Method for rapidly detecting MTHFR gene polymorphism based on complementary probe technology

Abstract

The invention discloses a method for rapidly detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism based on a complementary probe technology, which comprises the following steps: S1, acquiring a sample: extracting genome DNA (Deoxyribose Nucleic Acid) from a sample to be detected as an amplification template; s2, preparing a reaction system: adding the genome DNA, the upstream primer, the downstream primer, the C probe, the T probe, uracil-N-glycosylase, DNA polymerase, deoxyribonucleoside triphosphate and deoxyuridine triphosphate of the sample to be detected into the reaction system; s3, carrying out PCR (Polymerase Chain Reaction) amplification reaction under the conditions that the temperature is 90-98 DEG C, and the time is 5-20 minutes; performing denaturation at the temperature of 90-98 DEG C for 10-50 seconds; the temperature is 55-69 DEG C, and annealing is conducted for 60-120 s; the temperature is 68-72 DEG C, and extension is carried out for 15-300 s; carrying out 35 to 40 cycles; and S, genotype judgment. A test result is the same as a first-generation sequencing test result, which shows that the detection method of the scheme has relatively high accuracy. Meanwhile, compared with a sequencing method, the method has the advantages that a series of complex follow-up treatment does not need to be carried out on a PCR product, PCR amplification and detection are synchronously carried out, the detection time is greatly shortened, and the detection cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of MTHFR gene polymorphism detection technology, and more specifically, to a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology. Background Technology

[0002] Folic acid is a water-soluble B vitamin composed of pteridine, para-aminobenzoic acid, and glutamic acid residues, also known as vitamin BC or vitamin M. In the body, folic acid functions as tetrahydrofolate and acts as a one-carbon unit donor, participating in DNA oxidative damage repair, cell proliferation and tissue growth, aiding in protein metabolism, and working with vitamin B12 to promote the production and maturation of red blood cells.

[0003] Folic acid entering the body is converted to dihydrofolate by dihydrofolate reductase, and then to tetrahydrofolate. Tetrahydrofolate is activated to 5,10-methylenetetrahydrofolate by serine hydroxymethyltransferase; this reaction is reversible. 5,10-methylenetetrahydrofolate is then converted to 5-methyltetrahydrofolate by methylenetetrahydrofolate reductase. Homocysteine ​​and vitamin B12, under the action of methionine synthase, provide methyl esters from 5-methyltetrahydrofolate to synthesize methionine.

[0004] The MTHFR gene exhibits polymorphism in the general population. Mutations in this gene can alter enzyme activity, leading to abnormal folate metabolism, decreased levels of active folate, and increased levels of homocysteine ​​(Hcy), disrupting multiple fundamental biochemical processes and potentially causing neural tube defects, cardiovascular diseases, and other ailments. Fifteen point mutation types have been identified in human MTHFR, the most common and important being the C677T mutation, which manifests in three genotypes: CC, CT, and TT. The C677T mutation changes the encoded amino acid from alanine to valine, resulting in decreased heat tolerance and enzyme activity. The "Expert Consensus on the Diagnosis and Treatment of H-Type Hypertension" clearly states that individuals carrying the TT genotype have a 60% reduction in MTHFR activity.

[0005] Currently, methods for analyzing MTHFR gene polymorphism sites mainly include quantitative real-time PCR (qPCR), PCR-restriction fragment length polymorphism, and amplification arrest mutation system-PCR. However, these methods are not suitable for rapid testing on a large scale due to their numerous steps, time-consuming and labor-intensive nature, or the need for post-PCR processing which can lead to contamination. Sequencing is the gold standard for nucleic acid sequencing; although accurate, it is cumbersome and costly. Summary of the Invention

[0006] The purpose of this invention is to provide a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, so as to solve the technical problems existing in the background art.

[0007] This invention provides a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, comprising the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions are 90-98℃, pre-denaturation, 5-20 min; 90-98℃, denaturation, 10-50 s; 55-69℃, annealing, 60-120 s; 68-72℃, extension, 15-300 s; 35-40 cycles; S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

[0008] 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

[0009] In a preferred embodiment, both fluorescent group 1 and fluorescent group 2 are one of FAM, HEX, ROX, TAMRA, and Cy5, and are not used in the same detection.

[0010] In a preferred embodiment, the quenching group 1 and the quenching group 2 include one of BHQ-1, BHQ-2, BHQ-3, NFQ-MGB, and TAMRA.

[0011] In a preferred embodiment, the mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is (10-15):(4-6).

[0012] In a preferred embodiment, the PCR amplification reaction was performed under the following conditions: 92°C pre-denaturation for 10 min; 95°C denaturation for 30 s; 60°C annealing for 75 s; 70°C extension for 200 s; 35 cycles.

[0013] The beneficial effects of the technical solution of this invention are: The test results of this method are consistent with those of first-generation sequencing, indicating that the detection method has high accuracy. Furthermore, compared to sequencing, this method eliminates the need for complex post-processing of PCR products; PCR amplification and detection occur simultaneously, and the entire detection process does not require opening the lid, reducing the risk of PCR product contamination, significantly shortening detection time, and lowering detection costs. Detailed Implementation

[0014] The present invention will now be described in further detail. The embodiments of the invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose. Example

[0015] This invention provides a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, comprising the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions were 92℃, pre-denaturation, 10 min; 95℃, denaturation, 30 s; 60℃, annealing, 75 s; 70℃, extension, 200 s; 35 cycles; S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

[0016] 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

[0017] The fluorescent group 1 is FAM, and the fluorescent group 2 is HEX. Both the quenching group 1 and the quenching group 2 are BHQ-1.

[0018] The mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is 5:2. Example

[0019] This invention provides a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, comprising the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions were 90℃ pre-denaturation for 5 min; 90℃ denaturation for 10 s; 55-69℃ annealing for 60 s; 68℃ extension for 15 s; 35 cycles. S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

[0020] 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

[0021] The fluorescent group 1 is FAM and the fluorescent group 2 is ROX. The quenching group 1 and the quenching group 2 are both NFQ-MGB.

[0022] The mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is 10:3. Example

[0023] This invention provides a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, comprising the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions were 98℃ pre-denaturation for 20 min; 98℃ denaturation for 50 s; 69℃ annealing for 120 s; 72℃ extension for 300 s; 40 cycles; S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

[0024] 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

[0025] In a preferred embodiment, the fluorescent group 1 is TAMRA, and the fluorescent group 2 is HEX. Both the quenching group 1 and the quenching group 2 are BHQ-3.

[0026] In a preferred embodiment, the mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is 11:5. Example

[0027] This invention provides a method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, comprising the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions were 95℃ pre-denaturation for 15 min; 94℃ denaturation for 25 s; 55℃ annealing for 85 s; 69℃ extension for 120 s; 36 cycles. S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

[0028] 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

[0029] The fluorescent group 1 is Cy5, and the fluorescent group 2 is FAM. Both the quenching group 1 and the quenching group 2 are BHQ-3.

[0030] The mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is 13:6.

[0031] This protocol validated 15 samples covering the MTHFR gene C677T locus, including wild-type (CC), heterozygous mutant (CT), and homozygous mutant (TT). Both methods were used simultaneously: first-generation sequencing and the method described in this protocol. The concordance rate between the two methods was 100% (100% accuracy). The specificity for CC or AA genotyping was 100%, for CT genotyping it was 100%, and for TT genotyping it was 100%. See Table 1 below:

[0032] The test results from Examples 1-4 are the same as those from first-generation sequencing, indicating that the detection method presented in this scheme has high accuracy.

[0033] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A method for rapid detection of MTHFR gene polymorphism based on complementary probe technology, used for MTHFRC677T gene polymorphism genotyping, characterized in that, Includes the following steps: S1, Sample Acquisition: Extract genomic DNA from the sample to be tested as an amplification template; S2, Prepare the reaction system: Add the genomic DNA of the sample to be tested, upstream primer, downstream primer, C probe, T probe, uracil-N-glycosylation enzyme, DNA polymerase, deoxyribonucleoside triphosphate, and deoxyuridine triphosphate to the reaction system. S3, PCR amplification reaction: conditions are 90-98℃, pre-denaturation, 5-20 min; 90-98℃, denaturation, 10-50 s; 55-69℃, annealing, 60-120 s; 68-72℃, extension, 15-300 s; 35-40 cycles; S: Genotype determination. After the PCR reaction is completed, the polymorphism of the MTHFR gene in the sample to be tested is determined: wild type: CC; heterozygous mutant: CT; homozygous mutant: TT.

2. The method for rapid detection of MTHFR gene polymorphism based on complementary probe technology according to claim 1, characterized in that, 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'-fluorescent group 2-ATGAAATCGACTCCC-quenching group 2-3'.

3. The method for rapid detection of MTHFR gene polymorphism based on complementary probe technology according to claim 2, characterized in that, Both fluorescent group 1 and fluorescent group 2 are one of FAM, HEX, ROX, TAMRA, and Cy5, and are not used in the same detection.

4. The method for rapid detection of MTHFR gene polymorphism based on complementary probe technology according to claim 2, characterized in that, The quenching group 1 and the quenching group 2 include one of BHQ-1, BHQ-2, BHQ-3, NFQ-MGB, and TAMRA.

5. The method for rapid detection of MTHFR gene polymorphism based on complementary probe technology according to claim 1, characterized in that, The mass ratio of upstream primer to downstream primer is 1:1, the mass ratio of C probe to T probe is 1:1, and the mass ratio of the total amount of upstream primer to downstream primer to the total amount of C probe to T probe is (10-15):(4-6).

6. The method for rapid detection of MTHFR gene polymorphism based on complementary probe technology according to claim 1, characterized in that, PCR amplification reaction: conditions were 92℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s; 60℃ annealing for 75 s; 70℃ extension for 200 s; 35 cycles.

Images