotene and after the more than ten years cancer be negative correlation, wherein the most outstanding is lung cancer; 6. other, ACS classifies tuberculosis as one of pathogenic factors of lung cancer.Having the tuberculosis patient to suffer from risk of lung cancer is 10 times of normal population.Its main histological type is a gland cancer.In addition, factors such as virus infection, mycotoxins (flavus), body's immunity are low, endocrine disturbance and familial inheritance may also play certain comprehensive action to the generation of lung cancer.

In recent years studies show that the inactivation of the generation of lung cancer and activation of some oncogene and cancer suppressor gene is closely related.Verified several oncogene family comprises bcl-2, Kit that the ras family of mutagenesis, the myc family of amplifying gene, C-erB2, mechanism are not clear and by antioncogene p53, the p16 of wild-type variation and RB etc.Studies show that in a large number C-myc, L-myc, N-myc and RAF all have overexpression in small cell lung cancer.The then overexpression of Chang Kejian ras family gene in the nonsmall-cell lung cancer.These deep researchs will and be treated lung cancer and provide fundamental basis for the gene prevention.

SNP (single nucleotide polymorphism), promptly the single nucleotide polymorphism mark is the dna polymorphism that a class causes based on single nucleotide variation, is called third generation genetic marker by hereditary educational circles.Mainly be meant the dna sequence polymorphism that causes by the variation on the genome nucleotide level, comprise single base conversion, transversion, and the insertion/disappearance of single base etc.It is a class polymorphism mark that the most extensively exists in the genome, accounts for about 90%.The variation of these genome sequences can cause the difference of phenotype between individuality and Different Individual to disease, particularly the susceptibility of complex disease and to the difference of environmental factors, drug reaction.

The PARP1 gene is positioned at karyomit(e) 1q41-q42 position No. 1, total length 47.3kb, and mRNA total length 3861bp has 23 exons, the 1015 amino acid whose poly ADP ribose transferase proteins of encoding.This kind of enzyme is revised multiple nucleoprotein by poly-ADP ribosylation reaction.This correction participates in multiple important cellular activity based on DNA, for example: differentiation, propagation, metastases etc. also participate in the activity of molecular level simultaneously, for example DNA plerosis damaged cells or the like.The major function of PARP1 is described to 3 aspects usually: 1.DNA repairing effect (BER); 2. suppress transcribing of damaged dna; 3. exhaust the energy in the cell, cause apoptosis; 4. participate in the transcriptional control of portion gene.These functions can repair cell in impaired DNA, suppress transcribing of damaged DNA, even kill the cell that can't repair, reach the effect that cancer takes place that suppresses.Discover that the PARP1 afunction can improve mammary cancer, the sickness rate of cancers such as lung cancer.

Rs1136410 is that to be positioned on No. 1 karyomit(e) a T/C on the PARP1 gene polymorphic, causes that the 762nd amino acid of albumen of PARP1 genes encoding becomes Ala by Val.This polymorphic frequency distribution among the world crowd that NCBI announces, T accounts for 0.808, and C accounts for about 0.192, and heterozygosity is 0.311.Molecular biology research shows that last No. 762 amino acid of PARP1 are converted into Ala by Val, have reduced the activity of enzyme, causes the occurrence risk of multiple cancer to raise.The association analysis of the Chinese population of large sample shows that the polymorphism in this site is one of lung cancer influence factor important in the Chinese population.

XRCC1 is positioned at karyomit(e) 19q13.2-13.3 position No. 19, total length 32.3kb, and mRNA total length 2087bp has 17 exons, 634 the amino acid whose albumen of encoding.The XRCC1 encoded protein can play the effective for repairing effect to the dna single splitting of chain that causes because of ionizing rays and alkylide.XRCC1 encoded protein and dna ligase III, polymerase beta, poly ADP ribose transferring enzyme interaction, (Base Excision Repair, BER) path are repaired in the base excision that participates in DNA.Numerous studies show that, the polymorphism in site, XRCC1 gene top has confidential relation with cancer.The cancer relevant with XRCC1 mainly contains: mammary cancer, squamous cell carcinoma of the head and neck, the esophageal carcinoma, cancer of the stomach, colorectal carcinoma, carcinoma of the pancreas, lung cancer etc.

Rs25487 is that to be positioned at No. 19 G/A on the chromosome x RCC1 gene polymorphic, causes that the 399th amino acid of albumen of XRCC1 genes encoding becomes Gln by Arg.Arg399Gln is positioned at exon No. 10, XRCC1 protein B CRTI zone.This polymorphic frequency distribution in world crowd, G accounts for 0.72, and A accounts for about 0.28.Distribution G in the Chinese population is 0.68, and A is 0.32.Molecular biology research shows, the alternative reinforcement XRCC1 albumen of No. 399 amino acid Arg-Gln and the binding ability of DNA-carcinogens adducts on the XRCC1 gene, thereby the individual tumor susceptibility of influence.For the lung cancer case control study of large-scale crowd, the 399 amino acid mutation sites of XRCC1 take place relevant with lung cancer when doing single factor analysis by in a large number.

ERCC2 is positioned at karyomit(e) 19q13.3 position No. 19, has 23 exons, full length gene 19.0kb, and mRNA total length 2355bp, coding contains 761 amino acid whose albumen.Participate in transcribing the reparation of link coupled nucleotide excision by the ERCC2 encoded protein, it can be discerned and the irrelevant large-scale damage of repair structure, as big adducts and thymine dimer, and be the moiety of basic transcription factor mixture BTF2/TFIIH.This albumen has the dependent DNA of the ATP activity of untwisting, and belongs to RAD3/XPD helicase subfamily.The afunction of ERCC2 causes the basic transcription factor mixture can not correctly discern the dna damage site, thereby makes the base excision repair and can't correctly carry out, and causes the carcinogenic damage accumulation on the DNA.The disease that the sudden change of ERCC2 gene or inactivation may cause comprises: cancer, xeroderma pitmentosum, trichothiodystrophy, Ke Kaiyin syndrome.

Rs13181 is that to be positioned at the T/G of No. 23 exon section Lys751Gln of No. 19 karyomit(e) ERCC2 gene polymorphic.Frequency among the world crowd is about T: G=0.798: 0.202, and heterozygosity 0.322, the frequency in the Chinese population is about T: G=0.91: 0.09.Present multinomial studies show that, the amino acid replacement of Lys751Gln is positioned at ERCC2 PROTEIN C-terminal part, and the polymorphism in this site is relevant with diseases such as lung cancer, bladder cancer, mammary cancer, skin carcinomas.The polymorphism in ERCC2Lys751Gln site and the lung cancer particularly generation of lung squamous cancer exist significantly related.

Rs1799793 is that to be positioned at the G/A of No. 10 exon section Asp312Asn of No. 19 karyomit(e) ERCC2 gene polymorphic.Frequency among the world crowd is about G: A=0.778: 0.222, and the frequency in the Chinese population is about G: A=0.94: 0.06.Present multinomial studies show that, the performance that the polymorphic meeting in this site causes ERCC2 to participate in the transcription factor complex of formation weakens, thereby make the ability drop that dna damage is repaired, the ability of transcription factor also descends simultaneously, cause a series of such as lung cancer, the generation of diseases such as prostate cancer.ERCC2 Asp312Asn polymorphism and lung cancer particularly lung squamous cancer have remarkable relatedly, and the polymorphism in this site is considered to one of lung cancer inheritance susceptible factor in the Chinese population.

A large amount of association analysis both domestic and external shows that the occurrence probability of lung cancer increased when rs1136410 SNP loci gene type was C on the PARP1 gene; The occurrence probability of lung cancer increased when rs25487 SNP loci gene type was A on the XRCC1 gene; The occurrence probability of lung cancer increased when rs13181 SNP loci gene type was G on the ERCC2 gene; The occurrence probability of lung cancer increased when rs1799793 SNP loci gene type was A on the ERCC2 gene.The polymorphism in these four SNPs sites can be used for assessing individual susceptibility to lung cancer.

Summary of the invention

Can be used for assessing based on the polymorphism in rs13181 number on the rs1136410 SNP site on the PARP1 gene, rs25487 SNP site on the XRCC1 gene and the ERCC2 gene and rs1799793 SNP site on the basis of individual susceptibility to lung cancer, the invention provides a kind of test kit of detection of lung cancer susceptibility.

This test kit comprises: the Auele Specific Primer that detects the rs1136410 SNP site on the PARP1 gene is to reaching the specificity fluorescent probe, the Auele Specific Primer that detects the rs25487 SNP site on the XRCC1 gene is to reaching the specificity fluorescent probe, the Auele Specific Primer that detects the rs13181 SNP site on the ERCC2 gene is to reaching the specificity fluorescent probe, the Auele Specific Primer that detects the rs1799793 SNP site on the ERCC2 gene is to reaching the specificity fluorescent probe, the Taq enzyme, the dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

Auele Specific Primer described in this test kit to be meant at the rs1136410 SNP site on the PARP1 gene, on the XRCC1 gene rs25487 SNP site, rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene and design the dna fragmentation that can specific amplification goes out to harrow the site.Design this class primer to being that those skilled in the art can be unlabored.Preferably, comprise in the test kit primer with sequence shown in SEQ ID NO:1 and 2 to, primer with sequence shown in SEQ ID NO:3 and 4 to, primer with sequence shown in SEQ ID NO:5 and 6 to and to have a primer of sequence shown in SEQ ID NO:7 and 8 right.Auele Specific Primer synthesizes the synthetic technology of available routine.Those skilled in the art will appreciate that primer of the present invention is not limited to this four pairs of primers.

Specificity fluorescent probe described in this test kit be meant at the rs1136410 SNP site on the PARP1 gene, on the XRCC1 gene rs25487 SNP site, rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene and design, can go out the probe of these four SNPs site lung cancer tumor susceptibility gene types by the fluorescent quantitative PCR technique specific detection.Designing this class probe is that those skilled in the art can be unlabored.Preferably, comprise Taqman probe in the test kit with sequence shown in the SEQ ID NO:9,10,11 and 12.The specificity fluorescent probe can synthesize with the synthetic technology of routine.Those skilled in the art can understand, specificity fluorescent probe of the present invention is not limited to this four probes, and all probes that can be used for rs1136410 SNP site, the rs25487 SNP site on the XRCC1 gene, the rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene on the fluorescence quantitative PCR detection PARP1 gene all within the scope of the present invention.

The component and the content of test kit of the present invention comprise:

1 μ l 10X quantitative fluorescent PCR reaction buffer,

0.1 μ l 25mM dNTP mixed solution,

0.5 μ l 25mM MgCl ₂Solution,

0.02 μ l (5units/ μ l) Taq archaeal dna polymerase,

20 μ M Auele Specific Primers are to (eight) each 0.225 μ l,

Each 0.25 μ l of 10 μ M specificity fluorescent probes (four),

Deionized water 3.58 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment 1. detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: quantitative fluorescent PCR reaction

But use the fluorescent quantificationally PCR detecting kit of detection of lung cancer susceptibility, wherein, contain following primer to and fluorescent probe:

It is 58 ℃ that adopted primer 1:5 '-TTTGCCATTCACTGTGTTGG-3 ' (SEQ ID NO:1) Tm value is arranged

Antisense primer 1:5 '-ATCCTTGCTGCTATCATCA-3 ' (SEQ ID NO:2) Tm value is 54 ℃

It is 56 ℃ that adopted primer 2: 5 '-TAACTGGCATCTTCACTTCT-3 ' (SEQ ID NO:3) Tm value is arranged

Antisense primer 2:5 '-TCCAGATTCCTGGCATTGC-3 ' (SEQ ID NO:4) Tm value is 58 ℃

It is 58 ℃ that adopted primer 3:5 '-ACTCAGGAGTCACCAGGAA-3 ' (SEQ ID NO:5) Tm value is arranged

Antisense primer 3:5 '-TCTGTTCTCTGCAGGAGGA-3 ' (SEQ ID NO:6) Tm value is 58 ℃

It is 58 ℃ that adopted primer 4:5 '-ATCAAAGAGACAGACGAGCA-3 ' (SEQ ID NO:7) Tm value is arranged

Antisense primer 4:5 '-TCAGGAAGCCCAGGAAAT-3 ' (SEQ ID NO:8) Tm value is 54 ℃

Fluorescent probe 1:5 '-CAGGCCAAGGCGGAAATGCT-3 ' (SEQ ID NO:9) Tm value is 64 ℃

Fluorescent probe 2:5 '-CGGCTGCCCTCCCAGAGG-3 ' (SEQ ID NO:10) Tm value is 64 ℃

Fluorescent probe 3:5 '-AATCTGCTCTATCCTCTGCAGC-3 ' (SEQ ID NO:11) Tm value is 66 ℃

Fluorescent probe 4:5 '-TGCCCAACGAAGTGCTGCAG-3 ' (SEQ ID NO:12) Tm value is 64 ℃

Adopted primer 1 is arranged, and antisense primer 1 and fluorescent probe 1 are specifically at the rs1136410 SNP loci polymorphism that detects on the PARP1 gene;

Adopted primer 2 is arranged, and antisense primer 2 and fluorescent probe 2 are specifically at detecting rs25487 SNP loci polymorphism on the XRCC1 gene; Adopted primer 3 is arranged, and antisense primer 3 and fluorescent probe 3 are specifically at the rs13181 SNP loci polymorphism that detects on the ERCC2 gene; Adopted primer 4 is arranged, and antisense primer 4 and fluorescent probe 4 are specifically at the rs1799793 SNP loci polymorphism that detects on the ERCC2 gene.

The quantitative fluorescent PCR reaction system is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, the 0.5 μ l 25mM MgCl of 20ng/ μ l ₂The antisense primer 1,2,3 that adopted primer 1,2,3 and 4 each 0.225 μ l, 20 μ M are arranged of solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and the fluorescent probe 1,2,3 of 4 each 0.225 μ l, 10 μ M and 4 each 0.25 μ l, deionized water 3.58 μ l.

React on ABI9700 type pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on ABI7900 type quantitative real time PCR Instrument.

Step 3:SNP gene type assay

The final sample fluorescence volume and the normal control that show on the quantitative real time PCR Instrument are compared, the final fluorescent signal of fluorescent probe 1 is apparently higher than normal control, illustrate that rs1136410 SNP loci gene type carries the C type on the PARP1 gene of detected DNA, be lung cancer susceptible type; The final fluorescent signal of fluorescent probe 2 is apparently higher than normal control, illustrates that rs25487 SNP loci gene type carries the A type on the XRCC1 gene of detected DNA, is lung cancer susceptible type; The final fluorescent signal of fluorescent probe 3 is apparently higher than normal control, illustrates that the rs13181 SNP loci gene type on the ERCC2 gene of detected DNA carries the G type, is lung cancer susceptible type; The final fluorescent signal of fluorescent probe 4 is apparently higher than normal control, illustrates that the rs1799793 SNP loci gene type on the ERCC2 gene of detected DNA carries the A type, is lung cancer susceptible type.

Embodiment 2. instructs people initiatively to prevent the service of lung cancer

Carried out this service in Shanghai Zhujian Biological Engineering Co., Ltd at present.

Step 1:DNA extracts

The detected person is served preceding consulting, sign Informed Consent Form, fill in living and diet custom questionnaire by the detected person.Using the oral cavity sampling to wipe away according to the indication in the sampling box carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: genotype tests

Use test kit provided by the invention, fluorescence quantitative PCR detection is carried out in rs13181 SNP site on rs25487 SNP site, the ERCC2 gene on rs1136410 SNP site, the XRCC1 gene on the PARP1 gene of detected person DNA and the rs1799793 SNP site on the ERCC2 gene simultaneously, determine the genotype in these four SNPs sites.

Step 3: instruct people initiatively to prevent lung cancer

By to the genotypic analysis of detected person SNPs, provide genotype tests report and the report of detected person's individuation health guidance.The gene test report describes the height of detected person's lung cancer susceptible degree and ill probability size in detail.The report of individuation health guidance is based on detected person's genotype tests result, in conjunction with its living and diet custom survey result, the relative risk of assessment detected person lung cancer inheritance susceptible.Make the personalized healthy action scheme that is directed to the detected person simultaneously, scheme is included in the recommendation on improvement on food habits, the mode of life etc., and popularizes the health knowledge of prevention lung cancer for the detected person.

Sequence table

＜110〉Shanghai Zhujian Biological Engineering Co., Ltd

＜120〉test kit by the isogenic SNPs detection of lung cancer of XRCC1 susceptibility

<160>12

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>1

tttgccattc?actgtgttgg 20

<210>2

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>2

atccttgctg?ctatcatca 19

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3

taactggcat?cttcacttct 20

<210>4

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4

tccagattcc?tggcattgc 19

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

actcaggagt?caccaggaa 19

<210>6

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

tctgttctct?gcaggagga 19

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>7

atcaaagaga?cagacgagca 20

<210>8

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>8

tcaggaagcc?caggaaat 18

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>9

caggccaagg?cggaaatgct 20

<210>10

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>10

cggctgccct?cccagagg 18

<210>11

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>11

aatctgctct?atcctctgca?gc?22

<210>12

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉probe

<400>12

tgcccaacga?agtgctgcag 20

Claims (6)

1. the test kit of a detection of lung cancer susceptibility comprises: the Auele Specific Primer that detects the rs25487 SNP site on rs1136410 SNP site, the XRCC1 gene, the rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene on the PARP1 gene simultaneously to and specificity fluorescent probe, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, quantitative fluorescent PCR reaction buffer, deionized water.

2. test kit according to claim 1, it is characterized in that: described Auele Specific Primer is to being meant at rs1136410 SNP site on the PARP1 gene, rs25487 SNP site on the XRCC1 gene, rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene and design can go out to comprise rs1136410 SNP site on the PARP1 gene by specific amplification, rs25487 SNP site on the XRCC1 gene, the primer in rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene is right.

3. test kit according to claim 1, it is characterized in that: described specificity fluorescent probe is meant at the rs25487 SNP site on rs1136410 SNP site, the XRCC1 gene on the PARP1 gene, rs13181 SNP site on the ERCC2 gene and the rs1799793 SNP site on the ERCC2 gene and designs, can go out the probe of these four SNPs site lung cancer tumor susceptibility gene types by the fluorescent quantitative PCR technique specific detection.

4. test kit according to claim 1 is characterized in that: contained Auele Specific Primer to be selected from primer with sequence shown in SEQ ID NO:1 and 2 to, primer with sequence shown in SEQ ID NO:3 and 4 to, primer with sequence shown in SEQ ID NO:5 and 6 to and to have a primer of sequence shown in SEQ ID NO:7 and 8 right.

5. test kit according to claim 1 is characterized in that: contained specificity fluorescent probe is selected from the Taqman probe with sequence shown in the SEQ ID NO:9,10,11 and 12.

6. test kit according to claim 1 is characterized in that: the component of test kit and content comprise 1 μ l 10X quantitative fluorescent PCR reaction buffer, 0.1 μ l 25mM dNTP mixed solution, 0.5 μ l 25mM MgCl ₂Solution, 0.02 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M Auele Specific Primers are to (eight) each 0.225 μ l, and 10 μ M specificity fluorescent probes (four) are 0.25 μ l respectively, deionized water 3.58 μ l.This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.