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CN114107488A - Primer group and kit for detecting MTHFR gene polymorphism - Google Patents

Primer group and kit for detecting MTHFR gene polymorphism Download PDF

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CN114107488A
CN114107488A CN202111626470.5A CN202111626470A CN114107488A CN 114107488 A CN114107488 A CN 114107488A CN 202111626470 A CN202111626470 A CN 202111626470A CN 114107488 A CN114107488 A CN 114107488A
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mthfr
reactant
probe
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primer
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张祥林
张劲松
胡秋萍
魏鹏
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Shangai Meiji Yuhua Biomedicine Technology Co ltd
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Abstract

The invention relates to the field of gene detection, in particular to a primer group and a kit for detecting MTHFR gene polymorphism, wherein the kit comprises a PCR reactant and a quality control product, the PCR reactant comprises a first reactant and a second reactant, the first reactant comprises a first primer pair, an MTHFR 677C probe, an internal reference gene primer pair and an internal reference gene probe, and the second reactant comprises a second primer pair, an MTHFR 677T probe, an internal reference gene primer pair and an internal reference gene probe; the first primer pair comprises an upstream primer and a downstream primer for detecting MTHFR 677C; the second primer pair includes upstream and downstream primers for detecting MTHFR 677T. The kit disclosed by the invention does not need a complex nucleic acid extraction and purification step, directly uses the nucleic acid released from the blood card/whole blood sample for fluorescence PCR amplification, reduces the experimental detection step, the detection cost and the detection period, improves the safety, and meets the clinical requirements.

Description

Primer group and kit for detecting MTHFR gene polymorphism
Technical Field
The invention relates to the field of gene detection, in particular to a primer group and a kit for detecting MTHFR gene polymorphism.
Background
5, 10-methylenetetrahydrofolate reductase (MTHFR) can catalyze 5, 10-methylenetetrahydrofolate to generate 5-methyltetrahydrofolate, participates in methyl transmission and nucleotide synthesis, and is a key enzyme in the folate metabolism process. The MTHFR gene is located at the 1p36.3 position of chromosome 1, and the gene site mutation influences the activity and the heat stability of the enzyme, thereby influencing the metabolism of folic acid and methionine. The most common single nucleotide polymorphism of the gene is c.677C > T, and the incidence rate in China is 45.2%. The mutation of 677C > T site of MTHFR gene can cause the coded amino acid to change from alanine to valine, which causes the reduction of heat resistance and enzyme activity, the reduction of 30% of the enzyme activity of c.677 site (CT) of heterozygous MTHFR gene, and the reduction of more than 65% of the enzyme activity of c.677 site (TT) of homozygous MTHFR gene. In the environment of low folic acid, the c.677 site (TT) of the homozygous MTHFR gene can obviously increase the concentration of plasma homocysteine, thereby causing diseases such as venous embolism, pulmonary embolism, atherosclerosis, cerebral apoplexy and the like, and the risk that mothers carrying the genotype of the c.677 site (TT) of the MTHFR gene can grow sick children with neural tube defects in the environment of low folic acid is also increased. Therefore, the detection of the polymorphism of the c.677 locus of the MTHFR gene can find the folic acid absorption level of an individual in time, thereby realizing the high risk assessment of folic acid deficiency diseases and reducing the birth defect risk of newborns, and having important clinical significance.
Currently, methods for detecting the polymorphism of the c.677 site of the MTHFR gene include restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP), Sanger sequencing, pyrosequencing, fluorescence PCR, gene chips, fragment analysis, and high-resolution melting curve. Among them, Sanger sequencing method, pyrosequencing method, gene chip, fragment analysis method and high resolution melting curve method all require expensive equipment, are relatively complex in operation, long in detection time and high in detection cost, while PCR-RFLP method does not require expensive equipment, but the method is complex in operation, has many steps and is not suitable for clinical popularization. In addition, whole blood is a common clinical test sample type due to the advantages of stability, easy collection and storage and the like. The methods all need to extract and purify nucleic acid of a sample, and the nucleic acid extraction and purification process not only increases the experimental detection steps, the detection cost and the detection period, but also has the possibility of detection failure caused by misoperation. Therefore, it is difficult to meet the clinical requirements for simple, rapid and accurate detection.
In order to solve the above problems, there is a need in the art to establish a MTHFR genotype detection kit which is simple and convenient to operate, rapid in detection, low in cost, high in sensitivity and accurate in result, so as to meet the clinical detection requirements.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a primer set and a kit for detecting polymorphism of MTHFR gene, which are used for solving the problems in the prior art.
In order to achieve the above and other related objects, the present invention provides a primer set for detecting polymorphism of MTHFR gene, the primer set comprising one or more pairs of primers selected from the group consisting of:
1) a first primer pair: comprises an MTHFR 677C upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and an MTHFR 677C downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;
2) a second primer pair: comprises an MTHFR 677T upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and an MTHFR 677T downstream primer with a nucleotide sequence shown as SEQ ID NO. 5.
The invention also provides a kit for detecting the polymorphism of the MTHFR gene, which comprises a PCR reactant and a quality control product, wherein the PCR reactant comprises a first reactant and a second reactant, the first reactant comprises the first primer pair, and the second reactant comprises the second primer pair.
As described above, the primer set and the kit for detecting MTHFR gene polymorphism of the invention have the following beneficial effects:
1) the kit does not need a complex nucleic acid extraction and purification process, reduces extraction and purification links, and shortens detection time; the fluorescent PCR amplification detection can be carried out on the blood card and the whole blood sample.
2) The ARMS primers and the Taqman probe designed by the kit can specifically amplify and identify the genotype aiming at the MTHFR genotype, so that the accuracy of the result is ensured.
3) The detection process is closed tube detection, so that aerosol pollution caused by nucleic acid amplification is avoided, and the result is ensured to be real and credible;
4) a UNG enzyme anti-pollution system is added into the detection reaction system, so that the pollution possibility is obviously reduced;
5) and negative quality control and positive quality control are added, and false positive and reagent are monitored and operation effectiveness is improved.
6) The method has the advantages of high sensitivity, strong specificity, simple operation, low price and the like, can quickly and accurately detect the polymorphism of the MTHFR gene, is suitable for clinical analysis, detection and popularization of the polymorphism of the MTHFR gene, and meets the requirement of clinical diagnosis.
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FIG. 1 shows the result of wild-type detection of MTHFR c.677 locus CC in a blood card sample.
FIG. 2 shows the result of detection of MTHFR c.677 locus CT hybrid mutant of blood card sample.
FIG. 3 shows the result of detection of MTHFR c.677 locus TT homozygous mutant of blood card sample.
FIG. 4 shows the result of wild-type assay of MTHFR c.677 locus CC in whole blood samples.
FIG. 5 shows the result of MTHFR c.677 locus CT hybrid mutant assay in whole blood samples.
FIG. 6 shows the result of MTHFR c.677 locus TT homozygous mutant assay of whole blood samples.
Detailed Description
The invention provides a primer group for detecting MTHFR gene polymorphism, which comprises one or more pairs of primers in the following steps:
1) a first primer pair: comprises an MTHFR 677C upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and an MTHFR 677C downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;
2) a second primer pair: comprises an MTHFR 677T upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and an MTHFR 677T downstream primer with a nucleotide sequence shown as SEQ ID NO. 5.
In one embodiment, the primer set comprises a first primer pair and a second primer pair.
The primer group can be used for detecting polymorphism of site c.677 of MTHFR gene. In one embodiment, the MTHFR gene is a human MTHFR gene.
The specific base sequence of the primer pair may be obtained by replacing 1 or more bases with other bases or adding 1 or more bases to the 3 'end or 5' end, as long as the specific recognition regions can be specifically recognized under the conditions for carrying out PCR (preferably, annealing and self-annealing do not occur between primers used in a single reaction vessel). The number of the plurality is, for example, 2 to 3. When 1 or more bases are added to the primer, it is preferable to add the base to the 5' end of the primer.
The identity between a nucleotide sequence obtained by substituting 1 or more nucleotides in a nucleotide sequence specific to the primer set with another nucleotide and a nucleotide sequence before substitution (i.e., a nucleotide sequence represented by the sequence number) may be preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more.
The length of each primer is not particularly limited as long as it can specifically recognize the corresponding specific recognition region and hybridization does not occur between the primers, and is preferably 15 bases or more and 40 bases or less. The lower limit of the length of the primer is more preferably 16 bases or more, still more preferably 17 bases or more, and still more preferably 18 bases or more. More preferably, the upper limit of the length of the primer is 39 bases or less, still more preferably 38 bases or less, and still more preferably 37 bases or less.
The invention provides a kit for detecting MTHFR gene polymorphism, which comprises a PCR reactant and a quality control product, wherein the PCR reactant comprises a first reactant and a second reactant, the first reactant comprises a first primer pair, and the second reactant comprises a second primer pair.
In one embodiment, the first reactant further comprises one or more of a MTHFR 677C probe, a reference gene primer pair, and a reference gene probe.
In one embodiment, the second reactant further comprises one or more of a MTHFR 677T probe, a reference gene primer pair, and a reference gene probe.
The first reactant and the second reactant may be reagents in which a plurality of substances are independently packaged, or may be a mixture of the substances.
In a preferred embodiment, the first reactant is a mixture of the first primer pair, the MTHFR 677C probe, the reference gene primer pair and the reference gene probe. The second reactant is a mixed solution formed by the second primer pair, the MTHFR 677T probe, the reference gene primer pair and the reference gene probe. The concentration of each primer in the mixture is 100-400nM, and the concentration of each probe is 100-200 nM.
In one embodiment, the nucleotide sequence of the MTHFR 677C probe is shown in SEQ ID No. 3.
In one embodiment, the nucleotide sequence of the MTHFR 677T probe is shown in SEQ ID No. 6.
Specifically, one end of the MTHFR 677C probe and one end of the MTHFR 677T probe are modified with a fluorescent group, and the other end of the MTHFR 677C probe and the other end of the MTHFR 677T probe are modified with a quenching group. Specifically, the 5 'ends of the MTHFR 677C probe and the MTHFR 677T probe are modified with fluorescent groups, and the 3' ends are modified with quenching groups.
Specifically, the fluorescent group is selected from one of the following groups: FAM, HEX, VIC, CY3, ROX, 610, TEXAS RED, CY 5.
Specifically, the fluorescence quenching group corresponds to a fluorescence reporter group, and can quench the corresponding fluorescence reporter group. For example, when the fluorescence reporter is FAM, the fluorescence quencher is selected from one of the following: NFQ-MGB, BHQ1, Dabcyl, QYS-7, BHQ 2. In a preferred embodiment, the fluorescent group is selected from FAM and the quencher group is selected from NFQ-MGB.
In one embodiment, the sequences of the reference gene primer pairs in the first and second reactants are the same or different. In one embodiment, the sequences of the internal reference gene primer pair in the first reactant and the second reactant are the same, and the internal reference gene primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.7 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 8.
In one embodiment, the sequences of the reference gene probes in the first and second reactants are the same or different. In one embodiment, the sequences of the internal reference gene probes in the first reactant and the second reactant are the same, and the nucleotide sequences of the internal reference gene probes are shown as SEQ ID No. 9.
One end of the reference gene probe is modified with a fluorescent group, and the other end is modified with a quenching group. Specifically, the 5 'end of the reference gene probe is modified with a fluorescent group, and the 3' end is modified with a quenching group. The internal reference gene probe is different from the fluorescent groups of an MTHFR 677C probe and an MTHFR 677T probe. In one embodiment, the reference gene probe sequence is modified with VIC at the 5 'end and NFQ-MGB at the 3' end.
In one embodiment, the first and second reactants further comprise a DNA polymerase, a PCR buffer, a dNTP mix, and an aqueous medium.
The DNA polymerase is, for example, Taq DNA polymerase.
The buffer may generally provide the most suitable conditions for the enzymatic reaction to the PCR system. The buffer solution may be any buffer solution as long as it has the above-described effects.
The dNTP mixture is usually used as a raw material for DNA synthesis, and specifically may include dATP, dGTP, dTTP, dCTP, and the like.
The aqueous medium may be used to adjust the concentration of the components of the PCR system, and may generally act as a dilution solvent.
In a preferred embodiment, the DNA polymerase, PCR buffer, dNTP mixture and aqueous medium may be a commercial reagent, including all of the above reagents. The commercialized agent is, for example, Master MIX of England Weiji (Shanghai) trade Limited.
Specifically, the quality control product comprises a positive quality control product and a negative quality control product. The positive quality control product comprises a first positive quality control product and a second positive quality control product. The first positive quality control product comprises MTHFR 677C positive plasmid and reference gene positive plasmid. The second positive quality control product comprises MTHFR 677T positive plasmid and reference gene positive plasmid.
The MTHFR 677C positive plasmid is a plasmid comprising a specific sequence (shown as SEQ ID NO. 11) of MTHFR 677C site.
The MTHFR 677T positive plasmid is a plasmid comprising a specific sequence (shown as SEQ ID NO. 12) of MTHFR 677T sites.
In one embodiment, the reference gene positive plasmids in the first positive quality control and the second positive quality control are the same. The plasmid with positive reference gene is a plasmid containing specific sequence of reference gene. The reference gene is selected from human beta-actin, GAPDH or 18s-rRNA gene. In one embodiment, the reference gene is selected from GAPDH, and the nucleotide sequence of the reference gene specific sequence is shown in SEQ ID NO. 10.
The vector of the above plasmid may be a cloning vector commonly used in the art, for example, pUC57, pMD18-T, pMD19-T, etc.
In one embodiment, the negative control is nuclease-free water or TE buffer.
In one embodiment, the kit further comprises a nucleic acid releasing agent. The nucleic acid releasing agent is selected from one or more of sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether or sodium hydroxide. In a preferred embodiment, the nucleic acid releasing agent comprises sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether and sodium hydroxide, wherein the final concentration of the sodium dodecyl sulfate is 0.01-1.5% (w/w, g/g), the final concentration of the polyethylene glycol octyl phenyl ether is 0.1-2% (v/v, mL/mL) and the final concentration of the sodium hydroxide is 0.1M-1M based on the total volume of the nucleic acid releasing agent.
The nucleic acid releasing agent does not need complex nucleic acid extraction processes such as magnetic bead extraction and the like, does not need a nucleic acid purification process, can reduce extraction and purification links, and shortens detection time.
The invention also provides a method for detecting MTHFR gene polymorphism, which comprises the following steps:
1) pretreatment: pretreating a sample to be detected by using a nucleic acid releasing agent, and releasing nucleic acid in the sample to be detected;
2) preparing a reaction system, carrying out PCR amplification reaction, and collecting a fluorescent signal;
3) quality control and result analysis: the determination is made according to the following rules:
(1) the Ct value for each signal in the negative control group should be >35 or "underdetermined". Otherwise, the test result is invalid and the test is carried out again;
(2) in the positive control group, FAM and VIC channels should form a standard amplification curve, Ct values should be less than or equal to 32, otherwise, the experimental result is invalid, and re-detection is carried out;
(3) for the detection sample, if FAM and VIC channels do not form standard amplification curve or Ct value>35 or "underdetermined", the sample is negative. FAM and VIC channels form a standard amplification curve, 34 < Ct(VIC)If the detection result of the sample is less than or equal to 35, the detection result of the sample is invalid, and the sample is detected again; ct(VIC)Ct value Ct of the target signal amplification curve is calculated to be less than or equal to 34(FAM)And Ct(VIC)I.e. delta Ct ═ Ct(FAM)-Ct(VIC)The genotype was judged according to the following rules.
Figure BDA0003439803360000061
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1
Kit for preparing MTHFR gene polymorphism detection by direct amplification without nucleic acid extraction
1. Primers and probe sets for MTHFR 677C, MTHFR 677T and the reference gene were synthesized separately using prior art or third party companies as shown in the following table:
serial number Type (B) Sequence of SEQ ID NO
1 MTHFR 677C upstream primer GGTGTCTGCGGGAGC 1
2 MTHFR 677C downstream primer TCACAAAGCGGAAGAATGTGTC 2
3 MTHFR 677C Probe TTCATCATCACGCAGC 3
4 MTHFR 677T upstream primer AGGTGTCTGCGGGAGT 4
5 MTHFR 677T downstream primer TCACAAAGCGGAAGAATGTGTC 5
6 MTHFR 677T Probe TTCATCATCACGCAGC 6
7 Internal reference gene upstream primer AGATCCCTCCAAAATCAAGTGG 7
8 Internal reference geneDownstream primer GGCAGAGATGATGACCCTTTT 8
9 Internal reference gene probe ATCTTCCAGGAGTGAGT 9
2. The reagents of the kit were prepared according to the compositions and specifications shown in the following table:
Figure BDA0003439803360000071
master MIX was purchased from Weijie fundi (Shanghai) trade Co., Ltd, primer Committee worker bioengineering (Shanghai) Co., Ltd, and Probe Committee Weijie fundi (Shanghai) trade Co., Ltd. In the first and second reactions, the concentration of each primer was 150nM and the concentration of each probe was 150 nM. The first positive quality control product contains positive plasmid mixture with specific MTHFR 677C site sequence and specific reference gene sequence connected to pUC57 vector. The second positive quality control product contains positive plasmid mixture with specific MTHFR 677T site sequence and specific reference gene sequence connected to pUC57 vector. The chemical reagents are purchased from International medicine and health Limited of Chinese medicine (Shanghai).
Example 2
Method for detecting MTHFR gene polymorphism
1. Sample collection and processing
(1) Sample collection
a. Blood card sample: about 30. mu.L of whole blood anticoagulated with sodium or lithium heparin is added dropwise to the FTA card, and the blood should completely permeate the FTA card and be oven-dried at 60 ℃ for 10 minutes or naturally dried at room temperature for at least 4 hours. Blood spots are not required to be stacked and not contacted with other interfaces when blood card samples are collected, and the blood spots are placed into a sterile bag after being fully dried, so that mutual pollution among the samples is avoided.
b. Whole blood sample: peripheral blood was collected by a professional using a blood collection tube using non-heparin sodium or heparin lithium as an anticoagulant. After blood drawing, the mixture is turned upside down gently and mixed for 5-10 times to make the anticoagulant and the blood fully and uniformly mixed.
(2) Sample processing
a. Blood card sample pretreatment: and (3) disinfecting the outside of the sampler by using 75% alcohol, sampling the blood card by using the sampler, taking 3 blood cards in total, and pumping the blood card sample into a marked centrifugal tube of 0.2 mL. And 3 mu L of the negative quality control substance and 3 mu L of the positive quality control substance are respectively added into 2 centrifuge tubes with the volume of 0.2 mL. Add 15. mu.L of nucleic acid releasing agent to each 0.2mL centrifuge tube, mix well with shaking and centrifuge, incubate for 5 minutes in 95 ℃ metal bath. Centrifuging until no liquid exists on the tube cover and the side wall, shaking, mixing uniformly and centrifuging to obtain a sample to be detected, and detecting or temporarily storing at 2-8 ℃ for later use for no more than 7 days.
b. Peripheral blood sample pretreatment: taking 0.5 mu L of whole blood which takes non-heparin sodium or heparin lithium as anticoagulant, respectively taking 3 mu L of negative quality control product and positive quality control product, adding the negative quality control product and the positive quality control product into 3 centrifugal tubes with 0.2mL, respectively adding 15 mu L of nucleic acid releasing agent, fully oscillating, uniformly mixing and centrifuging, and placing in a metal bath at 95 ℃ for incubation for 5 minutes. Centrifuging until no liquid exists on the tube cover and the side wall, shaking, mixing uniformly and centrifuging to obtain a sample to be detected, and detecting or temporarily storing at 2-8 ℃ for later use for no more than 7 days.
2. Formulation system
(1) The processed blood card sample and the peripheral blood sample are respectively used as samples to be detected to prepare systems according to the following modes. Setting a typesetting mode according to detection requirements, adding a PCR reactant into 8-connected tubes according to typesetting, adding 22.5 mu L into each hole, respectively and sequentially adding 2.5 mu L of negative quality control substances, samples to be detected and positive quality control substances corresponding to reaction liquid into the reaction holes, and covering 8-connected tube covers after adding, wherein specific components in each tube in the experiment are as follows:
branch pipe Composition + volume (μ L)
First sample tube to be tested 22.5 muL of first reactant and 2.5 muL of sample to be detected
Second sample tube to be tested 22.5 μ L of the second reactant + 2.5 μ L of the sample to be tested
First positive control tube The first reactant 22.5. mu.L + MTHFR 677C positive quality control 2.5. mu.L
Second positive control tube The second reactant is 22.5 mu L + MTHFR 677T positive quality control substance 2.5 mu L
First negative control tube 22.5 muL of the first reactant and 2.5 muL of the negative quality control material
Second negative control tube The second reactant is 22.5 muL + the negative quality control substance is 2.5 muL
(2) Mix well and centrifuge.
3. Fluorescent PCR detection
And (3) placing the PCR reaction tube into a sample groove of an ABI 7300plus fluorescent PCR amplification instrument, and setting the names of samples to be detected according to the corresponding sequence. The conditions for the fluorescent quantitative PCR reaction were set as shown in the following table:
Figure BDA0003439803360000091
FAM and VIC channel fluorescence signals were collected simultaneously.
4. Analysis of the resulting data
And after the PCR program is operated, deriving experimental data, observing an amplification curve, analyzing a Ct value, and judging a result.
In this embodiment, ABI 7300plus is selected for amplification, but the present invention is not limited to ABI 7300plus, and it is suitable for other fluorescent quantitative PCR instruments with FAM and VIC fluorescent channels or for detecting different fluorescent probes according to different fluorescent quantitative PCR instruments.
5. Determination of results
(1) The Ct value for each signal in the negative control group should be >35 or "underdetermined". Otherwise, the test result is invalid and the test is carried out again;
(2) in the positive control group, FAM and VIC channels should form a standard amplification curve, Ct values should be less than or equal to 32, otherwise, the experimental result is invalid, and re-detection is carried out;
(3) for the detection sample, if FAM and VIC channels do not form standard amplification curve or Ct value>35 or "underdetermined", the sample is negative. FAM and VIC channels form a standard amplification curve, 34 < Ct(VIC)If the detection result of the sample is less than or equal to 35, the detection result of the sample is invalid, and the sample is detected again; ct(VIC)Ct value Ct of the target signal amplification curve is calculated to be less than or equal to 34(FAM)And Ct(VIC)I.e. delta Ct ═ Ct(FAM)-Ct(VIC)The genotype was judged according to the following rules.
Figure BDA0003439803360000092
Blood card and whole blood CC wild type, CT heterozygous mutant type and TT homozygous mutant type samples are obtained, detection is carried out according to the method, and the detection results are respectively shown in figures 1-6.
Example 3: performance evaluation of detection system of detection kit for detecting blood card and whole blood sample
In this embodiment, 20 blood cards and whole blood samples are collected respectively, fluorescence PCR amplification is performed according to the detection method described in the kit to detect the MTHFR genotype, and the detection result is directly compared with the "gold standard" first-generation sequencing result, thereby verifying the accuracy and the repeatability of the detection system of the detection kit of the present invention.
The specific implementation method comprises the following steps:
the detection method described by the kit is adopted to carry out sample collection and treatment, configuration of an experimental detection system, fluorescent PCR amplification detection and detection result data analysis according to steps. The results of MTHFR genotyping for samples of 20 blood cards and 20 whole blood are shown in the following table:
Figure BDA0003439803360000101
Figure BDA0003439803360000111
in 20 blood card samples, 9 were CC wild type, 7 were CT heterozygous mutant types, and 4 were TT homozygous mutant types. Of the 20 whole blood samples, 10 were CC wild type, 7 were CT heterozygous mutant, and 3 were TT homozygous mutant. By comparing with the result of 'gold standard' one-generation sequencing, the accuracy of the fluorescent PCR genotyping detection method is 100%. Therefore, the embodiment shows that the detection system of the detection kit has very good repeatability and accuracy in detecting MTHFR gene polymorphism in a blood card or whole blood sample. The detection kit can be stably used for directly amplifying and detecting MTHFR gene polymorphism of blood cards or whole blood samples without nucleic acid extraction.
The invention is used for detecting MTHFR gene polymorphism, avoids complex nucleic acid extraction and purification steps, directly uses nucleic acid released from a blood card/whole blood sample for fluorescence PCR amplification, reduces the problems of experiment detection steps, detection cost, detection period, safety and the like added in the nucleic acid extraction and purification steps, and meets the clinical requirements on simple, quick and accurate detection.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Shanghai Meiji-Rihua biomedical science and technology Co., Ltd
<120> primer group and kit for detecting MTHFR gene polymorphism
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggtgtctgcg ggagc 15
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcacaaagcg gaagaatgtg tc 22
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttcatcatca cgcagc 16
<210> 4
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aggtgtctgc gggagt 16
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcacaaagcg gaagaatgtg tc 22
<210> 6
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttcatcatca cgcagc 16
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
agatccctcc aaaatcaagt gg 22
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggcagagatg atgacccttt t 21
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atcttccagg agtgagt 17
<210> 10
<211> 225
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atggctgaga acgggaagct tgtcatcaat ggaaatccca tcaccatctt ccaggagcga 60
gatccctcca aaatcaagtg gggcgatgct ggcgctgagt acgtcgtgga gtccactggc 120
gtcttcacca ccatggagaa ggctggggct catttgcagg ggggagccaa aagggtcatc 180
atctctgccc cctctgctga tgcccccatg ttcgtcatgg gtgtg 225
<210> 11
<211> 301
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cctgtggtct cttcatccct cgccttgaac aggtggaggc cagcctctcc tgactgtcat 60
ccctattggc aggttacccc aaaggccacc ccgaagcagg gagctttgag gctgacctga 120
agcacttgaa ggagaaggtg tctgcgggag ccgatttcat catcacgcag cttttctttg 180
aggctgacac attcttccgc tttgtgaagg catgcaccga catgggcatc acttgcccca 240
tcgtccccgg gatctttccc atccaggtga ggggcccagg agagcccata agctccctcc 300
a 301
<210> 12
<211> 301
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cctgtggtct cttcatccct cgccttgaac aggtggaggc cagcctctcc tgactgtcat 60
ccctattggc aggttacccc aaaggccacc ccgaagcagg gagctttgag gctgacctga 120
agcacttgaa ggagaaggtg tctgcgggag tcgatttcat catcacgcag cttttctttg 180
aggctgacac attcttccgc tttgtgaagg catgcaccga catgggcatc acttgcccca 240
tcgtccccgg gatctttccc atccaggtga ggggcccagg agagcccata agctccctcc 300
a 301

Claims (10)

1. A primer group for detecting MTHFR gene polymorphism, which is characterized by comprising one or more pairs of primers selected from the following:
1) a first primer pair: comprises an MTHFR 677C upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and an MTHFR 677C downstream primer with a nucleotide sequence shown as SEQ ID NO. 2;
2) a second primer pair: comprises an MTHFR 677T upstream primer with a nucleotide sequence shown as SEQ ID NO.4 and an MTHFR 677T downstream primer with a nucleotide sequence shown as SEQ ID NO. 5.
2. A kit for detecting MTHFR gene polymorphism, which comprises a PCR reactant and a quality control product, wherein the PCR reactant comprises a first reactant and a second reactant, the first reactant comprises a first primer pair in the primer set according to claim 1, and the second reactant comprises a second primer pair in the primer set according to claim 1.
3. The kit of claim 2, wherein the first reactant further comprises one or more of an MTHFR 677C probe, a reference gene primer pair, and a reference gene probe; and/or the second reactant further comprises one or more of an MTHFR 677T probe, an internal reference gene primer pair and an internal reference gene probe.
4. The kit according to claim 3, wherein the nucleotide sequence of the MTHFR 677C probe is shown in SEQ ID No.3, and/or the nucleotide sequence of the MTHFR 677T probe is shown in SEQ ID No. 6; preferably, one end of the MTHFR 677C probe and one end of the MTHFR 677T probe are modified with a fluorescent group, and the other end of the MTHFR 677C probe and the other end of the MTHFR 677T probe are modified with a quenching group.
5. The kit according to claim 3, wherein the sequences of the internal reference gene primer pair in the first reactant and the second reactant are the same, and the internal reference gene primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID No.7 and a downstream primer with a nucleotide sequence shown as SEQ ID No. 8.
6. The kit according to claim 3, wherein the sequences of the internal reference gene probes in the first reactant and the second reactant are the same, and the nucleotide sequences of the internal reference gene probes are shown as SEQ ID No. 9; preferably, one end of the reference gene probe is modified with a fluorescent group, and the other end of the reference gene probe is modified with a quenching group; more preferably, the reference gene probe is different from the MTHFR 677C probe and the MTHFR 677T probe in the fluorescent group.
7. The kit of claim 2, wherein the first reactant and the second reactant each further comprise one of: a) a mixture of DNA polymerase, PCR buffer, dNTP mixture and aqueous medium; b) master MIX.
8. The kit of claim 2, wherein the quality control substances comprise positive quality control substances and negative quality control substances; preferably, the positive quality control substances comprise a first positive quality control substance and a second positive quality control substance; the first positive quality control product comprises an MTHFR 677C positive plasmid and an internal reference gene positive plasmid, and the second positive quality control product comprises an MTHFR 677T positive plasmid and an internal reference gene positive plasmid.
9. The kit according to claim 8, wherein the reference gene in the reference gene positive plasmid is selected from the group consisting of human β -actin, GAPDH, and 18 s-rRNA; and/or the negative quality control material is nuclease-free water or TE buffer solution.
10. The kit of claim 2, further comprising a nucleic acid releasing agent; preferably, the nucleic acid releasing agent is selected from one or more of sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether or sodium hydroxide.
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