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CN-121992086-A - Method for rapidly detecting MTHFR gene polymorphism based on complementary probe technology

CN121992086ACN 121992086 ACN121992086 ACN 121992086ACN-121992086-A

Abstract

The invention discloses a method for rapidly detecting MTHFR gene polymorphism based on a complementary probe technology, which comprises the following steps of S1, obtaining a sample, extracting genome DNA from a sample to be detected as an amplification template, S2, preparing a reaction system, adding the genome DNA of the sample to be detected, an upstream primer, a downstream primer, a C probe, a T probe, uracil-N-glycosylase, DNA polymerase, deoxyribonucleoside triphosphate and deoxyuridine triphosphate into the reaction system, S3, performing PCR amplification reaction under the conditions of 90-98 ℃, pre-denaturation, 5-20min, 90-98 ℃, denaturation, 10-50S, 55-69 ℃, annealing, 60-120S, 68-72 ℃ and extension, 15-300S, and 35-40 cycles, and S, judging the genotype. The test result is the same as the first generation sequencing test result, which shows that the detection method of the scheme has higher accuracy. Meanwhile, compared with a sequencing method, the method does not need to carry out a series of complex subsequent treatments on the PCR product, and the PCR amplification and detection are synchronously carried out, so that the detection time is greatly shortened, and the detection cost is reduced.

Inventors

  • WANG HUAIXIN
  • CHEN WEILI
  • JIN XIN

Assignees

  • 合肥安为康医学检验有限公司

Dates

Publication Date
20260508
Application Date
20251219

Claims (6)

  1. 1. A method for rapidly detecting MTHFR gene polymorphism based on a complementary probe technology is used for MTHFRC677T gene polymorphism typing detection, and is characterized by comprising the following steps: s1, obtaining a sample, namely extracting genome DNA from a sample to be detected and taking the genome DNA as an amplification template; S2, preparing a reaction system, namely adding genome DNA of a sample to be detected, an upstream primer, a downstream primer, a C probe, a T probe, uracil-N-glycosylase, DNA polymerase, deoxyribonucleoside triphosphate and deoxyuridine triphosphate into the reaction system; S3, PCR amplification reaction, wherein the conditions are 90-98 ℃, pre-denaturation, 5-20min, denaturation, 10-50S, 55-69 ℃, annealing, 60-120S, 68-72 ℃ and extension, and 15-300S and 35-40 cycles; s, judging genotype, judging polymorphism of MTHFR gene of a sample to be detected after PCR reaction is completed, wherein the wild type is CC, the heterozygous mutant type is CT, and the homozygous mutant type is TT.
  2. 2. The method for rapid detection of MTHFR gene polymorphism based on the complementary probe technique according to claim 1, wherein, The upstream primer is 5'-GCCTCAAAGAAAAGCTGCGT-3'; the downstream primer is 5'-TCTGAAGCACTTGAAGGAGAAGGT-3'; c probe, 5 '-fluorescent group 1-ATGAAATCGGCTCCC-quenching group 1-3'; t probe 5 '-fluorophore 2-ATGAAATCGACTCCC-quenching group 2-3'.
  3. 3. The method for rapid detection of an MTHFR gene polymorphism based on the complementary probe technology according to claim 2, wherein the fluorescent group 1 and the fluorescent group 2 are one of FAM, HEX, ROX, TAMRA, cy, and are not used in the same detection.
  4. 4. The method for rapid detection of MTHFR gene polymorphism based on the complementary probe technique according to claim 2, wherein the quenching group 1 and the quenching group 2 comprise one of BHQ-1, BHQ-2, BHQ-3, NFQ-MGB, TAMRA.
  5. 5. The method for rapidly detecting MTHFR gene polymorphism based on the complementary probe technique according to claim 1, wherein the mass ratio of the upstream primer to the downstream primer is 1:1, the mass ratio of the C probe to the T probe is 1:1, and the mass ratio of the total amount of the upstream primer to the downstream primer to the total amount of the C probe to the T probe is (10-15): 4-6.
  6. 6. The method for rapidly detecting MTHFR gene polymorphism based on the complementary probe technique according to claim 1, wherein the PCR amplification reaction is performed under 92 ℃, pre-denaturation, 10min, 95 ℃, denaturation, 30s, 60 ℃, annealing, 75s, 70 ℃, extension, 200s, 35 cycles.

Description

Method for rapidly detecting MTHFR gene polymorphism based on complementary probe technology Technical Field The invention relates to the technical field of MTHFR gene polymorphism detection, in particular to a method for rapidly detecting MTHFR gene polymorphism based on a complementary probe technology. Background Folic acid is a water-soluble B-group vitamin consisting of pteridine, para-aminobenzoic acid and glutamic acid residues, also known as vitamin BC or vitamin M. Folic acid acts in the form of tetrahydrofolate in vivo and is used as a donor of a carbon unit, participating in DNA oxidative damage repair, cell proliferation and tissue growth, helping protein metabolism, and promoting erythrocyte generation and maturation together with vitamin B12. Folic acid entering the organism is converted into dihydrofolic acid under the action of dihydrofolate reductase and then into tetrahydrofolic acid, the tetrahydrofolic acid is activated into 5, 10-methylene tetrahydrofolic acid under the action of serine hydroxymethyl transferase, the reaction is reversible, the 5, 10-methylene tetrahydrofolic acid is converted into 5-methyl tetrahydrofolic acid under the action of methylene tetrahydrofolic acid reductase, homocysteine and vitamin B12 are reacted with methionine synthetase, and 5-methyl tetrahydrofolic acid is used for providing methyl, so that methyl thiochloric acid is synthesized. The MTHFR gene has polymorphism in natural population, and mutation of the MTHFR gene can cause change of enzyme activity, cause abnormal folic acid metabolism, lower active folic acid level and raise homocysteine (Hcy) level, cause disorder of a plurality of basic biochemical processes of an organism, and further possibly cause various diseases such as nerve tube defect, cardiovascular and cerebrovascular diseases and the like. It has been found that there are 15 types of point mutations in human MTHFR, the most common of which is the mutation of C677T, which is represented by three genotypes, namely CC type, CT type, TT type. The mutation of C677T can cause the encoded amino acid to change from alanine to valine, resulting in reduced thermostability and enzyme activity. It is clearly indicated in the consensus of H-hypertension diagnosis and therapist that MTHFR activity of individuals carrying the TT genotype is reduced by 60%. The current methods for analyzing MTHFR gene polymorphism sites mainly comprise fluorescence quantitative PCR (qPCR), PCR-restriction fragment length polymorphism, amplification block mutation system-PCR and the like, but the methods are not suitable for large-scale rapid detection of crowds because of more operation steps, time and labor waste or need of PCR post-treatment and easy pollution generation. Sequencing is a gold standard for nucleic acid sequencing, but is accurate, cumbersome in operation steps and costly. Disclosure of Invention The invention aims to provide a method for rapidly detecting MTHFR gene polymorphism based on a complementary probe technology, which is used for solving the technical problems in the background technology. The technical scheme of the invention provides a method for rapidly detecting MTHFR gene polymorphism based on a complementary probe technology, which is used for MTHFRC677T gene polymorphism typing detection and comprises the following steps: s1, obtaining a sample, namely extracting genome DNA from a sample to be detected and taking the genome DNA as an amplification template; S2, preparing a reaction system, namely adding genome DNA of a sample to be detected, an upstream primer, a downstream primer, a C probe, a T probe, uracil-N-glycosylase, DNA polymerase, deoxyribonucleoside triphosphate and deoxyuridine triphosphate into the reaction system; S3, PCR amplification reaction, wherein the conditions are 90-98 ℃, pre-denaturation, 5-20min, denaturation, 10-50S, 55-69 ℃, annealing, 60-120S, 68-72 ℃ and extension, and 15-300S and 35-40 cycles; s, judging genotype, judging polymorphism of MTHFR gene of a sample to be detected after PCR reaction is completed, wherein the wild type is CC, the heterozygous mutant type is CT, and the homozygous mutant type is TT. In a preferred embodiment, the upstream primer is 5'-GCCTCAAAGAAAAGCTGCGT-3'; the downstream primer is 5'-TCTGAAGCACTTGAAGGAGAAGGT-3'; c probe, 5 '-fluorescent group 1-ATGAAATCGGCTCCC-quenching group 1-3'; t probe 5 '-fluorophore 2-ATGAAATCGACTCCC-quenching group 2-3'. In a preferred embodiment, both the fluorophore 1 and the fluorophore 2 are one of FAM, HEX, ROX, TAMRA, cy and are not used in the same assay. In a preferred embodiment, the quenching group 1 and the quenching group 2 comprise one of BHQ-1, BHQ-2, BHQ-3, NFQ-MGB, TAMRA. In a preferred embodiment, the mass ratio of the upstream primer to the downstream primer is 1:1, the mass ratio of the C probe to the T probe is 1:1, and the mass ratio of the total amount of the upstream primer to the downstream primer to the total amount of the C pro