CN110272988A

CN110272988A - Mankind's mthfr gene polymorphic detection kit

Info

Publication number: CN110272988A
Application number: CN201910530992.1A
Authority: CN
Inventors: 魏赵延; 李思慧; 徐倩; 曹丹枫
Original assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co Ltd
Current assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co Ltd
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2019-09-24

Abstract

The method of genetic polymorphism detection and its kit including specific probe and specific primer sequence are done with label probe and specific primer sequence the present invention relates to a kind of, is suitable for biotechnology and medical domain.Kit provides for while detecting the specific primer sequence and label probe of the mankind site mthfr gene C677T and A1298C loci polymorphism, wherein the kit contains Taq enzyme, specific primer, specific probe, interior mark system.Primer and kit provided by the present invention have the advantages such as high high specificity, susceptibility, quick, high throughput, safety easy to operate, result interpretation be objective for detecting the site mthfr gene C677T and A1298C loci polymorphism simultaneously.

Description

Mankind's mthfr gene polymorphic detection kit

Technical field

The present invention relates to field of biotechnology, and in particular to the mankind site mthfr gene C677T and the site A1298C are polymorphic Property detection kit and the preparation method and application thereof.

Background technique

Folic acid is a kind of water-soluble B family vitamin (Vitamin B9), it is not present in nature also inactive, It but is the precursor of biologically active folate (folate), active form in vivo is 5-methyltetrahydrofolate, energy A carbon-based group (methyl or formyl) is transmitted to deoxyuridylic acid, is allowed to become deoxythymidylic acid, and then synthetic DNA, is synthetic kernel Element necessary to acid is substance necessary to cell growth and tissue repair, even more indispensable in embryo development procedure Nutrient.Numerous studies have demonstrated that, folic acid is the indispensable nutrient of embryo growth and development in recent years, facilitates prevention mind Through defective tube, the generation including the birth defect very serious such as spina bifida and anencephalus.The clinical function of folic acid is in addition to prevention Outside Foetus neural tube defect, moreover it is possible to reduce pregnant woman's hypertension of pregnancy, spontaneous abortion and fetal intrauterine growth retardation, premature labor and The disease incidence such as newborn's under-weight, harelip, heart defect.

Methylenetetrahydrofolate reductase (methylenetetrahydrofolate red μ ctase, MTHFR) is cell The key enzyme of internal lobe acid balance and metabolism, irreversible catalysis 5- formyl tetrahydro leaf under the conditions of existing for the purine and thymidine Acid synthesis 5-methyltetrahydrofolate, the latter participate in the synthesis and methylation of DNA.There are two types of the common mutation of mthfr gene: 677 site C/T polymorphisms and 1298 site A/C polymorphisms.Wherein, the site C677T be the MTHFR that up to the present finds the most Common mutational site is 45.2% in the incidence of China.Studies have shown that after 677 sites sport T by C, the third of coding Propylhomoserin is substituted by valine, causes the thermal stability of MTHFR enzyme and enzymatic activity to reduce, and then can make homocysteine (Hcy) Metabolism is obstructed, and accumulation causes high homotype semicanal propylhomoserin mass formed by blood stasis in vivo.High homotype semicanal propylhomoserin mass formed by blood stasis and multiple miscarriage, eclampsia Early period, placental abruption, fetal growth restriction, fetal anomaly, stillborn foetus are related, and closely related with premature labor.In addition, 1298 site A After becoming C, glutamic acid is replaced by alanine, equally declines the enzymatic activity of MTHFR, causes Plasma Homocysteine It increases and the reduction of folate level, the site is up to 18.6% in the frequency of China's mutation.Inhibiting dihydro using methotrexate (MTX) When the activity of folic acid reductase, if there are the variations by patient, serious toxicity will be caused.Therefore mthfr gene is polymorphic Property detection for the individuation rational use of medicines have important directive significance.

At present to the detection of gene mutation and gene pleiomorphism, high-resolution fusion curve analytical technology (HRM common are Method), direct sequencing, DNA chip technology, PCR-RFLP method, Taqman sonde method etc..Due to high-resolution solubility curve method pair Equipment requirement is more special, and in clinical expansion, there are certain difficulties；PCR-PFLP technology relies on digestion with restriction enzyme, electricity , often there is result misjudgment phenomenon, influence its Detection accuracy in the reasons such as swimming analysis, and in addition its detection cycle is long, flux is lower, Also it is not suitable for the quick screening of a large amount of crowds.As the DNA direct sequencing of genetic test goldstandard, due to its expense compared with It is low, but time-consuming and sensitivity is low for operation, it is difficult to realize large-scale promotion.DNA chip technology is due to high-throughput, easy to operate fast The advantages that fast, is widely applied in SNP detection, but the technical costs is costly, complicated, poor repeatability, sensitivity are low, unfavorable In large-scale promotion.Quantitative fluorescent PCR-Taqman sonde method uses fluorescent quenching and double end-labellings, for SNP site Probe high sensitivity, the accuracy of variation design specificity are strong, and you can get it in 2-3 hour as a result, it is extensive therefore to obtain With and promote.

Summary of the invention

It is an object of the present invention to overcome the deficiencies of the prior art and provide single tube reaction while detecting mankind's mthfr gene The kit in the site C677T and A1298C loci polymorphism provides testing result to extracting for sample nucleic acid, it is only necessary to 2 ~3 hours, while also having result interpretation simple, detection flux is big, the low advantage of testing cost.It therefore can be in general molecule Laboratory and hospital laboratory complete corresponding detection；It carries out in clinical examination using the accurate of clinical diagnosis can be greatly increased Property, shorten the time that patient sees a doctor, it can also the reduction of medical health system cost.

A kind of method for detecting single nucleotide polymorphism based on Taqman sonde method of the invention, comprising:

(1) in same PCR amplification system, the enriching primer of two pairs of low temperature thermal oxidations is designed, it can be by PCR to wrapping respectively It is expanded containing 677 and 1,298 two site SNPs target templates, annealing temperature is 45~55 DEG C, the enriching primer Nucleotide sequence is as shown in SEQ ID No.14 and SEQ ID NO:15；

(2) in same PCR amplification system, 2 group-specific primers sequences are designed, using this two group-specific primers, into The AS-PCR that row carries out locus specificity differentiation to 677 and 1,298 two SNPs loci gene types is expanded；The core of specific primer Nucleotide sequence is as shown in SEQ ID No.1-6；

(3) in same PCR amplification system, 2 groups of SNP specific probes of design carry out specific detection purpose template, and described 2 The nucleotide sequence of group-specific probe is respectively SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and the specific probe 5 ' it is terminal modified be respectively FAM and HEX, Cy3 and Cy5,3 ' terminal modified have NFQ-MGB；

(4) in same PCR amplification system, while mark system and UNG enzyme decontamination system in introducing, can it is more accurate, Parting detection steadily is carried out to sample.

(5) preferably, present invention introduces interior mark system be conservative GAPDH gene, the end of internal standard probe 5 ' of design is repaired Decorations are ROX, and 3 ' terminal modified have NFQ-MGB.

The sequence table that mankind's mthfr gene polymorphic detection kit of the present invention is related to is as follows:

SEQ ID NO:1 5’-TCTGCGGGAGCCGATTTCA-3’

SEQ ID NO:2 5’-TCTGCGGGAGTCGATTTCA-3’

SEQ ID NO:3 5’-CAAAGCGGAAGAATGTGTCA-3’

SEQ ID NO:4 5’-AGCTGACCAGTGAAGAAAGTGT-3’

SEQ ID NO:5 5’-AGCTGACCAGTGAAGCAAGTGT-3’

SEQ ID NO:6 5’-GAACCAGGGTCCCCACTCCAG-3’

SEQ ID NO:7 5’-FAM-TGCGGGAGCCGATTTCA-NFQ-MGB-3’

SEQ ID NO:8 5’-HEX-TGCGGGAGTCGATTTCA-NFQ-MGB-3’

SEQ ID NO:9 5’-Cy3-CAGTGAAGAAAGTGTCTTTGA-NFQ-MGB-3’

SEQ ID NO:10 5’-Cy5-CAGTGAAGCAAGTGTCTTTGA-NFQ-MGB-3’

SEQ ID NO:11 5’-CATCGCTCAGACACCATGG-3’

SEQ ID NO:12 5’-GCAAGGCTCGTAGACGCGGTTC-3’

SEQ ID NO:13 5’-ROX-TCGGAGTCAACGGGTGAGTT-NFQ-MGB-3’

SEQ ID NO:14 5’-CAGCCTCTCCTGACTGTCATC-3’

SEQ ID NO:15 5’-GAGCAAGTCCCCCAAGGAG-3’

The present invention is based on Taqman sonde methods, using ARMS primer specificity amplification purpose template and SNP probe specificity Testing goal template method establishes the multiple fluorescence quantitative in two kinds of same gene different polymorphisms of same reaction tube detection PCR detection method, while mark system and UNG enzyme decontamination system in introducing, wherein UNG (μ racil-N-glycosylase) enzyme For uracil-N-glycosylase, its main feature is that optimum activity temperature is 50 DEG C, 95 DEG C of inactivations, action principle is optional water Uracil glycosidic bond in solution double-strand of the fracture containing dU or single stranded DNA, the DNA chain for having missing base of formation.It is reacted in PCR In, non-specific PCR amplification and pollution can be prevented using UNG enzyme, provided for two kinds of polymorphisms of specific detection same gene double Guarantee again.

Detailed description of the invention

Fig. 1 is the homozygous wild sample of 677C/C 1298A/A；

Fig. 2 is 677C/C 1298A/C heterozygous mutant sample；

Fig. 3 is 677C/T 1298A/C heterozygous mutant sample；

Fig. 4 is 677C/T 1298A/A heterozygous mutant sample；

Fig. 5 is 677T/T 1298A/A homozygous mutation sample；

Fig. 6 is 677T/T 1298C/C homozygous mutation sample.

Specific embodiment

In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below with reference to Specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention rather than limit this hair Bright range.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art can make the present invention Various changes or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

Fluorescence quantitative PCR instrument: LightCycler480 (Roche Holding Ag, Basel, Switzerland), ABI7500 (U.S.'s application life Object system house, New York, the U.S.), ABI7500FAST (Applied biosystems, New York, the U.S.), ABI7900HT (German Qiagen company, stops up by FAST (Applied biosystems, New York, the U.S.), QiagenRotor-Gene6000 Er Duofu, Germany), Idaho LightScanner (Idaho company, the U.S., the Idaho State, the U.S.).

Archaeal dna polymerase: ABI (U.S.), TaKaRa (Japan).

The present invention is to detect 2 groups of spies of Design for polymorphism in mankind's mthfr gene 677 and 1,298 two SNPS site simultaneously Specific primer, wherein the nucleotide sequence of the specific primer is as shown in SEQ ID No.1-6.2 groups of ARMS specific primers In the base pairing with type to be amplified respectively of 3 ' terminal bases, at the same in its 3 ' end 2-3 introducings reciprocal one or Two base mispairings, to enhance specificity when amplification.

Each specific primer sequence is listed below:

677C ARMS primer: 5 '-TCTGCGGGAGCCGATTTCA-3 '

677T ARMS primer: 5 '-TCTGCGGGAGTCGATTTCA-3 '

677 downstream primers: 5 '-CAAAGCGGAAGAATGTGTCA-3 '

1298A ARMS primer: 5 '-AGCTGACCAGTGAAGAAAGTGT-3 '

1298C ARMS primer: 5 '-AGCTGACCAGTGAAGCAAGTGT-3 '

1298 downstream primers: 5 '-GAACCAGGGTCCCCACTCCAG-3 '

The present invention is while detecting the polymorphism in the site mankind's mthfr gene 677 and 1,298 two SNPS and have also been devised 2 groups Specific probe, terminal modified the 5 ' of specific probe are respectively FAM, HEX, Cy3 and Cy5, and 3 ' terminal modified have NFQ-MGB.The base Group itself does not generate fluorescence, therefore can substantially reduce the intensity of background signal, while being also connected with MGB on the specific probe The Tm value of the probe can be improved 10 DEG C or so, therefore same Tm value by modification group, and MGB probe can be than common Taqman probe designs shorter, so that probe is when identification has Single nuclear polymorphism site, specificity is stronger.

Preferably, the nucleotide sequence of the 2 group-specific probe is respectively SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10

Each probe sequence is listed below:

5 '-FAM-TGCGGGAGCCGATTTCA-NFQ-MGB-3 ' of 677C detection probe

5 '-HEX-TGCGGGAGTCGATTTCA-NFQ-MGB-3 ' of 677T detection probe

5 '-Cy3-CAGTGAAGAAAGTGTCTTTGA-NFQ-MGB-3 ' of 1298A detection probe

5 '-Cy5-CAGTGAAGCAAGTGTCTTTGA-NFQ-MGB-3 ' of 1298C detection probe

In one embodiment of the invention, using the reaction of the internal standard system monitoring quantitative fluorescent PCR with the presence or absence of suppression False negative result caused by system, instrument error and human factor.Since certain ingredients in sample to be detected may contain PCR appearance is caused partially or completely to inhibit；Amplification instrument there may be higher than allowed band hole between it is poor, cause to expand between different pipes Efficiency variance；Artificial sample-adding mistake may also lead to the appearance of false negative result, therefore internal standard system is used in the embodiment of the present invention System ensure that the accuracy of testing result to eliminate above-mentioned hidden danger.The interior mark system preferably comprises interior label primer, internal standard probe.

Preferably, the interior mark system includes interior label primer and internal standard probe.The interior label primer is for human gene The conservative region design of gene GAPDH gene relatively conservative, nucleotide sequence such as SEQ ID NO:11 and SEQ in group Shown in ID NO:12, the internal standard probe sequence is SEQ ID NO:13；The internal standard probe 5 ' is terminal modified ROX, and 3 ' ends are repaired It is decorated with NFQ-MGB.

5 '-CATCGCTCAGACACCATGG-3 ' of internal standard forward primer F:5 '

Internal standard reverse primer R:5 '-GCAAGGCTCGTAGACGCGGTTC-3 '

Internal standard probe 5 '-ROX-TCGGAGTCAACGGGTGAGTT-NFQ-MGB-3 ' preferably, detection reagent of the invention Box further includes positive criteria reference substance and negative controls, and positive criteria reference substance and negative controls, which is arranged, can monitor and determine in real time Amount PCR reaction is normally carried out, and two kinds of polymorphisms of mthfr gene involved in the present invention are contained in the positive reference substance 5 kinds of plasmid mixed liquors of MTHFR 677, MTHFR 1298 and GAPDH gene；The negative controls are sterile water.

The another object of the embodiment of the present invention is to provide a kind of while detecting mankind's MTHFR and MTRR gene pleiomorphism Method, the method the following steps are included:

(1) sample to be tested genomic DNA is obtained；

(2) specific primer, specific probe, interior mark system, enriching primer, PCR reaction buffer, Taq are utilized Enzyme, UNG enzyme etc. carry out quantitative fluorescent PCR reaction, and single reaction pipe can detect the mthfr gene C677T of each sample simultaneously Site and A1298C polymorphism.

Preferably, it by taking 25 μ l reaction systems as an example, is added into reaction tube each in the mixture obtained after sample to be tested Component final concentration and content such as table 1:

Above-mentioned system is only illustrative, can proportionally expand or shrink in practical applications the volume of mixture and its Middle each component content.

Specifically, the program of the quantitative fluorescent PCR reaction are as follows:

Step 1: 50 DEG C of 2min, 95 DEG C of 10min；

Step 2: 95 DEG C of 15s, 45 DEG C~55 DEG C 1min, 5 circulations；

Step 3: 95 DEG C of 15s, 58 DEG C~62 DEG C 1min, 40 circulations；

And the fluorescence of channel FAM, HEX, Cy3, Cy5, ROX corresponding to acquisition probe are believed at 58 DEG C~62 DEG C of the phase III Number；

Program operation finishes, and PCR thin-walled reaction tube or the taking-up of eight unions are put into concave-convex bag, sealing is obturaged, by pollution Source processing.

Preferably, the program of the quantitative fluorescent PCR reaction are as follows:

Step 1: 50 DEG C of 2min, 95 DEG C of 10min；

Step 2: 95 DEG C of 15s, 48 DEG C~53 DEG C 1min, 5 circulations；

Step 3: 95 DEG C of 15s, 60 DEG C~62 DEG C 1min, 40 circulations；

And the fluorescence of channel FAM, HEX, Cy3, Cy5, ROX corresponding to acquisition probe are believed at 60 DEG C~62 DEG C of the phase III Number；

After above-mentioned PCR reaction, acquired results carry out result judgement by table 2:

The present invention is further described below by way of specific embodiment.

Embodiment 1

1, primer and probe synthesis:

Design and synthesize 2 group-specific primers SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3；SEQ ID NO: 4,SEQ ID NO:5；SEQ ID NO:6；2 group-specific probe SEQ ID NO:7, SEQ ID NO:8；SEQ ID NO:9, SEQ ID NO:10, and flag F AM, HEX, Cy3 and Cy5 fluorophor are held 5 ', 3 ' end label NFQ-MGB, which do not shine, is quenched base Group；2 enriching primer SEQ ID NO:14, SEQ ID NO:15.Primer, probe are prepared into 100 μM of mother liquor storage respectively.

1,1 pair of interior label primer for human genome design, the primer pair sequence mark system in preparing: are designed and synthesized For SEQ ID NO:11 and SEQ ID NO:12；Internal standard probe is designed and synthesized, which is SEQ ID NO:13.By primer, Probe is prepared into 100 μM of mother liquor storage respectively.

2, prepare other reagents: preparation PCR buffer, wherein the MgCl2 containing 1.0mM, dATP, dUTP, dGTP and Each 1.0mM of dCTP；Enzyme mixation is prepared, wherein containing 0.5 × 103 μ of Taq enzyme/ml, 0.1 × 103 μ of UNG enzyme/ml.

4, positive criteria reference substance and negative controls are prepared, are contained involved in the present invention in the positive reference substance 5 kinds of plasmid mixed liquors of two kinds of polymorphism MTHFR 677 of mthfr gene, MTHFR 1298 and GAPDH gene；The feminine gender is right It is sterile water according to product.

5, PCR reaction solution is prepared: the preparation of PCR reaction system is carried out according to the following table 3:

6, it assembles kit: including four components, PCR buffer, enzyme mixation, positive reference substance and feminine gender in kit Reference substance calculates 12 person-portions and each ingredient usage amount of 24 person-portions, two kinds of specifications, matches according to each ingredient usage amount of PCR reaction system It ingredient and is assembled in each pipe of kit processed.

Embodiment 2

Side sample is treated with mankind's mthfr gene polymorphic detection kit prepared by embodiment 1 to detect.

1, blood sample extracting genome DNA

With poba gene group pillar extracts kit, operating procedure, extracts human blood genome to specifications.With purple The concentration of outer spectrophotometer detection gained sample DNA solution, is then diluted to 10ng/ μ l for sample DNA, takes 2 μ l-5 μ respectively L is added into embodiment 1 in kit obtained and carries out the PCR reaction of next step.

2, the kit for the embodiment 1 for successively taking 2 μ l to be added separately to 23 μ l the DNA sample after diluting in step 1 In reaction system, and it is put into fluorescence quantitative PCR instrument, is carried out amplification reaction after PCR response procedures are set as described below:

50 DEG C of 2min, 95 DEG C of 10min；

95 DEG C of 15s, 48 DEG C~53 DEG C 1min, 5 circulations；

95 DEG C of 15s, 60 DEG C~62 DEG C 1min, 40 circulations；The glimmering of FAM, HEX, Cy3, Cy5, ROX is collected after each circulation Optical signal.

The Analysis of test results of sample: the testing result of 18 samples is as follows:

677C/C 1298A/A homozygous wild 3, sample, one of testing result is as shown in Figure 1；

677C/C 1298A/C heterozygous mutant 1, sample, one of testing result is as shown in Figure 2；

677C/T 1298A/C heterozygous mutant 5, sample, one of testing result is as shown in Figure 3；

677C/T 1298A/A heterozygous mutant 4, sample, one of testing result is as shown in Figure 4；

677T/T 1298A/A homozygous mutation 3, sample, one of testing result is as shown in Figure 5；

677T/T 1298C/C homozygous mutation 1, sample, one of testing result is as shown in Figure 6.

Above-mentioned 18 sample fluorescences quantitative PCR detection result is consistent with sequencing result.The above result shows that the present invention is implemented The detection kit that example provides is reliable for mankind's mthfr gene polymorphic detection result, reaches with direct Sequencing concordance rate 100%, and detection method high sensitivity is in traditional sequencing methods, it is easy to operate quickly, be conducive to large-scale promotion.

Sequence table

<110>create in the honest day in Jiangsu object Engineering Co., Ltd

<120>mankind's mthfr gene polymorphic detection kit

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<170> SIPOSequenceListing 1.0

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<213>artificial sequence (Artificial Sequence)

<400> 1

tctgcgggag ccgatttca 19

<210> 2

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

tctgcgggag tcgatttca 19

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

caaagcggaa gaatgtgtca 20

<210> 4

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

agctgaccag tgaagaaagt gt 22

<210> 5

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

agctgaccag tgaagcaagt gt 22

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<213>artificial sequence (Artificial Sequence)

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<400> 9

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<213>artificial sequence (Artificial Sequence)

<400> 10

cagtgaagca agtgtctttg a 21

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<213>artificial sequence (Artificial Sequence)

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catcgctcag acaccatgg 19

<210> 12

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

gcaaggctcg tagacgcggt tc 22

<210> 13

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

tcggagtcaa cgggtgagtt 20

<210> 14

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cagcctctcc tgactgtcat c 21

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gagcaagtcc cccaaggag 19

Claims

1. being used for while detecting the kit in the mankind site mthfr gene C677T and A1298C loci polymorphism, it is characterised in that It is located in box body equipped with box body, liner, PCR mixing liquid pipe, enzyme mixation pipe, positive control pipe and negative control pipe, liner, PCR mixing liquid pipe, enzyme mixation pipe, positive control pipe and negative control pipe are inserted into respectively in the hole of liner；PCR is mixed in liquid pipe Equipped with PCR mixed liquor, enzyme mixation pipe is provided with enzyme mixation, and positive control pipe is provided with positive criteria reference substance, negative right It looks after and is provided with negative standards' reference substance.

2. detection kit according to claim 1, which is characterized in that reacted in the detection kit comprising 1 × PCR Liquid, 3.0mM MgCl₂, dATP, dCTP, dGTP, dUTP, dTTP each 0.2mM, each 0.2 μM of probe, each 0.4 μM of primer；Institute Stating enzyme mixation includes 5 μ/μ L Taq archaeal dna polymerase, 0.1 μ/μ L μ NG enzyme.

3. detection kit according to claim 1, which is characterized in that contain 2 group-specifics in the detection kit Primer sequence, the nucleotide sequence of the specific primer is as shown in SEQ ID No.1-6.

4. detection kit according to claim 1, which is characterized in that also special containing 2 groups in the detection kit Property probe, the nucleotide sequence of the 2 group-specific probe are respectively SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO: 9 with SEQ ID NO:10, and the specific probe 5 ' it is terminal modified be respectively FAM and HEX, Cy3 and Cy5,3 ' terminal modified have NFQ-MGB。

5. detection kit according to claim 1, which is characterized in that the detection kit further includes interior mark system, The interior mark system includes interior label primer and internal standard probe, the nucleotide sequence of the primer such as SEQ ID NO:11 and SEQ ID Shown in NO:12, the nucleotide sequence of the internal standard probe is as shown in SEQ ID NO:13.

6. detection kit according to claim 3, which is characterized in that the terminal modified internal standard probe 5 ' is ROX, 3 ' ends It is modified with NFQ-MGB.

7. detection kit according to claim 3, which is characterized in that the detection kit further includes two low annealing The enriching primer of temperature is respectively used to the template in the enrichment site C677T and the site A1298C, the nucleotides sequence of the enriching primer Column are as shown in SEQ ID No.14 and SEQ ID NO:15.

8. kit according to any one of claims 1-7 detects the site mthfr gene C677T and A1298C simultaneously The method of loci polymorphism, which comprises the following steps:

(1) sample to be tested genomic DNA is obtained；

(2) using the specific primer, specific probe, interior mark system, enriching primer, PCR reaction buffer, Taq enzyme, UNG enzyme etc. carry out quantitative fluorescent PCR reaction, single tube reaction can detect simultaneously each sample the site mthfr gene C677T and A1298C polymorphism.

9. according to the method described in claim 8, it is characterized in that, the system of quantitative fluorescent PCR reaction are as follows:

PCR reaction buffer 12.5μl Each primer 0.2μM-1μM Each probe 0.1μM-0.5μM Taq enzyme 0.5μ-1.0μ UNG enzyme 0.1μ-0.5μ Sample DNA 2μl It adds water to 25μl

10. method according to claim 8 or claim 9, which is characterized in that the fluorescent quantitative PCR program are as follows:

Step 1: 50 DEG C of 2min, 95 DEG C of 10min；

Step 2: 95 DEG C of 15s, 45 DEG C~55 DEG C 1min, 5 circulations；

Step 3: 95 DEG C of 15s, 58 DEG C~62 DEG C 1min, 40 circulations；

And at 58 DEG C~62 DEG C of the phase III channel FAM, HEX, Cy3, Cy5, ROX corresponding to acquisition probe fluorescence signal；

Program operation finishes, and PCR thin-walled reaction tube or the taking-up of eight unions are put into concave-convex bag, sealing is obturaged, at pollution sources Reason.