CN107267645A

CN107267645A - Primer pair, probe and kit for detecting mthfr gene polymorphism

Info

Publication number: CN107267645A
Application number: CN201710643195.5A
Authority: CN
Inventors: 金京勋; 崔红; 易其; 易梅仙
Original assignee: Guangzhou Zhen Biological Technology Co Ltd
Current assignee: Guangzhou Zhen Biological Technology Co Ltd
Priority date: 2017-07-31
Filing date: 2017-07-31
Publication date: 2017-10-20
Anticipated expiration: 2037-07-31
Also published as: CN107267645B

Abstract

The present invention discloses a kind of primer pair, probe and kit for being used to detect mthfr gene polymorphism；The kit includes：For primer of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.1 and SEQ ID No.2,5 ' first base in end of the SEQ ID No.1 have amido modified group, and 5 ' first base in end of the SEQ ID No.2 have amido modified group；For probe of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.3 and SEQ ID No.4, a base of the SEQ ID No.3 has amido modified group, and a base of the SEQ ID No.4 has amido modified group.Compared with prior art, the time required to kit of the present invention can significantly shorten detection, reduce non-specific amplification, SNPs and point mutation, reduction design difficulty and testing cost can be detected, and does not influence diagnostic accuracy, realize method of the single reaction pipe without detection mutation of uncapping, Aerosol Pollution is avoided to greatest extent, facilitates Clinical practice, reduces error caused by operation.

Description

Primer pair, probe and kit for detecting mthfr gene polymorphism

Technical field

The present invention relates to technical field of molecular biology, more particularly to a kind of it is used to detect drawing for mthfr gene polymorphism Thing is to, probe and kit.

Background technology

PCR (PCR) is a kind of Protocols in Molecular Biology for being used to amplify the specific DNA fragmentation of amplification.It is glimmering Light PCR (qPCR) is detected, is the real-time detection technique in round pcr base growth, is monitored in real time using fluorescence signal accumulation whole Individual PCR processes, quantitative analysis is carried out to unknown template or according to fluorescence signal accumulation detection target base finally by standard curve Cause.The technology realizes leaps of the PCR from qualitative to quantitative, and compared with Standard PCR, it has specificity stronger, effective PCR pollution problems are solved, the features such as automaticity is high, to being widely used at the beginning of 21 century.

Although PCR detection method has quick feature, it is quickly, relative to other inspections, such as to cultivate, current state Inside and outside outpatient service detection has increasing need for faster going out result, and current round pcr each circulates needs and heats cooling, reaches quick The method of PCR purposes is concentrated mainly on shortening heating cool time, and shortening chronergy is limited, and PCR reactions need each in itself 75 seconds or so time is circulated, in order to shorten detection time, period can only be reduced, cause fast PCR sensitivity decrease.

SNP (single nucleotide polymorphisms, SNPs), is primarily referred to as in genome As the DNA sequence polymorphism caused by the variation of single nucleotide acid in level, SNPs is as third generation genetic marker, with known Property, hereditability, detectability, positioning for disease gene, clone and identify, SNPs have an effect on body physiological characteristic, Medicament metabolism ability, pathological conditions, the neurological susceptibility for being usually used in studying or diagnosing the illness, personalized medicine, such as mthfr gene The SNPs in 677 sites is related to folic acid metabolism, and folic acid ability is metabolized as 100% using MTHFR677 sites wild type C/C homozygotes When heterozygote C/T types metabolic capability be 65%, saltant type homozygote T/T metabolic capabilities are 30%, and folic acid metabolism and homotype Cysteine metabolism is closely related, and the disease relationship such as congenital abnormality, hypertension is close.

Point mutation is one kind of gene mutation, refers to only one of which base-pair and changes.Broad sense point mutation can be base Replace, single base insertion or base deletion；Narrow sense point mutation is also referred to as single base and replaces (base substitution).Base Replace and be divided into conversion (transitions) and the class of transversion (transversions) two again.Point mutation has very high back mutation Rate.

Point mutation and tumour are related closely, such as non-small cell lung cancer-adenocarcinoma patients' epidermal growth factor receptor (epidermal growth factor receptor:EGFR) gene appearance mutation patient is occupied more than 30%, with targeting medicine The appearance of thing, detection targeted drug effectively mutation can effectively distribute medical resource, medical treatment is shot the arrow at the target, reduce and waste.

Detection SNPs or the method for point mutation mainly have at present：1) PCR-SSCP methods；2) Heteroduplex analysis (HA)；3) Mutant-enriched PCR (mutant-enriched PCR)；4) allele specific oligonucleotide analytic approach (allele- specific oligonucleotide,ASO)；5) DNA chip technology (DNA chip)；6) it is mutated amplification system (amplification refractory mutation system,ARMS)；7) method such as pyrosequencing method.

PCR-SSCP methods, Heteroduplex analysis, Mutant-enriched PCR etc. need first step PCR anti-in the above method Test tube cap is opened after should terminating, reaction product pollution air is easily caused, forms aerosol, cause false positive results；And be sequenced Method needs to configure expensive tester, and test agent is expensive, it is difficult to popularize, while PCR sequencing PCR relies on the overlapping and height of crest Degree is judged, causes to be difficult to detect that low-copy is mutated in height copy wild type signal background；Chip method is needed before carrying out PCR is expanded, and easily produces Aerosol Pollution, detection principle is mutant probe and wild-type probe fluorescent value comparing calculation, and is surveyed Sequence method equally has to be difficult to detect the problem of low-copy is mutated in height copy wild type signal background；ARMS-PCR methods are ancient Allusion quotation method is two sets of reaction systems of design, is judged by the Ct value differences in two peaceful systems of method are different, and reaction system needs anti- Multiple optimization, while needing two reaction tubes, or needs to design scorpion type primer, annular region and linear areas need to use high Expensive space son modification, while designing numerous and diverse, it is necessary to verify the primed probe of multiple sequences, causes research and development feasibility reduction, inspection Survey cost increase；Defect and design difficulty the influence Clinical practice scope and clinical expansion of these technologies, need a kind of new PCR badly Detection method.

The content of the invention

Based on this, it is necessary to the problems such as easily being polluted for prior art testing cost height, reaction product, mesh of the invention Be provide that a kind of testing cost is low, single reaction pipe close lid can complete detection be used for detect mthfr gene polymorphism Primer pair, probe and kit.

To achieve these goals, the technical solution adopted by the present invention is as follows：

It is an object of the invention to provide a kind of primer pair for being used to detect mthfr gene polymorphism, include：For Primer of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.1 and SEQ ID No.2.

In wherein some embodiments, 5 ' first base in end of the SEQ ID No.1 have amido modified group, institute Stating SEQ ID No.2 5 ' first base in end has amido modified group.

Another object of the present invention is to provide a kind of probe for being used to detect mthfr gene polymorphism, include：For Probe of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.3 and SEQ ID No.4.

In wherein some embodiments, a base of the SEQ ID No.3 has amido modified group, the SEQ An ID No.4 base has amido modified group.

In wherein some embodiments, 5 ' the 4th bases in end of the SEQ ID No.3 have amido modified group, Or/and 5 ' the tenth bases in end of the SEQ ID No.4 have amido modified group.

Another object of the present invention is to provide a kind of kit for being used to detect mthfr gene polymorphism, include：

For the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.1 and SEQ ID No.2 Primer, 5 ' first base in end of the SEQ ID No.1 have an amido modified group, the 5 ' ends of the SEQ ID No.2 the One base has amido modified group；

For the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.3 and SEQ ID No.4 Probe a, base of the SEQ ID No.3 has amido modified group, and a base of the SEQ ID No.4 has Amido modified group.

Another object of the present invention is to provide a kind of fluorescence PCR method for detecting mthfr gene polymorphism, including it is as follows Step：

(1) DNA of testing sample is extracted；

(2) using the DNA as template, fluorescent PCR amplified reaction is carried out using primer, probe, wherein：

The primer is included for mthfr gene base sequence such as SEQ ID No.1 and the SEQ ID that c.677 site is designed Primer shown in No.2,5 ' first base in end of the SEQ ID No.1 have amido modified group, the SEQ ID No.2 5 ' first base in end have amido modified group,

The probe is included for mthfr gene base sequence such as SEQ ID No.3 and the SEQ ID that c.677 site is designed Probe shown in No.4 a, base of the SEQ ID No.3 has amido modified group, the one of the SEQ ID No.4 Individual base has amido modified group.

In wherein some embodiments, the parameter of the fluorescent PCR amplified reaction is：95 DEG C, after 3-5min, by 95 DEG C, 2-10s, 60-63.2 DEG C, 10-30s, 35-50 circulation.

Compared with prior art, the invention has the advantages that：

Kit of the present invention, below 20bp particularly 15-20bp primer sequence is designed for the site of mthfr gene 677 The particularly 15-20bp probe sequence of row and below 20bp, by many experiments from numerous primer sequences, probe sequence In filtered out the sequence of high specific as primer pair, probe, and modified using amino group the primer pair filtered out and Probe, the time required to significantly shortening detection, reduction non-specific amplification, reduction primer, probe design difficulty and testing cost, no Influence diagnostic accuracy, realize single reaction pipe close lid can while detect site C/C, C/T, T/T genotype of mthfr gene 677, Aerosol Pollution is avoided to greatest extent, facilitates Clinical practice, reduces error caused by operation.

The primer of the present invention, uses amino group to modify 5 ' ends of the length for below 20bp particularly 15-20bp primers, 60 DEG C or so are reached to improve primer Tm, institute's expanding fragment length is shorter than 60bp situation；The fluorescence probe of the present invention, is used Amino group modification length is a base of below 20bp particularly 15-20bp probes, and 68- is reached to improve probe Tm values 70 DEG C, this design ensure that the hybridization efficiency of probe probe in 60 DEG C of annealing extensions is very low, along with allele has two Bar probe, ensures that the probe with a base hybridizes to mould to 100% homotactic probe prior to other one always in reaction Plate, the hybridization of the homotactic probe of Reverse transcriptase non-100%, is finally reached specific detection SNPs purpose.So as to realize The detection kit can mthfr gene c.677 high specific, high-precision detection are realized in site, and make detection time Shorten.In addition, by the restriction to fluorescence probe decorating site, detection sensitivity is further lifted.

The detection method of the present invention, based on the above-mentioned restriction to primer pair, probe, compared with existing detection method, denaturation Time only needs 2-10s, and annealing extension of time only needs 2-30s, and whole testing process shortens 20-35min；Further, react The shortening of time also further shorten non-100% identical sequence probes hybridization time so that the mismatch probe of inefficiency is miscellaneous The amount of hybridization of friendship is more reduced, and is finally reached the purpose of specific detection point mutation.

Embodiment

In order to be more clearly understood that the technology contents of the present invention, described in detail especially exemplified by following examples.It should be understood that this A little embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.Unreceipted actual conditions in the following example Experimental method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York: ColdSpring Harbor Laboratory Press, 1989) described in condition, or according to the bar proposed by manufacturer Part.Used various conventional chemical reagent, are commercially available prod in embodiment.

Unless otherwise defined, the technical field of all technologies used in the present invention and scientific terminology with belonging to the present invention The implication that is generally understood that of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality The purpose of example is applied, the limitation present invention is not used in.Term "and/or" used in the present invention includes one or more related listed The arbitrary and all combination of project.

Embodiment 1, the primer pair for detecting mthfr gene polymorphism

For mthfr gene, c.677 site devises primer pair to the present embodiment.

Primer pair is designed according to rs1801133 sequences (SEQ ID No.5), primer pair sequence is as follows：

Forward primer (SEQ ID No.1)：5 ' C (amino group) TTGAAGGAGAAGGT 3 '；

Reverse primer (SEQ ID No.2)：5 ' C (amino group) AAAGCGGAAGAAT 3 '.

During preparing above-mentioned primer pair, using ABI companies primer-design software primer express respectively to ammonia The Tm values of the base sequence after base sequence, amino group modification before base base group modification are tested, as a result as follows：

Before modification：The Tm values of forward primer base sequence are 36.8 DEG C, and the Tm values of reverse primer base sequence are 39.4 DEG C；

After modification：The Tm values of forward primer base sequence are 59.3 DEG C, and the Tm values of reverse primer base sequence are 61.2 DEG C；

The Tm values of base sequence are lower than conventional fluorescent PCR primer temperature before modification, by 5 ' the first base amino bases of end Tm values after group's modification are obviously improved.

The present invention is used to detect that the primer pair commission Guangzhou Ai Ji bio tech ltd of mthfr gene polymorphism to be closed Into.

Embodiment 2, the probe for detecting mthfr gene polymorphism

For mthfr gene, c.677 site devises fluorescence probe to the present embodiment.

Segment design wild-type probe and saltant type probe are mutated according to rs1801133 sequences (SEQ ID No.5)：

Wild-type probe (SEQ ID No.3)：

The ends of 5 ' FAM-CGGG (amino group) AGCCGATTTCAT-BHQ1 3 ', 5 ' start the 4th base (G) and use ammonia Base base group modification, it is SNP site that 5 ' ends, which start the 7th base,.

Saltant type probe (SEQ ID No.4)：

5 ' Cy5-CGGGAGTCGA (amino group) TTTCATCATC-BHQ2 3 '；

5 ' ends are started the tenth base (A) and modified using amino group, and it is SNP site that 5 ' ends, which start the 7th base,.

During above-mentioned probe, amino group is repaiied respectively using ABI companies primer-design software primer express The Tm values of the base sequence after base sequence, amino group modification before decorations are tested, as a result as follows：

Before modification：The Tm values of wild-type probe base sequence are 53.8 DEG C, and the Tm values of saltant type probe base sequence are 52.9℃；

After modification：The Tm values of wild-type probe base sequence are 70.8 DEG C, and the Tm values of saltant type probe base sequence are 70.6℃。

The present invention is used for the probe commission Guangzhou Ai Ji bio tech ltd synthesis for detecting mthfr gene polymorphism.

Embodiment 3, the kit for detecting mthfr gene polymorphism

For mthfr gene, c.677 site is devised containing the primer pair of embodiment 1 and the probe of embodiment 2 the present embodiment Kit.

Embodiment 4, structure plasmid

Build the site SNPs wild types of mthfr gene 677 and mutant plasmids：

NCBI-dbSNPs is retrieved, rs1801133 sequences (referring to SEQ ID No.6), the auspicious brilliant biological section in commission Shanghai is found Skill Co., Ltd builds wild plasmid and mutant plasmids, and wild plasmid sequence is as shown in SEQ ID No.6, saltant type matter Grain sequence is as shown in SEQ ID No.7.

Embodiment 5, the optimization of the reaction temperature of kit for detecting mthfr gene polymorphism

In order to verify primer, probe and find optimal reaction temperature, thermograde PCR tests are carried out, Bole is used (Biorad) CFX fluorescent PCRs instrument carries out thermograde fluorescent PCR.

1st, reaction system is as follows：

End reaction system 25ul.

The reaction solution needed for 48 reactions is proportionally prepared, 48 PCR of dispensing white centrifuge tubes of 200ul add DNA Template.

Wherein, DNA profiling (WT is that wild plasmid, MT are mutant plasmids) is referring to such as table 1 below：

Table 1

2nd, response procedures are：95 DEG C, 3min, 1 circulation；95 DEG C, 2s, 60-68 DEG C of thermograde, 30s, 45 circulations.

3rd, reaction result

Reaction result refers to table 2, as can be seen from Table 2：Wild-type template can be detected at 60-63.2 DEG C, and is mutated Pattern plate has no fluorescence, and optimum temperature is 60 DEG C, and all temperature do not detect saltant type template, thus substantially determines reaction temperature Degree and condition.

Table 2

Embodiment 6, the kit provided using embodiment 3 carry out wild-type template and saltant type template detection

1st, ABI7500 fluorescent PCRs amplification instrument

(1) reaction system is as follows：

Wherein, DNA profiling (WT is that wild plasmid, MT are mutant plasmids) is shown in Table 3：

Table 3

	7	8	9	10
					A	MT-10³	MT-10⁷	WT-10³	WT-10⁷
B	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					C	MT-10³	MT-10⁷	WT-10³	WT-10⁷
D	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					E	MT-10³	MT-10⁷	WT-10³	WT-10⁷
F	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					G	MT-10³	MT-10⁷	WT-10³	WT-10⁷
H	Blank	Blank	Blank	Blank

(2) response procedures are：95 DEG C, 5min, 1 circulation；95 DEG C, 10s, 60 DEG C, 30s, 45 circulation (67 points of total time-consumings Clock).

(3) reaction result

Reaction result refers to table 4, as seen from Table 4：The inventive method can be with effective detection high copy number and low copy number Wild type and saltant type MTHFR plasmids, and without non-specific performance signals.

Table 4

During using ABI7500 fluorescent PCR amplification instruments, denaturation temperature is 95 DEG C, denaturation time is 10 seconds, elongating temperature of annealing For 60 DEG C, annealing extension of time be 30 seconds, each recycle ratio Standard PCR (denaturation time 15 seconds, annealing extension of time 60 seconds) contracting Short reaction time 35 seconds, carries out 35-45 circulation, at least shortens 20 minutes total time.

2nd, Bio-Rad CFX96Touch real-time fluorescent PCR amplifications instrument

(1) reaction system is as follows：

Wherein, DNA profiling (WT is that wild type, MT are mutability plasmid) is shown in Table 5：

Table 5

(2) response procedures are：95℃、3min；95 DEG C, 2s, 60 DEG C, 10s, 50 circulations (total time-consuming 45 minutes).

(3) reaction result

Reaction result is shown in Table 6, as seen from Table 6：Kit of the present invention can also effective detection height on Bio-Rad instrument The wild type and saltant type MTHFR plasmids of copy number and low copy number, and without non-specific performance signals.

Table 6

During using Bio-Rad CFX96Touch real-time fluorescent PCR amplification instrument, denaturation temperature is 95 DEG C, denaturation time is 2 Second, annealing elongating temperature is 60 DEG C, annealing extension of time is 10 seconds, each 58 seconds shortening time of circulation, carries out 35-45 and follows Ring, total time at least shortens 35 minutes than Standard PCR (denaturation time 15 seconds, annealing extension of time 60 seconds).

In the above embodiment of the present invention, as a result interpretation foundation is：Mthfr gene C677T wild-type probe fluorescence report collection Group is that FAM fluorophors, saltant type probes report group are Cy5 fluorophors, and wild-type homozygote (C/C) can only be detected FAM fluorescence, saltant type homozygote (T/T) can only detect Cy5 fluorescence, heterozygous (C/T) can detect simultaneously FAM fluorescence and Cy5 fluorescence.

In the above embodiment of the present invention, after tested, wild-type probe and 100% homotactic hybridization temperature and 1 mispairing Hybridization temperature differs 17.1 DEG C；Anomaly probe and 100% homotactic hybridization temperature and 1 mismatch hybridization temperature difference 16.2 ℃.Thus wild-type probe and saltant type template hybridization temperature can be calculated：T=70.8-17.1=53.7 DEG C of Tm- Δs, than moving back Fiery elongating temperature is 60 DEG C low 6.3 DEG C；Saltant type probe and wild-type template hybridization temperature Tm- Δs t=70.6-16.2=54.4 DEG C, it is 60 DEG C lower 5.6 DEG C than annealing elongating temperature.

The above embodiment of the present invention, is limited using the design of unconventional taqman probes and primer design method and time PCR method processed, quick and convenient effectively low cost detection SNPs, detection method is realized and detected simultaneously in single PCR reaction tubes Site C/C, C/T, T/T genotype of mthfr gene 677, facilitates clinical manipulation, reduces Aerosol Pollution, avoids to greatest extent False positive and false negative.This method is directed to diagnostic purpose flexible Application, while available for other purposes：Scientific research, genetic analysis etc..

The above embodiment of the present invention, the primer modified using amino group, probe, and length are in below 20bp, amplification Product total length is less than 60bp, the time required to thus reducing amplification, realizes fast PCR purpose, probe Tm temperature is about 70 DEG C of left sides The right side, and the Tm values after one base of mispairing are less than annealing elongating temperature, this design ensure that probe in 60 DEG C of annealing extensions The hybridization efficiency of probe is very low, along with allele has two probes, ensures that 100% homotactic probe is first in reaction always The probe in other one with a base hybridizes to template, the hybridization of the homotactic probe of Reverse transcriptase non-100%, most Reach specific detection SNPs purpose eventually, and time restriction PCR programs are when artificially shortening non-100% identical sequence probes hybridization Between so that the amount of hybridization of the mismatch probe hybridization of inefficiency is more reduced, and is finally reached the purpose of specific detection point mutation.

Comparative example 1

This comparative example is the comparative example of the part 1 of embodiment 6 (i.e. using the part of ABI7500 fluorescent PCR amplification instruments), right It is only that in the probe of kit that amino group decorating site is different than part, it is specific as follows：

The base sequence of wild-type probe is same as Example 6, the difference is that only the decorating site of amino group not Together, it is specially：5 ' FAM-CGGGAG (amino group) CCGATTTCAT-BHQ1 3 ', i.e., 5 ' ends, which start the 6th base (G), to be made Modified with amino group.

The base sequence of saltant type probe is same as Example 6, the difference is that only the decorating site of amino group not Together, it is specially：5 ' Cy5-CGGGAGTCGAT (amino group) TTCATCATC-BHQ2 3 ', i.e., 5 ' ends start the 11st base (T) modified using amino group.

Reaction system and response procedures are identical with embodiment.

(1) reaction system is as follows：

Wherein, DNA profiling (WT is that wild plasmid, MT are mutant plasmids) is shown in Table 7：

Table 7

	6	7	8	9
					A	MT-10³	MT-10⁷	WT-10³	WT-10⁷
B	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					C	MT-10³	MT-10⁷	WT-10³	WT-10⁷
D	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					E	MT-10³	MT-10⁷	WT-10³	WT-10⁷
F	MT-10³	MT-10⁷	WT-10³	WT-10⁷
					G (positive control)	MT-10³	MT-10⁷	WT-10³	WT-10⁷
H	Blank	Blank	Blank	Blank

(2) response procedures are：95℃、5min；95 DEG C, 10s, 60 DEG C, 30s, 45 circulations (total time-consuming 67 minutes).

(3) reaction result

Reaction result refers to table 8, as seen from Table 8：The inventive method (positive control) surveys high copy number and low copy number Wild type and saltant type MTHFR plasmids can detect fluorescence signal, and change the primed probe detection of amido modified position High copy number masterplate Ct values move to right 2-3 or so, and low copy number masterplate fails to detect fluorescence signal, shows sensitivity decrease.

Table 8

The gene tester of the present invention is also applied for the detection of non-mutation gene, and now reaction time range and temperature be more Extensively, 90-96 DEG C of denaturation temperature scope, reaction of degeneration (RD) time range 2-15 seconds or longer, annealing extension temperature range is in 51- 65 DEG C, about 2-60 seconds or longer time.

Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

SEQUENCE LISTING

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ccccagatgc tgcccagcca gtgctaactg tagcattttc tcttttctat ggccaccaag 120

tgcaggcctg atttgcttgg ctgctcaagg caggacagtg tgggagtttg gagcaatcca 180

cccccactct tggaactggg ctctgagcca cctcccctga gagtcatctc tggggtcaga 240

agcatatcag tcatgagccc agccactcac tgttttagtt caggctgtgc tgtgctgttg 300

gaaggtgcaa gatcagagcc cccaaagcag aggactctct ctgcccagtc cctgtggtct 360

cttcatccct cgccttgaac aggtggaggc cagcctctcc tgactgtcat ccctattggc 420

aggttacccc aaaggccacc ccgaagcagg gagctttgag gctgacctga agcacttgaa 480

ggagaaggtg tctgcgggag tcgatttcat catcacgcag cttttctttg aggctgacac 540

attcttccgc tttgtgaagg catgcaccga catgggcatc acttgcccca tcgtccccgg 600

gatctttccc atccaggtga ggggcccagg agagcccata agctccctcc accccactct 660

caccgcaccg tcctcgcaca ggctgggggc tctgggtgga gtgctgagtt cgctgagttc 720

ttcccagatc tcctctcagg tccagaactt gcacagcgtt gcttggccac cccattttgg 780

ttacctctaa ttttcccccc aaaacccagc aacagtgtct gttgaggggt ttgttgtact 840

ttggccaaca agcatcacca aaagggattc taattctcat tacaaatcct gcttaaatca 900

gtgtttccca aaggtggctg tcatcagaac cacttgataa gctttttcaa aaagtggatc 960

tccaggtccc acccctggag gttctcactc agtaaatctg a 1001

Claims

1. the primer pair for detecting mthfr gene polymorphism, it is characterised in that include：For mthfr gene c.677 position Primer of the base sequence of point design as shown in SEQ ID No.1 and SEQ ID No.2.

2. the primer pair according to claim 1 for being used to detect mthfr gene polymorphism, it is characterised in that the SEQ ID No.1 5 ' first base in end have amido modified group, and 5 ' first base in end of the SEQ ID No.2 have ammonia Base modification group.

3. the probe for detecting mthfr gene polymorphism, it is characterised in that include：For mthfr gene c.677 site Probe of the base sequence of design as shown in SEQ ID No.3 and SEQ ID No.4.

4. the probe according to claim 3 for being used to detect mthfr gene polymorphism, it is characterised in that the SEQ ID A No.3 base has amido modified group, and a base of the SEQ ID No.4 has amido modified group.

5. being used for according to claim 3 or 4 detects the probe of mthfr gene polymorphism, it is characterised in that the SEQ ID No.3 5 ' the 4th bases in end have an amido modified group, or/and the SEQ ID No.4 5 ' the tenth bases in end With amido modified group.

6. the kit for detecting mthfr gene polymorphism, it is characterised in that include：

For primer of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.1 and SEQ ID No.2, 5 ' first base in end of the SEQ ID No.1 have amido modified group, 5 ' first alkali in end of the SEQ ID No.2 Base has amido modified group；

For probe of the mthfr gene base sequence that c.677 site is designed as shown in SEQ ID No.3 and SEQ ID No.4, A base of the SEQ ID No.3 has amido modified group, and there is a base of the SEQ ID No.4 amino to repair Adorn group.

7. the kit according to claim 6 for being used to detect mthfr gene polymorphism, it is characterised in that the SEQ ID No.3 5 ' the 4th bases in end have an amido modified group, or/and the SEQ ID No.4 5 ' the tenth bases in end With amido modified group.

8. a kind of fluorescence PCR method for detecting mthfr gene polymorphism, it is characterised in that comprise the following steps：

(1) DNA of testing sample is extracted；

9. the fluorescence PCR method of detection mthfr gene polymorphism according to claim 8, it is characterised in that the SEQ ID No.3 5 ' the 4th bases in end have an amido modified group, or/and the SEQ ID No.4 5 ' the tenth bases in end With amido modified group.

10. the fluorescence PCR method of detection mthfr gene polymorphism according to claim 8, it is characterised in that described glimmering The parameter of light pcr amplification reaction is：95 DEG C, after 3-5min, by 95 DEG C, 2-10s, 60-63.2 DEG C, 10-30s, 35-50 are followed Ring.