CN100533120C

CN100533120C - Multiple colour fluorescent composite amplification kit for 11 pulmonary cancers susceptibility related SNP sites

Info

Publication number: CN100533120C
Application number: CNB2007101127724A
Authority: CN
Inventors: 王瑞恒
Original assignee: Liaoning Normal University
Current assignee: Liaoning Normal University
Priority date: 2007-03-24
Filing date: 2007-06-17
Publication date: 2009-08-26
Anticipated expiration: 2027-06-17
Also published as: CN101158633A

Abstract

The invention relates to a disposable multicolor fluorescence combined augmentation reagent kit for 11 SNP gene loci which are related to lung cancer/munity for extracorporeal use, comprising separately packed primer mixture, DNA polymerase, buffer solution, quality control DNA specimen and allele parting normal mixture. Wherein, the primer mixture consists of 11 sharing primers marked by fluorescence and 22 length specificity primers specific to different alleles, the invention particularly consists of 2.0ml of primer mixture (therein gene locus has different primer consistency, 30nM-240nM), 1000u of DNA polymerase, 2.0ml of buffer solution, 1ml of the total amount of allele parting normal mixture, and 0.1ml (10ng/ul) of quality control DNA. Under the condition of best primer mixture ratio, the invention can rapidly and accurately obtain the genotypes of the 11 SNP points related to the lung cancer munity.

Description

A multiple colour fluorescent composite amplification 11 lung cancer susceptibility related SNP site kit

Technical field

The present invention relates to a kind of kit that is used for 11 SNP locus that lung cancer susceptibility is relevant on the external disposable composite amplification chromosome, belong to the invention of lung cancer susceptibility detection range.

Background technology

Along with finishing of the accurate sequence chart of the Human Genome Project (Human Genome Project), the clone of functional gene and evaluation, the multifarious research of human genome have become next scientific and technological commanding elevation.Restriction fragment length polymorphism (restriction fragment length polymorphism RFLP) and microsatellite polymorphism (microsatellite polymorphisms) be as the first generation and second generation genetic marker, takes at the splicing of the structure of physical map and genetic map, sequence base figure and dress and once played conclusive effect in the process.

The E.Lander of the U.S. in 1996 propose first to be referred to as " third generation genetic marker " " single nucleotide polymorphism " (single nucleotide polymorphism, SNP).SNP is meant the dna sequence polymorphism that the variation of single nucleotide on chromogene group level causes, and the wherein minimum frequency of a kind of allele in colony is not less than 1%.It comprises the forms such as conversion, transversion, insertion and disappearance of single base.SNP has following characteristics: at first, the SNP site is very abundant.Almost be distributed in whole genome, 1 SNP will appear in about average every 1000bp in the genome, and sum has 3,000,000 approximately.Secondly, the mutation rate of SNP is lower, and per generation, every base mutation rate was about 2 * 10 ^-8Especially being in (coding region) SNP (claiming cSNP again) of code area, is high stability, and the feasible genetic analysis to colony of the high mutation rate of the microsatellite locus that series connection repeats encounters difficulties.The 3rd, representative.CSNP often causes the polymorphic variation of expressing protein, and and then influences their function, characteristic.The 4th, the amplification segment is short, is easy to meet amplification, easily realizes the robotization of analyzing.

At present, the research work of human genome enters the genome times afterwards comprehensively (Post genome Era), researchist's emphasis is in the functional genomics field, comprising disease gene evaluation, gene expression adjusting, human diversity and every research work such as evolution, protein structure and function.In this case, as a genoid research tool, SNP is widely used in the research of aspects such as genetic disease, human evolution, biotic population diversity.

The research of SNP and various diseases relation more and more is subjected to researchist's attention.If draw particular combinations and specified disease, the particular locality morbidity crowd of some SNP or some SNP obvious correlativity is arranged, just can be with this SNP as molecular genetic marker, carry out the prevention of molecular marker assisted selection and disease forecasting, in epidemiology survey, determine disease patient's people at highest risk, and the evaluation of carrying out ill hazard level, set up effective early warning system, reach effective primary prevention purpose.

Studies show that in recent years, have strong correlation between many tumor susceptibilities and the SNP, such as: the Lys allele of the T allele of the Met/Met genotype of XRCC3 (Thr241Met), LIG4 (T1977C), the C allele of RAD51 (G135C), XPD (Lys751G1n) increases individual prostate cancer, breast cancer, SCCHN, the risk for lung cancer suffered from respectively.Carry the easier trouble tumour of individuality of this kind genotype or haplotype.The T allele of GSTM4 (T2517C) increases the individual possibility of suffering from lung cancer, and the CC genotype of NQO1 (C609T ') increases the individual risk for lung cancer of suffering from, and the GG genotype of hOGG1 (A11656G) increases the neurological susceptibility of lung cancer; LIG4 Ile658Val, Tp53 Arg72Pro, PLOI (RAD3OB) Thr706Ala, REV1 Phe257Ser SNP suddenly change all relevant with lung cancer susceptibility; AXIN2Pro50Ser, XPD Lys751Gln, XPC Lys939Gln, MPO-463G-〉SNP sudden changes such as A, XRCC1 194Trp/Arg, MBD4Glu346Lys, LRMPV141L, RASSF1 Alal33Ser, CYP1B1 Val432Leu, ADPRTVal762Ala all increase the neurological susceptibility of lung cancer.These specific markers can be used as the molecular labeling of judging individual certain tumor susceptibility, carry out disease forecasting, thereby can determine to suffer from the people at highest risk of certain tumour.

The research of SNP locus mostly one by one the site carry out, detection means or just use sequencing technologies perhaps adopts order-checking or adopts the method for digestion with restriction enzyme, the former technical sophistication, expense costliness; Latter's complex operation, poor accuracy.Composite amplification (Multiplex PCR) is called multiplex PCR again, promptly uses many cover primers in a reaction system, the process that increases at the zones of different of a plurality of dna profilings or same template.It can simplify procedures significantly, save time and reagent, can satisfy the needs of analyzing the different genes site simultaneously simultaneously.

Based on the difference of detection architecture, in the world composite amplification mainly can be divided into two big classes at present: cma staining composite amplification and fluorescent composite amplification.The former is that the common electrophoresis tank of the product utilization behind the composite amplification separates, and carries out the method that cma staining detects, and method is simple, and cost is lower, can carry out this project in common lab.But because the just single color of planting of gained result, so the number of sites of its composite amplification reagent kit can not be too many, the site of general 1 composite amplification can not surpass 4, otherwise can be difficult to distinguish the genotype of sample because the amplified production of different loci overlaps each other.Add the gained result and detect by an unaided eye, so its sensitivity is subjected to certain restriction.

Fluorescent composite amplification is that wherein primer one end at common composite amplification PCR adds fluorescence labeling, utilizes automatic laser fluorescence Genetic Detection instrument to detect pcr amplification product, and the result is accurate, highly sensitive, automaticity is high, repeatability is strong etc.With fluorescein-labelled one group of 3 or 4 gene loci of a kind of color (its amplified fragments can not overlaid), 3 or 4 gene locis of fluorescein-labelled another group of another kind of color, multiple fluorescein then can the different gene locis of the many groups of mark.Automatic laser fluorescence Genetic Detection instrument can detect tens kinds of fluoresceins at present, but in a reaction system, can detect 5 kinds of fluoresceins simultaneously at most, except that wherein a kind of as the interior mark, all the other four kinds of fluoresceins can be used for marked product, add together, just can reach the purpose of the gene loci of 1 detection more than 10.

Fluorescent composite amplification has become present advanced person's composite amplification method.

Summary of the invention

The present invention has screened 11 through studies have shown that the SNP locus relevant with lung cancer susceptibility, and according to the principle of primer specificity PCR, each SNP locus has designed 3 primers.In each SNP locus side 100-280bp scope, designed a primer,, and selected a kind of fluorescent material mark for use at primer 5 ' end as the shared primer of this locus; Not iso-allele at the SNP locus, designed again respectively and SNP locus two primers being complementary of iso-allele not, as Auele Specific Primer, primer 3 ' last terminal base is mated with the not homoallelic base of SNP locus respectively, but can not suppression of amplification owing to have only 3 ' last terminal base often not match, so in order to prevent the mistake amplification, base mismatch of the artificial introducing of 3 ' terminal the 3rd or 4 bit bases, this is the most key part of design of primers; At last for the different allele of automatic distinguishing when detecting, two primer lengths differ 4bp (if two primer Tm values have big difference during the design primer, then artificially introduce four bases of TATA, this is another key of primer difference design, because TATA is minimum to the annealing temperature influence of primer, satisfied simultaneously the different requirement of amplification fragment length again), all primer length 18-28bp, for the ease of composite amplification, the Tm value of all primers remains on 60 ± 0.5 ℃, for the competition that reduces between the primer suppresses, primer concentration to different SNP site in the reaction system is adjusted, obtain best primer concentration proportioning, prevented the generation of non-specific amplification segment.Utilize this kit, just can carry out composite amplification to 11 relevant SNP locus of lung cancer susceptibility simultaneously easily, obtain the genotype result of each locus; Simultaneously, the allelic ladder that utilizes this kit to provide utilizes genetic analysis software (Genetyper2.5.2), can also detect automatically easily, analysing amplified result.

Description of drawings

Fig. 1 is the structural formula of blue-fluorescence label 6-FAM.

Fig. 2 is the structural formula of yellow fluorescence label 6-TAMRA (Tetramethyl-rhodamine).

Fig. 3 be green fluorescence label JOE (6-carboxy-4 ', 5 '-dichloro-2 ', 7 '-dimethoxy-fluorescein) structural formula.

Fig. 4 is the structural formula of red fluorescence label 6-ROX (X-RHODAMINE).

Embodiment

This kit is to be made of 11 SNP site primer mixtures that separate packing, archaeal dna polymerase, reaction buffer (containing MgCl2) and 11 SNP site allelic ladders and Quality Control DNA.The primer mixture 2.0ml of 11 SNP locus (different genes seat primer concentration is inequality, and from 30nM-240nM, consumption is 2ul in the composite PCR reaction system of each 20ul); Archaeal dna polymerase 1000u (consumption is 1u in the composite PCR reaction system of each 20ul of 5u/p1); Damping fluid 2.0ml (10 times of concentrated damping fluids, consumption is 2ul in the composite PCR reaction system of each 20ul); Allelic ladder amount of the mixture 1ml (40nM detects use for 1000 times), the allelic ladder equivalent of contained each locus in the allelic ladder potpourri; Quality Control DNA0.1ml (10ng/ul).

The technological core of fluorescent composite amplification mainly is the concentration proportioning of primer sequence and fluorescently-labeled position and various primers.In kit of the present invention, 11 SNP sites are there are some researches show and the closely-related locus of lung cancer susceptibility that its reference sequences all can be consulted by NCBI, and the register in 11 SNP sites is:

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11 lung cancer susceptibility related SNP site primer sequences are:

RASSF1-pub-5 '-F1-GAAGCCCTGGGTTCCTCAA-3 ' (primer is shared in the RASSF1 site, and F1 is fluorescein-labelled)

RASSF1-spel-5 '-GATCTTCTGCTCAATCTCGGC-3 ' (RASSF1 locus specificity primer 1 is at C allele)

RASSF1-spe2-5 '-TATAGATCTTCTGCTCAATCTCGGA-3 ' (RASSF1 locus specificity primer 2 is at A allele)

MBD4-pub-5 '-F1-TTTCCTGGTTGGTGAGCAGTT-3 ' (primer is shared in the MBD4 site, and F1 is fluorescein-labelled)

MBD4-spe1-5 '-GCCAAAGACTCAGAACACACCA-3 ' (MBD4 locus specificity primer 1 is at A allele)

MBD4-spe2-5 '-TATATCCAAAGACTCAGAACACATCG-3 ' (MBD4 locus specificity primer 2 is at G allele)

XPD-pub-5 '-F1-CGGACATCTCCAAATTCATTCA-3 ' (primer is shared in the XPD site, and F1 is fluorescein-labelled)

XPD-spe1-5 '-CTGAGCAATCTGCTCTATCCTGTT-3 ' (XPD locus specificity primer 1 is at T allele)

XPD-spe2-5 '-TATACTGAGCAATCTGCTCTATCCTATG-3 ' (XPD locus specificity primer 2 is at G allele)

XRCC1-pub-5 '-F1-TCCAGACAAAGATGAGGCAGAG-3 ' (primer is shared in the XRCC1 site, and F1 is fluorescein-labelled)

XRCC1-spel-5 '-CTGGGGATGTCTTGTTGAACC-3 ' (XRCC1 locus specificity primer 1 is at G allele)

XRCC1-spe2-5 '-TATATTGGGGATGTCTTGTTGAGCT-3 ' (XRCC1 locus specificity primer 2 is at A allele)

ADPRT-pub-5 '-F2-CAGCAGGAGGGTTTGCCA-3 ' (primer is shared in the ADPRT site, and F2 is fluorescein-labelled)

ADPRT-spel-5 '-AGCAGGTTGTCAAGCATTTACG-3 ' (ADPRT locus specificity primer 1 is at C allele)

ADPRT-spe2-5 '-TATAAGCAGGTTGTCAAGCATTTACA-3 ' (ADPRT locus specificity primer 2 is at T allele)

CYP1B1-pub-5 '-F2-GAGGGACCGTCTGCCTTGTA-3 ' (primer is shared in the CYP1B1 site, and F2 is fluorescein-labelled)

CYP1B1-sepl-5 '-CCGGGTTAGGCCACTTGAG-3 ' (CYP1B1 locus specificity primer 1 is at C allele)

CYP1B1-sep2-5 '-TATACCGGGTTAGGCCACTTTAC-3 ' (CYP1B1 locus specificity primer 2 is at G allele)

REV1-pub-5 '-F2-AGTCAATGGCATGAACAGTTGG-3 ' (primer is shared in the REV1 site, and F2 is fluorescein-labelled)

REV1-spel-5 '-CCTTATCCTCCTCCTGGGAAA-3 ' (REV1 locus specificity primer 1 is at T allele)

REV1-spe2-5 '-TATACCTTATCCTCCTCCTGGGTAG-3 ' (REV1 locus specificity primer 2 is at C allele)

XPC-pub-5 '-F2-TGATTACTAACCCTCGCCTGTG-3 ' (primer is shared in the XPC site, and F2 is fluorescein-labelled)

XPC-spel-5 '-GGCGCTCAGCTCACAGGTT-3 ' (XPC locus specificity primer 1 is at A allele)

XPC-spe2-5 '-TATAAGCGCTCAGCTCACAGTTG-3 ' (XPC locus specificity primer 2 is at C allele)

GSTM4-pub-5 '-F3-GGAAAGGCGTCCAAGCAGT-3 ' (primer is shared in the GSTM4 site, and F3 is fluorescein-labelled)

GSTM4-sepl-5 '-GGCCATGGTTTGTTGGACAC-3 ' (GSTM4 locus specificity primer 1 is at C allele)

GSTM4-sep2-5 '-TATAGGCCATGGTTTGTTGGATAT-3 ' (GSTM4 locus specificity primer 2 is at T allele)

AXIN2-pub-5 '-F4-CAGCAGCAGCTTCCGTGAG-3 ' (primer is shared in the AXIN2 site, and F4 is fluorescein-labelled)

AXIN2-spel-5 '-CCTGGTGTTGGAAGAGACTGG-3 ' (AXIN2 locus specificity primer 1 is at C allele)

AXIN2-spe2-5 ' TATACCTGGTGTTGGAAGAGACCGA-3 ' (AXIN2 locus specificity primer 2 is at T allele)

PLOI-pub-5 '-F4-CAGATAGCCATAAGCAAACAGTAGC-3 ' (primer is shared in the PLOI site, and F4 is fluorescein-labelled)

PLOI-spel-5 '-ATGTGGAAATCTGATCCGGC-3 ' (PLOI locus specificity primer 1 is at G allele)

PLOI-spe2-5 '-TATAATGTGGAAATCTGATCCGGT-3 ' (PLOI locus specificity primer 2 is at A allele)

The title and the structure of four kinds of fluoresceins are respectively:

F1: blue-fluorescence label 6-FAM, structural formula are Fig. 1.

F2: yellow fluorescence label 6-TAMRA (Tetramethyl-rhodamine), structural formula are Fig. 2.

F3: green fluorescence label JOE (6-carboxy-4 ', 5 '-dichloro-2 ', 7 '-dimethoxy-fluorescein), structural formula is Fig. 3.

F4: red fluorescence label 6-ROX (X-RHODAMINE), structural formula are Fig. 4.

Need to prove that four kinds of fluorescent markers can exchange.

Above-mentioned primer design is very crucial, and its design needs to consider the key element of the following aspects: (1), all sites obtain amplification simultaneously, and promptly the right annealing temperature of all primers must be close; (2) do not form primer dimer between the primer; (3), do not form secondary structure.

All primers that comprised above-mentioned 11 SNP locus in the kit of the present invention, 3 primers in each site, wherein one is to share with primer, use different fluorescein-labelled respectively, the two other primer is at different SNP allele designs, match the different allele of amplification respectively with shared primer, because primer length difference, so different amplified allele product length difference, generally differ 4bp, detect, just can distinguish different allele according to the difference of product length by automatic laser fluorescence Genetic Detection instrument, if homozygote can only obtain 1 product peak; And if heterozygote then can obtain two product peaks.

For the allelic detection of SNP, the most direct method the most accurately is a gene sequencing, but this method is carried out in the site inefficiency, technical requirement height and expense costliness one by one; Secondly method commonly used is the allele according to the SNP site, seek the restriction enzyme that is fit to, difference according to digestion with restriction enzyme afterproduct length, distinguish different allele, but this method also one by one site SNP detect, and not exclusively derive a wrong conclusion owing to the digestion of enzyme sometimes, so have the shortcoming of inefficiency, poor accuracy.This method detects the amplification simultaneously in a reaction tube of 11 SNP sites simultaneously, and efficient improves greatly, and (GenoTyper2.5.2) can analyze the result automatically in conjunction with genetic analysis software, can finish the detection of routine samples up to a hundred in one day; Because fluoroscopic examination has high sensitivity,, once only need 5-10ng so detect extremely trace of needed sample size; Not homoallelic amplified production length differs 4bp, can satisfy the requirement that detects fully; By the debugging primer concentration, obtained best primer concentration proportioning, can effectively suppress the appearance of non-specific amplification product; The SNP site of selecting in this kit is position lung cancer susceptibility related gene site all, can play a significant role in the prediction of lung cancer susceptible risk, reaches the purpose of primary prevention.

When carrying out the composite amplification reaction, can in the PCR reaction system, add the four kinds of required deoxynucleoside triphosphates of primer mixture, archaeal dna polymerase, damping fluid and popular response that separate packing in this kit simultaneously, just can carry out pcr amplification effectively.Concrete reaction system is as follows:

Sample DNA (5-10ng) 1ul

Primer mixture 2ul

dNTPs 1.6ul

Damping fluid 2ul

Archaeal dna polymerase 0.2ul (1u)

Aseptic deionized water is supplemented to 20ul

The Quality Control DNA sample and 11 the SNP site allelic ladders (Ladder) that are used for standard different experiments chamber assay also are provided in the kit of the present invention, Quality Control DNA sample be through order-checking proved conclusively according to the accurate DNA sample of somatotype of the operation instructions of this kit, can be used as the use of positive control or standard DNA sample; Allelic ladder is that the inventor has collected all allelic amplified productions of 11 SNP sites, use as standard when being used for genetic analysis software automatic parting direction, adopt this allelic ladder, can be fast, accurately testing result is carried out somatotype; Simultaneously as long as with allelic ladder and the synchronous electrophoresis of sample pcr amplification product, different laboratories, different equipment, different electrophoresis methods, all can obtain consistent repeatable result, can be used for the testing result of standard different experiments chamber, avoid the error that causes because of electrophoresis.

Kit of the present invention can carry out composite amplification to the relevant SNP locus of 11 lung cancer susceptibilities expeditiously, detect somatotype by automatic fluorescence sequenator, reach the high automation level, the purpose that experimental result is accurate, reproducibility is good, kit of the present invention are used for the lung cancer prediction and the risk evaluation has very big advantage and potentiality.

Claims

1, a kind of 11 SNP sites multiple colour fluorescent composite amplification reagent kits, this kit comprises a) primer mixture, b) archaeal dna polymerase c) contains MgCl ₂Damping fluid, d) Quality Control DNA, e) allelic ladder potpourri, primer mixture is shared primer and 22 by 11 fluorescence labelings and is formed at not homoallelic length Auele Specific Primer, respectively at 11 lung cancer related SNP gene locis, be respectively RASSF1 (Ala133Ser), MBD4 (Glu346Lys), XPD (Lys751Gln), XRCC1 (194Trp/Arg), ADPRT (Val762Ala), CYP1B1 (Val432Leu), REV1 (Phe257Ser), XPC (Lys939Gln), GSTM4 (T2517C), AXIN2 (Pro50Ser), PLOI (Thr706Ala), 11 SNP gene loci primer sequences are: RASSF1-pub-5 '-F1-GAAGCCCTGGGTTCCTCAA-3 ', primer is shared in the RASSF1 site, RASSF1-spe1-5 '-GATCTTCTGCTCAATCTCGGC-3 ', the RASSF1 site is at the allelic Auele Specific Primer of C, RASSF1-spe2-5 '-TATAGATCTTCTGCTCAATCTCGGA-3 ', the RASSF1 site is at the allelic Auele Specific Primer of A, MBD4-pub-5 '-F1-TTTCCTGGTTGGTGAGCAGTT-3 ', primer, MBD4-spe1-5 '-GCCAAAGACTCAGAACACACCA-3 ' are shared in the MBD4 site, the MBD4 site is at the allelic Auele Specific Primer of A, MBD4-spe2-5 '-TATATCCAAAGACTCAGAACACATCG-3 ', the MBD4 site is at the allelic Auele Specific Primer of G, XPD-pub-5 '-F1-CGGACATCTCCAAATTCATTCA-3 ', primer is shared in the XPD site, XPD-spe1-5 '-CTGAGCAATCTGCTCTATCCTGTT-3 ', the XPD site is at the allelic Auele Specific Primer of T, XPD-spe2-5 '-TATACTGAGCAATCTGCTCTATCCTATG-3 ', the XPD site is at the allelic Auele Specific Primer of G, XRCC1-pub-5 '-F1-TCCAGACAAAGATGAGGCAGAG-3 ', primer, XRCC1-spe1-5 '-CTGGGGATGTCTTGTTGAACC-3 ' are shared in the XRCC1 site, the XRCC1 site is at the allelic Auele Specific Primer of G, XRCC1-spe2-5 '-TATATTGGGGATGTCTTGTTGAGCT-3 ', the XRCC1 site is at the allelic Auele Specific Primer of A, ADPRT-pub-5 '-F2-CAGCAGGAGGGTTTGCCA-3 ', primer is shared in the ADPRT site, ADPRT-spe1-5 '-AGCAGGTTGTCAAGCATTTACG-3 ', the ADPRT site is at the allelic Auele Specific Primer of C, ADPRT-spe2-5 '-TATAAGCAGGTTGTCAAGCATTTACA-3 ', the ADPRT site is at the allelic Auele Specific Primer of T, CYP1B1-pub-5 '-F2-GAGGGACCGTCTGCCTTGTA-3 ', primer, CYP1B1-sep1-5 '-CCGGGTTAGGCCACTTGAG-3 ' are shared in the CYP1B1 site, the CYP1B1 site is at the allelic Auele Specific Primer of C, CYP1B1-sep2-5 '-TATACCGGGTTAGGCCACTTTAC-3 ', the CYP1B1 site is at the allelic Auele Specific Primer of G, REV1-pub-5 '-F2-AGTCAATGGCATGAACAGTTGG-3 ', primer is shared in the REV1 site, REV1-spe1-5 '-CCTTATCCTCCTCCTGGGAAA-3 ', the REV1 site is at the allelic Auele Specific Primer of T, REV1-spe2-5 '-TATACCTTATCCTCCTCCTGGGTAG-3 ', the REV1 site is at the allelic Auele Specific Primer of C, XPC-pub-5 '-F2-TGATTACTAACCCTCGCCTGTG-3 ', primer, XPC-spe1-5 '-GGCGCTCAGCTCACAGGTT-3 ' are shared in the XPC site, the XPC site is at the allelic Auele Specific Primer of A, XPC-spe2-5 '-TATAAGCGCTCAGCTCACAGTTG-3 ', the XPC site is at the allelic Auele Specific Primer of C, GSTM4-pub-5 '-F3-GGAAAGGCGTCCAAGCAGT-3 ', primer is shared in the GSTM4 site, GSTM4-sep1-5 '-GGCCATGGTTTGTTGGACAC-3 ', the GSTM4 site is at the allelic Auele Specific Primer of C, GSTM4-sep2-5 '-TATAGGCCATGGTTTGTTGGATAT-3 ', the GSTM4 site is at the allelic Auele Specific Primer of T, AXIN2-pub-5 '-F4-CAGCAGCAGCTTCCGTGAG-3 ', primer, AXIN2-spe1-5 '-CCTGGTGTTGGAAGAGACTGG-3 ' are shared in the AXIN2 site, the AXIN2 site is at the allelic Auele Specific Primer of C, AXIN2-spe2-5 ' TATACCTGGTGTTGGAAGAGACCGA-3 ', the AXIN2 site is at the allelic Auele Specific Primer of T, PLOI-pub-5 '-F4-CAGATAGCCATAAGCAAACAGTAGC-3 ', primer is shared in the PLOI site, PLOI-spe1-5 '-ATGTGGAAATCTGATCCGGC-3 ', the PLOI site is at the allelic Auele Specific Primer of G, PLOI-spe2-5 '-TATAATGTGGAAATCTGATCCGGT-3 ', the PLOI site is at the allelic Auele Specific Primer of A, and the title of four kinds of fluoresceins is respectively: F1: blue-fluorescence label FAM; F2: yellow fluorescence label TAMRA; F3: green fluorescence label JOE; F4: red fluorescence label ROX; Four kinds of fluorescent markers can exchange.