CN103205488B - Method and reagent for prediction of ankylosing spondylitis susceptibility - Google Patents

Abstract

The invention discloses a method and a reagent for prediction of ankylosing spondylitis susceptibility. According to the invention, genomic DNA of a host cell is extracted and the genotype of site + 879 in a second intron zone of the AIF1 gene of a subject is determined so as to predicate susceptibility of the subject to ankylosing spondylitis; if the genotype of the single nucleotide polymorphism site + 879 is CC, lowest ankylosing spondylitis susceptibility is obtained, and if the site + 879 contains a T allele, i.e., the genotype is CT or TT, highest susceptibility is obtained. The invention has the following advantages: correlation between the AIF1 gene polymorphism site and the ankylosing spondylitis is elaborated for the first time; the method for predicting ankylosing spondylitis susceptibility is provided; and the method is applicable to prevention, auxiliary diagnosis and treatment of ankylosing spondylitis and can also be used for research and development of a novel drug.

Description

A kind of method and reagent predicting susceptibility of ankylosing spondylitis

Technical field

The present invention relates to a kind of method and the reagent of predicting susceptibility of ankylosing spondylitis, predict the susceptibility of experimenter for ankylosing spondylitis by measuring with the polymorphism of ankylosing spondylitis genes involved AIF1 in particular, the method can be used for auxiliary diagnosis and the new drug development of disease, belongs to biological technical field.

Background technology

Ankylosing spondylitis (Ankylosing spondylitis, AS) is a kind of autoimmune disorder, and be mainly in the person between twenty and fifty in 16-40 year, the ill ratio of men and women is about 4 ~ 10: 1.Pathology Chang Shouxian betides articulatio sacroiliaca, and minority patient with severe symptoms shows as whole rigid spine.In addition, some patients is with the outer pathology of the backbones such as hip joint in various degree, eye, lung, cardiovascular, kidney.The sickness rate of AS in white race crowd is about 1% ~ 3%, China's AS morbidity is about 0.2-0.6%, wherein the patient of more than 60% gets involved with hip joint, cause more than 20% AS patient disabilities, inflammation mainly involves the bone attachment point of joint capsule, tendon and ligament, cause local joint accretion tetanic, limitation of activity.So far the medicine can obviously alleviating and control disease progression is still lacked clinically.AS belongs to multigenic disease, has obvious genetic predisposition, although it has been generally acknowledged that heredity and immune factor play a leading role in AS falls ill, definite cause and onset of disease mechanism is still unclear.

Carry out the research of AS inherited pathogenic factor at present, the SNP that adopts, as the association analysis method of genomic marker, is effective, is proven more.SNP refers to the DNA sequence polymorphism that in chromogene group level, single nucleotide diversity causes, frequency in crowd needs > 1%, SNPs is biallelic marker, 70.1% is had for the conversion between homotype base: as G/A or T/C, 29.1% for occurring in the transversion between purine and pyrimidine in this single base change.C (cytosine(Cyt)) is the most labile site in human genome, because great majority are methylated cytosines, can be converted to T (thymus pyrimidine) by spontaneous deaminizating, SNP contains the 80-90% of known polymorphism, is modal heritable variation.

Because the selective pressure of existence causes the distribution of SNP in term single gene and whole genome to be ununiformity.SNPs is 4 times of coding region in the quantity of gene non-coding region, and sum can reach 300 ten thousand.SNP with its density high (average every 1kb just have 1), representative strong (SNP being arranged in gene internal may directly affect protein structure or expression level), genetic stability good (with microsatellite polymorphism comparatively speaking), be easy to the features such as automated analysis (because SNP mostly is biallelic marker crowd, can simply with " +/-or 1/0 " direct somatotype) and become good genetic marker.

1973, first Brewerton etc. found and the Human leukocyte antigen-B27 of AS strong correlation (HLA-B27).Along with progress of research, other tumor susceptibility genes relevant to AS such as tumor necrosis factor-alpha (TNF-α), il-1 (IL-1) are identified successively.

AIF1 (allograft inflammatory factor-1; Allograft Inflammatory Factor 1) gene is positioned at 6q21.3; total length 1692bp; containing 4 exons; its coded product is a kind of hormonelike cytokine; molecular weight is 17kDa; containing 146 amino-acid residues; N end is acetylation; primarily of activate scavenger cell and T Expressions In Lymphocytes, secretion; the assignment of genes gene mapping is in human leucocyte antigen III region, closely related with generation development of organ transplant rejection, self property Immunological diseases and tumour etc.

There is no any about AIF1 gene and the report be associated between autoimmune disorder at present, also have no the result of study that AIF1 gene is associated with AS.

Summary of the invention

Main purpose of the present invention is to provide a kind of method detecting susceptibility of ankylosing spondylitis gene.

Second object of the present invention is to provide a kind of reagent detecting susceptibility of ankylosing spondylitis gene, comprises PCR primer and the test kit containing this primer.

For achieving the above object, the present invention is by the following technical solutions:

Detecting a nucleotide sequence for susceptibility of ankylosing spondylitis, is nucleotide sequence shown in sequence table SEQ ID No.1.

Described nucleotides sequence is classified as the nucleotide fragments that AIF1 gene intron 2 district comprises+879 mononucleotide polymorphism sites.+ 879 is variant sites, indicates with letter " Y ".This nucleotide sequence is AIF1 full length gene sequence.Fig. 1 is the schematic diagram in AIF1 gene structure and polymorphic variation site thereof, and include 4 exons in accompanying drawing ,+879 sites are marked on the corresponding position in intron 2 district in AIF1 gene map.

When the genotype of described+879 mononucleotide polymorphism sites is CC, susceptibility of ankylosing spondylitis is minimum; During containing T allelotrope, when namely genotype is CT or TT, susceptibility of ankylosing spondylitis is higher.

One group is detected the primer of susceptibility of ankylosing spondylitis, can increase and obtain the nucleotide fragments that AIF1 gene intron 2 district comprises+879 mononucleotide polymorphism sites.

The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

A detection method for susceptibility of ankylosing spondylitis gene, comprises the steps:

(1) genomic dna of extracting sample, amplification AIF1 gene intron 2 district comprises the nucleotide fragments of+879 mononucleotide polymorphism sites;

(2), when in detecting step (1) product, the genotype of+879 mononucleotide polymorphism sites is CC, susceptibility of ankylosing spondylitis is minimum; Containing T allelotrope, when namely genotype is CT or TT, susceptibility of ankylosing spondylitis is higher.

Described amplification AIF1 gene intron 2 district comprises the nucleotide fragments of+879 mononucleotide polymorphism sites, and the nucleotide sequence of one group of primer of use is respectively shown in sequence table SEQ ID No.2 and SEQ ID No.3.

The invention provides a kind of diagnostic kit detecting susceptibility of ankylosing spondylitis, the general components, reagent, damping fluid etc. of the primer pair wherein containing specific amplification AIF1 gene+879 site of the present invention and the test kit for pcr amplification detection, those skilled in the art know these general components and detection method.Whole components in test kit of the present invention, content, source and using method are as follows:

10 μ L 10 × PCR damping fluids; (Pharmacia)

2 μ L 10mM dNTP mixed solutions; (Pharmacia)

2 μ L Taq archaeal dna polymerases, 2unit/ μ L; (Takara)

1 μ L F1 primer, be the nucleotide sequence shown in SEQ ID No.2, concentration is 10pmol/ μ L;

1 μ L R1 primer, be the nucleotide sequence shown in SEQ ID No.3, concentration is 10pmol/ μ L;

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuffs; (American I daho company)

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.

Using method:

1) pcr amplification: by the intron 2 district Partial Fragment of pcr amplification AIF1 gene, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 1 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, oligonucleotide internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds pure water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ L is added, to prevent from evaporating before PCR.

2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light Scanner TMHR-I96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

Nucleotide sequence shown in sequence table SEQ ID No.1 and the nucleotide fragments comprising+879 mononucleotide polymorphism sites thereof are preparing the purposes in the reagent or medicine of diagnosing or treating ankylosing spondylitis.

The purposes of AIF1 gene intron 2+879, district mononucleotide polymorphism site in the reagent preparing diagnosis or treatment ankylosing spondylitis or medicine.

Measuring method of the present invention determines the genomic dna deriving from people, and sample source is unrestricted, as: body fluid (blood, ascites and urine etc.), histocyte (as hepatic tissue) etc.Genomic dna can be prepared by these samples of Isolation and purification.The concentration of adjustment genomic dna, makes it consistent as much as possible.Take genomic dna as template, the nucleic acid fragment containing AIF1 gene mutation site can be amplified, to obtain the great amount of samples of mensuration.This sample obtained containing the DNA fragmentation of AIF1 genovariation point by amplification, is particularly suitable for as mensuration material.

When carrying out gene auxiliary diagnosis, the present invention is preferably applied in the auxiliary diagnostic measuring and exist according to the mutation type of AIF1 gene, auxiliary diagnostic comprises the particular agent as neccessary composition, and it corresponds to the method for measuring AIF1 gene mutation type.Suitable particular agent is selected, as DNA fragmentation and/or the primer for pcr amplification step by the measuring method adopted.

Advantage of the present invention is: the present invention illustrates the dependency of AIF1 gene polymorphism sites and AS first, provides a kind of method predicting AS susceptibility, and the method can be used for the prevention of AS, auxiliary diagnosis and treatment, can also be used for new drug development.

Below in conjunction with the drawings and specific embodiments, the present invention is further described; so that the public has a better understanding summary of the invention; not limitation of the present invention, the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.

Accompanying drawing explanation

Fig. 1 is the schematic diagram in AIF1 gene structure and polymorphic variation site thereof.

Fig. 2 is the gene type figure of AIF1 genovariation site through HRM method, and transverse axis is temperature, and the longitudinal axis is the fluorescent value of normalization.Unimodal curve represents CC genotype individuals, and bimodal curve represents CT genotype individuals.

Fig. 3 is the backward sequencing figure of AIF1 gene+879, and left figure genotype is CC type, and right figure is CT genotype (adopting backward sequencing, so be shown as G and the A base of matching with C, T on peak figure).

Embodiment

As follows for representing the english abbreviation of reagent in the following example:

10 × PCR damping fluid: 10mM Tris-HCl (pH=8.3), 500mM Repone K (KCl), 10mM magnesium chloride (MgCl2), 0.01% (W/V) gelatinum

DNTP: deoxynucleoside triphosphate

EDTA: disodium ethylene diamine tetraacetate

TE：10mM?Tris-HCl(pH＝7.5)，1mM?EDTA(pH＝8.0)

Embodiment 1: the extraction of blood sample collection and genomic dna

By the New York Revised diagnostic criteria MethodsThe cases enrolled of 1984, choose the AS patient 118 example (age: 14-44 year from Jilin Area consanguinity-less relation altogether, average 24 years old), with area normal healthy controls volunteer 148 example (age: 39-72 year, average 46 years old).All persons under inspection are Han nationality and signature Written informed consent, this research obtains Beijing Hospital, the accreditation of ethics audit committee of Gerontological Research Center institute of the Ministry of Health, meets " World Medical Association's Declaration of Helsinki ": the ethic principle of human medical research.

According to following method, preparation human gene group DNA.1. first in the 1.5mLEP pipe of label, add 1000 μ L erythrocyte cracked liquids, after add 400 μ LEDTA anticoagulations (anticoagulation puts upside down mixing 3-5 time before adding), put upside down mixing, standing 10 minutes of room temperature; 2. 13000rpm is after centrifugal 30 seconds, removing supernatant liquor; 3. in gained precipitation, adding 480 μ L nucleic acid cleavage liquid, attack tube wall, fully adding 20 μ L Proteinase Ks (diluting 20 times of diluted protein enzyme K with splitting karyolymph) after mixing, put upside down mixing, 65 DEG C of incubators 10 minutes, (period mixes frequently up and down, guarantees without grumeleuse); 4. be down to room temperature after taking out, add 300 μ L albumen precipitation liquid, fully put upside down mixing, leave standstill 10 minutes, centrifugal 2 minutes of 13000rpm; 5. moved to by supernatant liquor in new EP pipe, add the Virahol of 670 μ L precoolings, fully put upside down mixing (more than 10 times), visible linear DNA forms little agglomerate gradually, centrifugal 2 minutes of 13000rpm; 6. abandon supernatant liquor and guarantee that precipitation is stayed in EP pipe, adding 670 μ L70% ethanol, mixing of turning upside down, centrifugal 2 minutes of 13000rpm; 7. abandon supernatant, make ethanol volatilization in pipe clean; 8. add TE solution (400 μ L), fully dissolve, the genomic dna extracted is carried out to the analysis of concentration and purity, draw part DNA solution as working fluid, concentration correction to 20ng/ μ L, be placed in 4 DEG C for subsequent use, residue genomic dna put-20 DEG C of Refrigerator stores.

The identification qualification of embodiment 2 SNP

The present invention adopts PCR-high resolving power melting curve (HRM) analytical method and PCR sequencing technologies to detect the genotype in+879 sites (its loci is C/T) in the intron 2 district of AIF1 gene simultaneously.Fig. 2 is AIF1 genovariation site is the sequencer map in AIF1 genovariation site through gene type figure, Fig. 3 of HRM method.

1) determination of PCR-HRM primer

From Genebank, look into the DNA base sequence (SEQ ID No.1) got near+879, design of primers completes under Oligo7.0 software.Object fragment is positioned at AIF1 gene intron 2 district, total length 58bp, and determine positive-sense strand F1 (+848bp-+867bp) and antisense strand R1 (+887bp-+905bp), specific primer sequence is as follows:

F1：5’-GGTGTGCAGGACTAAGAAGA-3’(SEQ?ID?No.2)

R1：5’-CAAATCGTGAGGAATGGAG-3’(SEQ?ID?No.3)

2) PCR-HRM reaction system and condition

By pcr amplification AIF1 gene intron 2 district Partial Fragment, PCR reaction system is: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 10 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, oligonucleotide internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds deionized water to 10 μ L.In each system, add 20 μ L paraffin oils during PCR, prevent from evaporating.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.

Table 1 high and low temperature oligonucleotide internal reference primer sequence, annealing temperature and product sheet segment length

3) HRM judges genotype

PCR primer is moved in special 96 orifice plates of HRM, Light Scanner TMHR-I 96 carries out HRM analysis: from 40 DEG C, melting curve is gathered with the slope of 0.3 DEG C/s, to 98 DEG C of end, with Light Scanner CallIT software, the curve (Fig. 2) after collection is analyzed, judge genotype.

4) sequence verification

From the individuality of the different genotype of gained, randomly draw 3 routine samples respectively carry out sequence verification.Order-checking sample re-starts pcr amplification, sequencing primer sequence is: F2:5 '-GGCAACCCCTTCCTCAGTC-3 ' (SEQID No.8), R2:5 '-GTTTCTCCAGCATTCGTTTC-3 ' (SEQ ID No.9), the long 349bp of amplified fragments.PCR reaction is totally 30 μ L, comprises: genomic dna 2 μ L, 10 × PCRBuffer3 μ L, 10mMdNTP0.5 μ L, Taq DNA polymerase (5U/ μ L) 0.5 μ L, upstream and downstream primer (10pM/ μ L) each 0.5 μ L, pure water is supplemented to cumulative volume 30 μ L.PCR reaction conditions is: 95 DEG C of denaturation 5min laggard enter major cycle, 95 DEG C of sex change 30s, 64 DEG C of annealing 30s, 72 DEG C extend 20s, 35 circulations, 72 DEG C of extension 2min.PCR primer detects through 8% polyacrylamide gel electrophoresis, send Hua Da gene sequencing portion to carry out sequence verification (Fig. 3) after gel imaging system observation is qualified.

The dependency of embodiment 3 embodiment 3 gene SNP and AS

Statistical method: the populational representation using Hardy-Weinberg balance check research sample.Pearson chi square test in SPSS11.0 software is utilized to calculate allelotrope, the distribution frequency of genotype between AS case group and Normal group of AIF1 gene+879 pleomorphism site, the risk OR value of AS and 95%CI credibility interval thereof are significance of difference standard with P < 0.05.

Result: on the AIF1 gene being positioned at 6p21.3 region, the genotype of SNP+879 polymorphic site and the distribution of gene frequency between case and control group refer to table 2.

The genotype of table 2AIF1 gene+879C/T polymorphic site and the distribution of gene frequency between case-control group

Note: OR: odds ratio; CI: credibility interval.* T allelotrope is the risk allelotrope of easily suffering from AS.Experimenter is divided into risk allelotrope (CT) carrier of AS, and non-risk allelotrope (CC) carrier.

From table 2, the T allelotrope of AIF1 gene+879, namely be A allelotrope on its DNA complementary strand, distribution frequency in PATIENT POPULATION is significantly higher than its genotypes distribution and allele frequencies (7.2%vs.2.0%) in healthy normal population, there is significant difference (P=0.004), and the OR value in T site is 3.752,95%CI:1.455-9.673; In risk allelotrope (CT) carrier and non-risk allelotrope (CC) carrier of AS, the distribution frequency of risk genotype in case group is significantly higher than (P < 0.05) in control group, all show AIF1 gene+879 site and AS is ill is proportionate, the risk increasing AS morbidity may be had.

Embodiment 4 detection kit

Preparation detects the test kit of AS relevant risk, includes the primer pair that can amplify AIF1 gene SNP+879 site, and other PCR-HRM corresponding reagent.Test kit of the present invention detects application for 10 person-portions, and keep in Dark Place in-20 DEG C, its component, content and source comprise:

10 μ L 10 × PCR damping fluid (Pharmacia),

2 μ L 10mM dNTP mixed solution (Pharmacia),

2 μ L Taq archaeal dna polymerases (2unit/ μ L) (Takara),

1μL?F1(SEQ?ID?NO.2)(10pmol/μL)，

1 μ L R1 (SEQ ID NO.3) (10pmol/ μ L) primer, (the raw work synthesis in Shanghai)

8 μ L 10 × LC-green PLUS saturated fluorescence dyestuff (American I daho company),

2 μ L oligonucleotide internal references (10pmol/ μ L) (each 0.5 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer), (sequence is in table 1),

64 μ L pure water.

After PCR-HRM detects, the polymorphism of AIF1 gene intron 2 district SNP+879 can be detected easily.

Proof test: adopt this test kit, random selecting AS clinical samples 10 example, control group sample 10 example, detects the single nucleotide polymorphism in site, AIF1 gene intron 2 district+879 through PCR-HRM.

One. method

1) pcr amplification: by the intron 2 district Partial Fragment of pcr amplification AIF1 gene, prepare mixed solution: 10 × PCR reaction buffer 1 μ L, 10mM/LdNTP0.2 μ L, Taq DNA polymerase 0.2 μ L, 10pM/L upstream primer 0.1 μ L, 10pM/L downstream primer 0.1 μ L, 1 × LC-Green Plus saturated fluorescence dyestuff 0.8 μ L, oligonucleotide internal reference 0.2 μ L (each 0.05 μ L of high and low temperature oligonucleotide internal reference upstream and downstream primer) (sequence is in table 1), genomic dna 1 μ L, adds pure water to 10 μ L.PCR reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 5s, altogether 45 circulations, 72 DEG C of total elongation 2min.Before carrying out high-resolution fusion curve analysis, carry out denature and renature process: 95 DEG C of 30s, 25 DEG C of 2min, 94 DEG C of 30s, 24 DEG C of 4min.In each system, the paraffin oil of 20 μ L is added, to prevent from evaporating before PCR.

2) genotype judges: PCR primer moved in special 96 orifice plates of HRM, Light Scanner TMHR-I96 carries out HRM analysis, analyze the curve after collection with Light Scanner Call IT software, the difference according to melting curve judges genotype.

Two. result:

Result shows, and the CT genotype frequency in site, AS clinical samples AIF1 gene intron 2 district+879 is significantly higher than the CT genotype in control group sample+879 site.

The present invention has the illustration of practicality:

The detection method of AIF1 gene pleiomorphism of the present invention can be used for the T allelotrope of the rare variant sites on the AIF1 gene in analyst's euchromosome 6p21.3 district, namely be A allelotrope on its DNA complementary strand, be applied to the complementary diagnosis of AS and the individual AS risk of assessment how, be beneficial to early intervention and the treatment of carrying out AS.

Utilize the present invention to set forth the nucleotide variation in AIF1 gene+879 site, as one of biomarker, can be used as the screening of the molecular target of medicinal design, to help to find the bioactive molecule having and regulate AIF1 to express, promote AS new drug development.

The nucleotide sequence of detection AIF1 gene pleiomorphism that the present invention sets up and AS related locus, can highly sensitive, the specific test kit being applied to AS gene auxiliary diagnosis.

As above told, reached a conclusion, the polymorphism in AIF1 gene+879 site and AS tool significant correlation.Therefore, measure this polymorphism according to the present invention, can be used for the gene auxiliary diagnosis of AS.

Invention describes the new mutant site that AIF1 Gene A S-phase is closed, and provide a kind of method measuring AIF1 gene pleiomorphism, and, according to the present invention, only need a small amount of DNA sample to be just enough to measure AIF1 gene polynorphisms.

The invention provides a kind of gene aided diagnosis method measuring AS related gene polymorphism.

Claims (2)

1. one group is detected the primer of susceptibility of ankylosing spondylitis, it is characterized in that: can increase and obtain the nucleotide fragments that AIF1 gene intron 2 district comprises+879 mononucleotide polymorphism sites; The nucleotide sequence of described primer is respectively shown in sequence table SEQ ID No.2 and sequence table SEQ ID No.3.

2. detect a test kit for ankylosing spondylitis tumor susceptibility gene, it is characterized in that being made up of following reagent:

10 μ L10 × PCR damping fluids;

2 μ L10mM dNTP mixed solutions;

2 μ L Taq archaeal dna polymerases, 2unit/ μ L;

1 μ L F1 primer, be the nucleotide sequence shown in SEQ ID No.2, concentration is 10pmol/ μ L;

1 μ L R1 primer, be the nucleotide sequence shown in SEQ ID No.3, concentration is 10pmol/ μ L;

8 μ L10 × LC-green PLUS saturated fluorescence dyestuffs;

The each 0.5 μ L of 2 μ L oligonucleotide internal reference primer, concentration is 10pmol/ μ L, wherein low temperature internal reference primers F is the nucleotide sequence shown in SEQ ID NO.4, low temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.5, high temperature internal reference primers F is the sequence shown in SEQ ID NO.6, and high temperature internal reference primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μ L pure water.