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Folic acid metabolism gene polymorphism detecting primer and kit

A folic acid metabolism gene and polymorphism detection technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of elevated homocysteine ​​levels, changes in enzyme activity, folic acid Insufficient and other problems, to achieve the effect of simple and convenient operation, high accuracy, and prevention of product pollution

Active Publication Date: 2019-01-11
江门市妇幼保健院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in this gene site can cause changes in the activity of the enzyme, resulting in folate deficiency or increased homocysteine ​​levels in the body

Method used

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  • Folic acid metabolism gene polymorphism detecting primer and kit
  • Folic acid metabolism gene polymorphism detecting primer and kit
  • Folic acid metabolism gene polymorphism detecting primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] An embodiment of the polymorphism detection kit of the folic acid metabolism gene of the present invention includes the following primers:

[0060] (1) Detection primers for the polymorphic site of the MTHFR gene (rs1801133, C677T): the upstream primer shown in SEQ ID No.1, the downstream primer shown in SEQ ID No.2, and the downstream primer shown in SEQ ID No.3 the probe;

[0061] (2) Detection primers for polymorphic sites of the MTHFR gene (rs1801131, A1298C): upstream primers shown in SEQ ID No.4, downstream primers shown in SEQ ID No.5 and SEQ ID No.6 the probe;

[0062] (3) Detection primers for polymorphic sites of MTRR gene (rs1801394, A66G): upstream primers shown in SEQ ID No.7, downstream primers shown in SEQ ID No.8 and SEQ ID No.9 the probe;

[0063] The fluorescent reporter group connected to the 5' end of the probe shown in SEQ ID No.3 is ROX, and the fluorescent quencher group connected to the 3' end is BHQ2;

[0064] The fluorescent reporter group ...

Embodiment 2

[0077] In this example, the kit described in Example 1 is used to detect the polymorphism of the folic acid metabolism gene.

[0078] 1. Reaction system configuration

[0079] Configure the reaction system according to the components and component final concentrations shown in Table 1:

[0080] Table 1 reaction system

[0081]

[0082]

[0083] The DNA template is DNA extracted from peripheral blood cells of the patient.

[0084] 2. Reaction procedure

[0085] The PCR reaction program is shown in Table 2:

[0086] Table 2 Reaction program

[0087]

[0088]3. Judgment criteria for results

[0089] The kit of the present invention can complete MTHFR gene (rs1801133, C677T) polymorphic sites, MTHFR gene (rs1801131, A1298C) polymorphic sites and MTRR gene (rs1801394, A66G) polymorphic sites in a single-tube PCR system simultaneously Click the genotyping test of the three loci, and the test result judgment criteria are as follows:

[0090] (1) Wild-type polymorphic ...

Embodiment 3

[0111] In this example, polymorphisms of folic acid metabolism genes in 10 clinical samples were detected. Take the peripheral blood sample of the subject to be tested, extract the DNA, use the kit described in Example 1, and perform detection according to the detection method described in Example 2, and at the same time perform the first-generation sequencing on the sample. The test results are shown in Table 3:

[0112] Table 3 Test results

[0113]

[0114]

[0115] The above results show that the kit of the present invention has good accuracy when used for detecting polymorphisms of folic acid metabolism genes, and the detection results are completely consistent with the sequencing results.

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Abstract

The invention discloses a folic acid metabolism gene polymorphism detecting primer, comprising (1) detection primers for polymorphic sites of MTHFR gene (rs1801133, C677T): an upstream primer as shownin SEQ ID No. 1, a downstream primer as shown in SEQ ID No. 2 and a probe as shown in SEQ ID No. 3; (2) detection primers for polymorphic sites of MTHFR gene (rs1801131, a1298C): upstream primers asshown in SEQ ID No. 4, downstream primers as shown in SEQ ID No. 5 and probes as shown in SEQ ID No. 6; (3) detection primers for polymorphic sites of MTRR gene (rs1801394, a66G): upstream primers asshown in SEQ ID No. 7, downstream primers as shown in SEQ ID No. 8 and probes as shown in SEQ ID No. 9. The invention also discloses the use of the detecting primer in preparing a folic acid metabolism gene polymorphism detecting reagent and a folic acid metabolism gene polymorphism detecting reagent kit. The detecting primer provided by the invention can simultaneously complete the genotyping ofthree loci in a single-tube PCR system, and has the advantages of simple and convenient operation, strong specificity, high sensitivity, high accuracy and easy interpretation.

Description

technical field [0001] The invention belongs to the technical field of gene mutation detection, and in particular relates to a detection primer and a kit for folic acid metabolism gene polymorphism. Background technique [0002] Folic acid is composed of three components: pteridine, p-aminobenzoic acid and glutamic acid. It is a necessary substance for the human body to utilize sugar and amino acids, and is a necessary substance for the growth and reproduction of body cells. As the coenzyme of the one-carbon unit transferase system in the biochemical reaction in vivo, it acts as a one-carbon unit transfer body. After FL carrying one carbon unit enters the folic acid cycle, it immediately participates in the transfer and conversion of one carbon unit in the molecule. 5-formyltetrahydrofolate and 10-formyltetrahydrofolate are converted to 5,10-methyltetrahydrofolate, which is then reduced to 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate reductase reduces 5,10-met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2537/143C12Q2527/107
Inventor 唐佳曾钦龙徐进美谭洁亮
Owner 江门市妇幼保健院
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