CN107267645B

CN107267645B - Primer pair, probe and kit for detecting MTHFR gene polymorphism

Info

Publication number: CN107267645B
Application number: CN201710643195.5A
Authority: CN
Inventors: 金京勋; 崔红; 易其; 易梅仙
Original assignee: Guangzhou Dezhen Biotechnology Co ltd
Current assignee: Guangzhou Dezhen Biotechnology Co ltd
Priority date: 2017-07-31
Filing date: 2017-07-31
Publication date: 2021-10-08
Anticipated expiration: 2037-07-31
Also published as: CN107267645A

Abstract

The invention discloses a primer pair, a probe and a kit for detecting MTHFR gene polymorphism; the kit comprises: primers with base sequences shown in SEQ ID No.1 and SEQ ID No.2 are designed aiming at a c.677 site of an MTHFR gene, wherein the first base at the 5 'end of the SEQ ID No.1 is provided with an amino modification group, and the first base at the 5' end of the SEQ ID No.2 is provided with an amino modification group; the probe with base sequences shown in SEQ ID No.3 and SEQ ID No.4 is designed aiming at the c.677 site of the MTHFR gene, wherein one base of the SEQ ID No.3 is provided with an amino modification group, and one base of the SEQ ID No.4 is provided with an amino modification group. Compared with the prior art, the kit can obviously shorten the time required by detection, reduce non-specific amplification, detect SNPs and point mutation, reduce the design difficulty and the detection cost, does not influence the diagnosis precision, realizes a method for detecting mutation without uncovering a single reaction tube, avoids aerosol pollution to the maximum extent, is convenient for clinical use, and reduces errors caused by operation.

Description

Primer pair, probe and kit for detecting MTHFR gene polymorphism

Technical Field

The invention relates to the technical field of molecular biology, in particular to a primer pair, a probe and a kit for detecting MTHFR gene polymorphism.

Background

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify a specific DNA fragment. Fluorescence PCR (qPCR) detection is a real-time detection technology developed on the basis of PCR technology, the whole PCR process is monitored in real time by using fluorescence signal accumulation, and finally, an unknown template is subjected to quantitative analysis by a standard curve or a target gene is detected according to the fluorescence signal accumulation. The technology realizes qualitative to quantitative leap of PCR, and compared with the conventional PCR, the technology has the characteristics of stronger specificity, effective solution of PCR pollution, high automation degree and the like, and is widely applied to the beginning of the 21 st century.

Although the PCR detection method has the characteristic of rapidness, the rapidness is that compared with other examinations such as culture, the current clinic detection at home and abroad needs to obtain a result faster, and the current PCR technology needs heating and cooling every cycle, so that the method for achieving the purpose of rapid PCR mainly focuses on shortening the heating and cooling time and limiting the time shortening effect, the PCR reaction needs about 75 seconds every cycle, and only the number of cycles can be reduced in order to shorten the detection time, so that the sensitivity of rapid PCR is reduced.

Single Nucleotide Polymorphisms (SNPs) mainly refer to DNA sequence polymorphisms caused by single nucleotide variation at the genome level, SNPs are used as third-generation genetic markers, have known properties, heritability and detectability, and are used for positioning, cloning and identifying disease genes, SNPs also affect physiological characteristics, drug metabolism capability and pathological conditions of organisms, are commonly used for researching or diagnosing disease susceptibility and personalized medicine, for example, SNPs at the site of the MTHFR677 are related to folate metabolism, and the metabolic capability of a heterozygote C/T type is 65% when the metabolic capability of a wild type C/C homozygote at the site of the MTHFR677 is 100%, the metabolic capability of a mutant homozygote T/T is 30%, while the folate metabolism is closely related to homocysteine metabolism, and is closely related to congenital malformation, hypertension and other diseases.

A point mutation is a type of genetic mutation, meaning that only one base pair is changed. The generalized point mutation can be base substitution, single base insertion or base deletion; a point mutation in the narrow sense is also referred to as a single base substitution (base mutation). Base substitutions are further classified into transitions and transversions. Point mutations have a high reversion rate.

The point mutation is closely related to tumorigenesis, for example, more than 30% of patients with Epidermal Growth Factor Receptor (EGFR) gene mutation in non-small cell lung cancer-adenocarcinoma patients are suffering from the EGFR gene mutation, and with the occurrence of targeted drugs, medical resources can be effectively allocated by detecting the effective mutation of the targeted drugs, so that the medical treatment is targeted, and the waste is reduced.

The current methods for detecting SNPs or point mutations mainly comprise: 1) PCR-SSCP method; 2) heteroduplex Analysis (HA); 3) mutant-enriched PCR method (mutant-enriched PCR); 4) allele-specific oligonucleotide Assay (ASO); 5) DNA chip technology (DNA chip); 6) mutation amplification system (ARMS); 7) pyrosequencing and the like.

In the method, a PCR-SSCP method, a heteroduplex analysis method, a mutant enrichment PCR method and the like need to open a test tube cover after the first-step PCR reaction is finished, so that reaction products are easy to pollute air, aerosol is formed, and a false positive result is caused; the sequencing method needs to be provided with an expensive tester, the price of a testing reagent is high, the popularization is difficult, and meanwhile, the sequencing method judges by depending on the overlapping and height of wave crests, so that the low-copy mutation is difficult to detect in the background of a high-copy wild-type signal; the chip method needs pre-PCR amplification, aerosol pollution is easy to generate, the detection principle is that fluorescence values of a mutation probe and a wild probe are compared and calculated, and the chip method has the problem that low-copy mutation is difficult to detect in a high-copy wild signal background as a sequencing method; the ARMS-PCR method is characterized in that two sets of reaction systems are designed, the difference of Ct values in the two systems is used for judgment, the reaction systems need to be repeatedly optimized, two reaction test tubes are needed simultaneously, or scorpion primers need to be designed, expensive space modifiers need to be used for annular regions and linear regions, the design is complicated, primer probes with multiple sequences need to be verified, the research and development feasibility is reduced, and the detection cost is increased; the defects and design difficulty of the technologies affect the clinical application range and clinical popularization, and a new PCR detection method is urgently needed.

Disclosure of Invention

Accordingly, it is necessary to solve the problems of high detection cost, easy contamination of reaction products, and the like in the prior art, and an object of the present invention is to provide a primer set, a probe, and a kit for detecting a polymorphism of the MTHFR gene, which are capable of completing detection with a single reaction tube closed at low detection cost.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention aims to provide a primer pair for detecting MTHFR gene polymorphism, which comprises the following components: primers with base sequences shown in SEQ ID No.1 and SEQ ID No.2 are designed aiming at the c.677 site of the MTHFR gene.

In some embodiments, the first base at the 5 'end of SEQ ID No.1 has an amino modifying group and the first base at the 5' end of SEQ ID No.2 has an amino modifying group.

Another object of the present invention is to provide a probe for detecting polymorphism of MTHFR gene, comprising: probes with base sequences shown in SEQ ID No.3 and SEQ ID No.4 designed aiming at the site c.677 of the MTHFR gene.

In some of these embodiments, one base of SEQ ID No.3 has an amino modifying group and one base of SEQ ID No.4 has an amino modifying group.

In some embodiments, the fourth base at the 5 'end of SEQ ID No.3 has an amino modifying group, or/and the tenth base at the 5' end of SEQ ID No.4 has an amino modifying group.

Another object of the present invention is to provide a kit for detecting polymorphism of MTHFR gene, comprising:

primers with base sequences shown in SEQ ID No.1 and SEQ ID No.2 are designed aiming at a c.677 site of an MTHFR gene, wherein the first base at the 5 'end of the SEQ ID No.1 is provided with an amino modification group, and the first base at the 5' end of the SEQ ID No.2 is provided with an amino modification group;

the probe with base sequences shown in SEQ ID No.3 and SEQ ID No.4 is designed aiming at the c.677 site of the MTHFR gene, wherein one base of the SEQ ID No.3 is provided with an amino modification group, and one base of the SEQ ID No.4 is provided with an amino modification group.

Another object of the present invention is to provide a fluorescent PCR method for detecting polymorphism of MTHFR gene, comprising the steps of:

(1) extracting DNA of a sample to be detected;

(2) and (2) performing a fluorescent PCR amplification reaction by using the DNA as a template and using a primer and a probe, wherein:

the primer comprises primers with base sequences shown in SEQ ID No.1 and SEQ ID No.2 designed aiming at a c.677 site of an MTHFR gene, wherein the first base at the 5 'end of the SEQ ID No.1 is provided with an amino modification group, the first base at the 5' end of the SEQ ID No.2 is provided with an amino modification group,

the probe comprises probes with base sequences shown in SEQ ID No.3 and SEQ ID No.4, wherein the base sequences are designed aiming at a c.677 site of an MTHFR gene, one base of the SEQ ID No.3 is provided with an amino modification group, and one base of the SEQ ID No.4 is provided with an amino modification group.

In some of these embodiments, the parameters of the fluorescent PCR amplification reaction are: after 3-5min at 95 deg.C, 2-10s at 95 deg.C, 60-63.2 deg.C, 10-30s, and 35-50 cycles.

Compared with the prior art, the invention has the following beneficial effects:

the kit provided by the invention designs a primer sequence of less than 20bp, particularly 15-20bp, and a probe sequence of less than 20bp, particularly 15-20bp, aiming at the site 677 of the MTHFR gene, a large number of experiments are carried out to screen out a high-specificity sequence from a plurality of primer sequences and probe sequences to serve as a primer pair and a probe, and an amino group is adopted to modify the screened primer pair and probe, so that the time required by detection is obviously shortened, non-specific amplification is reduced, the design difficulty and the detection cost of the primer and probe are reduced, the diagnosis precision is not influenced, the C/C, C/T, T/T genotype of the site 677 of the MTHFR gene can be simultaneously detected by closing a single reaction tube, the aerosol pollution is avoided to the maximum extent, the clinical use is convenient, and the error caused by operation is reduced.

The primer of the invention adopts amino group to modify the 5' end of the primer with the length of less than 20bp, particularly 15-20bp, so as to improve the Tm value of the primer to reach about 60 ℃, and the length of the amplified fragment is shorter than 60 bp; the fluorescent probe of the invention uses an amino group to modify a basic group with the length of less than 20bp, particularly a basic group of a probe of 15-20bp, so as to improve the Tm value of the probe to 68-70 ℃, the design ensures that the hybridization efficiency of the probe is very low when the probe is annealed and extended at 60 ℃, in addition, the allele has two probes, the reaction always ensures that 100 percent of the probes with the same sequence are hybridized to a template before the other probe with a basic group, and the purpose of specifically detecting SNPs is finally achieved. Therefore, the detection kit can realize high specificity and high precision detection of the c.677 site of the MTHFR gene, and shorten the detection time. In addition, the detection sensitivity is further improved by limiting the modification sites of the fluorescent probe.

Based on the limitations of the primer pair and the probe, compared with the existing detection method, the detection method has the advantages that the denaturation time is only 2-10s, the annealing extension time is only 2-30s, and the whole detection process is shortened by 20-35 min; furthermore, the reaction time is shortened, so that the hybridization time of the probe with the non-100% identical sequence is further shortened, the hybridization amount of the mismatched probe hybridization with low efficiency is further reduced, and the aim of detecting the point mutation specifically is fulfilled.

Detailed Description

In order to clearly understand the technical contents of the present invention, the following examples are given in detail. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1 primer set for detecting polymorphism of MTHFR Gene

This example designs a primer pair for the c.677 site of MTHFR gene.

Designing a primer pair according to the rs1801133 sequence (SEQ ID No.5), wherein the sequences of the primer pair are 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'.

In the process of preparing the primer pair, Tm values of the base sequence before amino group modification and the base sequence after amino group modification were measured using primer express of ABI corporation, respectively, and the results were as follows:

before modification: the Tm value of the base sequence of the forward primer is 36.8 ℃ and the Tm value of the base sequence of the reverse primer is 39.4 ℃;

after modification: the Tm value of the base sequence of the forward primer is 59.3 ℃ and the Tm value of the base sequence of the reverse primer is 61.2 ℃;

the Tm value of the base sequence before modification is lower than that of the conventional fluorescent PCR primer, and the Tm value after modification by the amino group of the first base at the 5' end is obviously improved.

The primer pair for detecting the MTHFR gene polymorphism is synthesized by Guangzhou Egyki Biotechnology Co.

Example 2 Probe for detecting polymorphism of MTHFR Gene

This example designs a fluorescent probe for site c.677 of MTHFR gene.

Designing a wild probe and a mutant probe according to the rs1801133 sequence (SEQ ID No.5) mutant fragment:

wild-type probe (SEQ ID No. 3):

5 'FAM-CGGG (amino group) AGCCGATTTCAT-BHQ 13', the fourth base (G) from the 5 'end is modified by the amino group, and the seventh base from the 5' end is an SNP site.

Mutant probe (SEQ ID No. 4):

5 'Cy 5-CGGGAGTCGA (amino group) TTTCATCATC-BHQ 23';

the tenth base (A) from the 5 'end is modified with an amino group, and the seventh base from the 5' end is a SNP site.

In the above-mentioned probe procedure, Tm values of the base sequence before modification of the amino group and the base sequence after modification of the amino group were measured using primer express of ABI corporation, respectively, and the results were as follows:

before modification: the Tm value of the base sequence of the wild-type probe is 53.8 ℃ and the Tm value of the base sequence of the mutant-type probe is 52.9 ℃;

after modification: the Tm value of the base sequence of the wild-type probe was 70.8 ℃ and the Tm value of the base sequence of the mutant-type probe was 70.6 ℃.

The probe for detecting MTHFR gene polymorphism is synthesized by Guangzhou Egyki Biotechnology Co.

Example 3 kit for detecting polymorphism of MTHFR Gene

This example designs a kit containing the primer pair of example 1 and the probe of example 2 for the c.677 site of MTHFR gene.

Example 4 construction of plasmids

Constructing wild type plasmids and mutant plasmids of SNPs at 677 site of MTHFR gene:

NCBI-dbSNPs are searched, rs1801133 sequences (see SEQ ID No.6) are found, Shanghai Ruiying Biotech limited company is entrusted to construct wild type plasmids and mutant type plasmids, the wild type plasmid sequences are shown as SEQ ID No.6, and the mutant type plasmid sequences are shown as SEQ ID No. 7.

Example 5 reaction temperature optimization of kit for detecting polymorphism of MTHFR Gene

To validate the primers, probes and find the optimal reaction temperature, a temperature gradient PCR test was performed using a berle (Biorad) CFX fluorescence PCR instrument.

1. The reaction system is as follows:

the final reaction system was 25 ul.

The reaction solution required by 48 reactions was prepared in proportion, and dispensed into 48 PCR 200ul white centrifuge tubes, and then DNA template was added.

Wherein, the DNA template (WT is a wild type plasmid, MT is a mutant type plasmid) is shown in the following table 1:

TABLE 1

2. The reaction procedure is as follows: 95 ℃, 3min,1 cycle; 95 ℃, 2s, 60-68 ℃ temperature gradient, 30s, 45 cycles.

3. Results of the reaction

See table 2 for reaction results, from table 2 it can be seen that: the reaction temperature and conditions were determined approximately by detecting the wild-type template at 60-63.2 ℃ and the mutant template without fluorescence, preferably at 60 ℃ and detecting no mutant template at all.

TABLE 2

Example 6 detection of wild type template and mutant template Using the kit provided in example 3

1. ABI7500 fluorescent PCR (polymerase chain reaction) amplification instrument

(1) The reaction system is as follows:

wherein, the DNA template (WT is a wild type plasmid, MT is a mutant type plasmid) 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 space	Blank space	Blank space	Blank space

(2) The reaction procedure is as follows: 95 ℃, 5min, 1 cycle; 95 ℃, 10s, 60 ℃, 30s, 45 cycles (total time takes 67 minutes).

(3) Results of the reaction

See table 4 for reaction results, from table 4: the method of the present invention can detect wild type and mutant MTHFR plasmid with high copy number and low copy number effectively and has no non-specific energy signal.

TABLE 4

When an ABI7500 fluorescent PCR amplification instrument is adopted, the denaturation temperature is 95 ℃, the denaturation time is 10 seconds, the annealing extension temperature is 60 ℃, the annealing extension time is 30 seconds, the reaction time is shortened by 35 seconds for each cycle compared with the conventional PCR (the denaturation time is 15 seconds, the annealing extension time is 60 seconds), 35-45 cycles are carried out, and the total time is shortened by at least 20 minutes.

2. Bio-Rad CFX96Touch real-time fluorescence PCR amplification instrument

(1) The reaction system is as follows:

wherein, the DNA template (WT is wild type, MT is mutant plasmid) is shown in Table 5:

TABLE 5

(2) The reaction procedure is as follows: 95 deg.C for 3 min; 95 ℃, 2s, 60 ℃, 10s and 50 cycles (total time consumption 45 minutes).

(3) Results of the reaction

The results are shown in Table 6, from which it can be seen that: the kit can also effectively detect wild type and mutant MTHFR plasmids with high copy number and low copy number on a Bio-Rad instrument, and has no non-specific performance signals.

TABLE 6

When a Bio-Rad CFX96Touch real-time fluorescent PCR amplification instrument is adopted, the denaturation temperature is 95 ℃, the denaturation time is 2 seconds, the annealing extension temperature is 60 ℃, the annealing extension time is 10 seconds, the time of each cycle is shortened by 58 seconds, 35-45 cycles are carried out, and the total time is at least 35 minutes shorter than that of the conventional PCR (the denaturation time is 15 seconds, and the annealing extension time is 60 seconds).

In the above embodiments of the present invention, the result interpretation criterion is: the MTHFR gene C677T fluorescent reporter group of the wild-type probe is FAM fluorescent group, the reporter group of the mutant-type probe is Cy5 fluorescent group, the wild-type homozygote (C/C) can only detect FAM fluorescence, the mutant homozygote (T/T) can only detect Cy5 fluorescence, and the heterozygote (C/T) can simultaneously detect FAM fluorescence and Cy5 fluorescence.

In the above-mentioned embodiment of the present invention, the hybridization temperature between the wild-type probe and 100% of the homologous sequence and the 1 mismatch hybridization temperature were tested to be different by 17.1 ℃; the hybridization temperature between the variant probe and the 100% homologous sequence and the hybridization temperature between 1 mismatch differ by 16.2 ℃. Therefore, the hybridization temperature of the wild-type probe and the mutant template can be calculated: tm-delta t is 70.8-17.1 is 53.7 ℃, and is 6.3 ℃ lower than the annealing extension temperature of 60 ℃; the hybridization temperature Tm-delta t of the mutant probe and the wild template is 70.6-16.2-54.4 ℃, and is 5.6 ℃ lower than the annealing extension temperature of 60 ℃.

According to the embodiment of the invention, the SNPs are quickly, conveniently, effectively and low-cost detected by using the unconventional taqman probe design and primer design method and the time-limited PCR method, the detection method realizes the simultaneous detection of the C/C, C/T, T/T genotype at the 677 site of the MTHFR gene in a single PCR reaction tube, facilitates clinical operation, reduces aerosol pollution and avoids false positive and false negative to the maximum extent. The method is flexible for diagnostic purposes, while being useful for other purposes: scientific research, gene analysis and the like.

In the above embodiment of the invention, the primer and probe modified by amino group are adopted, the length is below 20bp, the total length of the amplification product is less than 60bp, thereby reducing the time required by amplification, realizing the purpose of rapid PCR, ensuring that the Tm temperature of the probe is about 70 ℃, the Tm value after mismatching one base is lower than the annealing extension temperature, the design ensures that the hybridization efficiency of the probe is very low when the probe is annealed and extended at 60 ℃, in addition, the allele has two probes, the reaction always ensures that 100 percent of the probes with the same sequence are hybridized to the template before the other probe with one base, the hybridization of the probes with the same sequence which is not 100 percent is competitively inhibited, and the aim of specifically detecting the SNPs is finally achieved, and the time limit PCR program artificially shortens the hybridization time of the probe with the non-100 percent of the same sequence, so that the hybridization amount of the mismatched probe hybridization with low efficiency is reduced, and finally the purpose of detecting point mutation specifically is achieved.

Comparative example 1

This comparative example is a comparative example of part 1 of example 6 (i.e., the part using the ABI7500 fluorescent PCR amplification apparatus) compared only in that the site of amino group modification in the probe of the kit is different as follows:

the base sequence of the wild-type probe is the same as that of example 6, and the difference is only that the modification site of the amino group is different, specifically: 5 ' FAM-CGGGAG (amino group) CCGATTTCAT-BHQ 13 ', i.e., the sixth base (G) from the 5 ' end was modified with an amino group.

The base sequence of the mutant probe was the same as that of example 6, except that the modification site of the amino group was different, specifically: 5 ' Cy5-CGGGAGTCGAT (amino group) TTCATCATC-BHQ 23 ', i.e., the eleventh base (T) from the 5 ' end was modified with an amino group.

The reaction system and the reaction procedure were the same as in the examples.

(1) The reaction system is as follows:

wherein, the DNA templates (WT is a wild type plasmid, MT is a mutant type plasmid) are 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 space	Blank space	Blank space	Blank space

(2) The reaction procedure is as follows: 95 deg.C for 5 min; 95 ℃, 10s, 60 ℃, 30s, 45 cycles (total time takes 67 minutes).

(3) Results of the reaction

See table 8 for reaction results, from table 8: the wild type and mutant MTHFR plasmids with high copy number and low copy number measured by the method (positive control) can detect fluorescent signals, the Ct value of a template with high copy number detected by a primer probe with a changed amino modification position is shifted to the right by about 2-3, and the fluorescent signals cannot be detected by the template with low copy number, so that the sensitivity is reduced.

TABLE 8

The gene detection method of the invention is also suitable for non-point mutation gene detection, and the reaction time range and temperature are wider, the denaturation temperature range is 90-96 ℃, the denaturation reaction time range is 2-15 seconds or longer, the annealing extension reaction temperature range is 51-65 ℃, and the annealing extension reaction time is about 2-60 seconds or longer.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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<223> Artificial sequence

<400> 7

ttcagtgtta cattaaaaac aatgtttaat ccggtgccta gagaaaagtc aagcttacta 60

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. A kit for detecting MTHFR gene polymorphism, comprising:

a forward primer and a reverse primer which are designed aiming at a c.677 site of an MTHFR gene and have base sequences shown as SEQ ID No.1 and SEQ ID No.2, wherein the first base at the 5 'end of the SEQ ID No.1 has an amino modification group, and the first base at the 5' end of the SEQ ID No.2 has an amino modification group;

the base sequences designed aiming at the c.677 site of the MTHFR gene are shown as a wild type probe and a mutant type probe shown in SEQ ID No.3 and SEQ ID No.4, the fourth base at the 5 'end of the SEQ ID No.3 is provided with an amino modifying group, and the tenth base at the 5' end of the SEQ ID No.4 is provided with an amino modifying group.

2. The kit for detecting MTHFR gene polymorphism according to claim 1, wherein the forward primer has a Tm of 59.3 ℃.

3. The kit for detecting MTHFR gene polymorphism according to claim 1, wherein the reverse primer has a Tm of 61.2 ℃.

4. The kit for detecting MTHFR gene polymorphism according to claim 1, wherein the Tm of the wild-type probe is 70.8 ℃.

5. The kit for detecting polymorphism of MTHFR gene according to claim 1, wherein Tm of said mutant-type probe is 70.6 ℃.

6. The kit for detecting MTHFR gene polymorphism according to claim 1, wherein the wild-type probe is labeled with FAM at the 5 'end and BHQ1 at the 3' end.

7. The kit for detecting MTHFR gene polymorphism according to claim 1, wherein the fluorescence of the mutant probe is Cy5 and BHQ2 is labeled at the 3' end.

8. The kit for detecting MTHFR gene polymorphism according to any one of claims 1 to 7, wherein the forward primer, the reverse primer, the wild-type probe and the mutant-type probe are designed based on a sequence shown as SEQ ID No. 5.