Method for detecting polymorphism of MTHFR gene through fluorescent quantitative PCR

A technology of gene polymorphism and fluorescence quantification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of amplification product contamination, cumbersome operation steps, and long detection cycle. Achieve the effects of improving detection efficiency, simple operation, and improving specificity

Inactive Publication Date: 2020-03-13
昆明和合医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the most commonly used sequencing method can directly detect the position and type of the mutation site, but this method has cumbersome operation steps, long detection cycle, low sensitivity, and the amplification product is prone to contamination

Method used

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  • Method for detecting polymorphism of MTHFR gene through fluorescent quantitative PCR
  • Method for detecting polymorphism of MTHFR gene through fluorescent quantitative PCR
  • Method for detecting polymorphism of MTHFR gene through fluorescent quantitative PCR

Examples

Experimental program
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Effect test

Embodiment 1

[0031] A method for preparing a DNA sample to be tested in a method for detecting MTHFR gene polymorphisms, comprising the steps of: extracting genomic DNA with a blood / cell / tissue genomic DNA extraction kit (DP304), and using NP80-touch (Germany IMPLEN) Determine the concentration and purity of the DNA, and then save the genomic DNA.

Embodiment 2

[0033] 1. Primer and Probe Design

[0034] The present invention designs specific primers and specific fluorescent probes respectively according to the base sequences of c.677C>T and c.1298A>C sites of the MTHFR gene, and the specific primers are designed at both ends of the SNP sites by Primer Premier 5.0 Primers, select the most suitable primers through software analysis; specific probes are located in the region between a pair of primers, where the Tm value should be between 60-70°C, usually 5-10°C higher than the primers. The 5' end of the probe for detecting the wild type is labeled with a fluorescent reporter group (FAM), the 5' end of the probe for detecting the mutant type is labeled with a fluorescent reporter group (VIC), and the 3' end is labeled with a non-fluorescent quencher group ( NFQ) label, and the MGB modification group is also linked to the probe; in actual operation, the fluorescent reporter group can be replaced according to the actual situation, as long ...

Embodiment 3

[0050] Embodiment 3: the configuration of kit

[0051] According to the above experimental results, the present embodiment provides a preferred kit for the detection of MTHFR gene polymorphisms by fluorescent quantitative PCR, which includes the following reagents:

[0052] 1. 2×TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (including dUTP);

[0053] 2. Probe Mix: two sets of fluorescent probes, the final concentration is 0.6μM;

[0054] 3. Primer Mix: two sets of primer pairs, F upstream primer and R downstream primer have a final concentration of 0.6 μM;

[0055] 4. Sample DNA extraction reagents;

[0056] 5. Sample collection storage box (such as containing oral swabs and swab storage boxes / tubes);

[0057] 6. Ultrapure water.

[0058] The reagents are properly placed in the kit, and then put into the instructions for use of the kit (optionally reconfigure PCR test tubes or orifice plates or pipette guns). The instructions for use include the col...

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Abstract

The invention provides a primer probe combination for detecting the polymorphism of an MTHFR gene through fluorescent quantitative PCR. The primer probe combination comprises two groups of primers anddouble probes as shown in a sequence table; meanwhile, the invention provides a method for detecting the polymorphism of the MTHFR gene through fluorescent quantitative PCR. According to the invention, the polymorphism of the MTHFR gene is detected by adopting a real-time fluorescent quantitative PCR Taqman-MGB probe method, and a wild type and mutant type double-probe detection method is adopted, so specificity is improved, and cost is reduced; and a Taqman-MGB probe is superior to an ordinary probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of experimental results. Thus, the MTHFR gene polymorphism method established by the invention has the advantages of accurate result, high specific sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a combination of primers and probes and a detection method for detecting MTHFR gene polymorphisms by fluorescent quantitative PCR. Background technique [0002] Folic acid is an important nutrient required by the human body and belongs to the B vitamins. Folic acid is reduced to tetrahydrofolate with physiological activity in the body. As a donor of one-carbon units, it is mainly involved in the repair of DNA oxidative damage, cell proliferation and tissue growth. In the folic acid metabolic pathway, a variety of enzymes are involved in the transport and metabolism of folic acid, among which 5,10-methylenetetrahydrofolate reductase (methionine synthase reductase, MTHFR), methionine synthase reductase (methylenetetrahydrofolate Reductase, MTRR) two enzymes are the most critical. These enzyme genes have some single nucleotide polymorphisms (SNPs) in the natural population, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 任静静智慧芳倪君君
Owner 昆明和合医学检验所有限公司
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