Human MTHFR (methylene tetrahydrofolate reductase) gene polymorphism test kit as well as preparation method and application thereof

A detection kit and gene technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of long experiment time, low sensitivity of PCR-sequencing method, and low detection sensitivity, etc. Achieve the effect of preventing false negative and false positive results, improving specificity and accuracy, and simplifying reaction procedures

Active Publication Date: 2018-12-11
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The PCR-sequencing method has low sensitivity and takes a long time for the experiment, so it is not suitable for clinical promotion; the chip hybridization method has low detection sensitivity and poor specificity, and is prone to false positive results; the high-resolution melting curve method has special requirements for equipment and is not suitable for clinical promotion. Detection sensitivity is not high
The Taqman-qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and it is genotyped by the Genotyping method, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting mutation frequency too low position

Method used

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  • Human MTHFR (methylene tetrahydrofolate reductase) gene polymorphism test kit as well as preparation method and application thereof
  • Human MTHFR (methylene tetrahydrofolate reductase) gene polymorphism test kit as well as preparation method and application thereof
  • Human MTHFR (methylene tetrahydrofolate reductase) gene polymorphism test kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 prepares the MTHFR gene detection kit of the present invention

[0059] 1. Primer design and synthesis:

[0060] MTHFR gene rs1801133 site, MTHFR gene rs1801131 2 sites Each system contains 6 primers; two sense chain upstream primers F1 and F2, two antisense chain downstream primers R1 and R2, F1 and R1 are common primers , F2 and R2 are specific ARMs primers with fluorophore and tag sequence, screened by primers and PCR reaction conditions to ensure that the primer pair F1R1 enriches the template, while F2 and R1, F1 and R2 can normally amplify the band Fluorescent specific genotype PCR products; at the same time, internal reference primers are added to the gene locus.

[0061] The specific sequence is as follows:

[0062] MTHFR 677-F1: 5'-CTCTCCTGACTGTCATCCCTAT-3' SEQ ID NO.1

[0063] MTHFR 677-F2: 5'-FAM-AAGATACATTGATGAGGGAG T CG-3' SEQ ID NO.2

[0064] (Specific recognition of mutation template)

[0065] MTHFR 677-R1: 5'-GCGGAAGAATGTGTCAGCCT-3' SE...

Embodiment 2

[0101] The human MTHFR gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested. In this example, 100 cases of EDTA anticoagulated venous whole blood samples were collected, genomic DNA was extracted, and human MTHFR gene polymorphism detection kit was used to detect MTHFR 677 and MTHFR 1298 gene polymorphisms. The specific operation process was as follows:

[0102] (1) Genomic DNA extraction from blood samples: Use a commercial extraction kit to extract genomic DNA. After the extraction is complete, use TE buffer to elute the DNA and measure the DNA concentration; dilute the genomic DNA to 20ng / μl;

[0103] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 5 minutes for pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C ...

Embodiment 3

[0117] Use the kit prepared in Example 1 to detect and verify the minimum detection line of this kit, specifically including the following steps:

[0118] (1) Using samples of the known MTHFR gene rs1801133 locus and rs1801131 corresponding locus genotype in Example 2, each locus is selected from each of the wild-type genome, mutant genome and heterozygous genome samples, and the above-mentioned samples Dilute to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.001ng / μL;

[0119] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, collect fluorescence; each concentration gradient of each sample is repeated 20 times;

[0120] (3) After the PCR reaction is completed,...

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Abstract

The invention belongs to the biotechnology field, and particularly relates to a human MTHFR (methylene tetrahydrofolate reductase) gene polymorphism test kit as well as preparation method and application thereof. The kit comprises a PCR (polymerase chain reaction) premix reaction solution, a positive control and a negative control for respectively amplifying MTHFR gene rs1801133 and MTHFR gene rs1801131 on two sites, wherein the PCR premix reaction solution contains a specific primer sequence set, a probe set and a PCR solution for amplifying each site, and the specific primer set comprises acommon external primer and a fluorescent-tagged specific ARMs (amplification refractory mutation system) primer. The kit disclosed by the invention is used for testing human MTHFR gene polymorphism, has the advantages including high sensitivity, high specificity, simple operation and reliable results, and can complete a test within 1 hour, and the result interpretation is simple and objective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human MTHFR gene polymorphism detection kit and its preparation method and application. Background technique [0002] Human methylenetetrahydrofolate reductase (MTHFR, 5,10-methylenetetrahydrofolatereductase, methylenetetrahydrofolate reductase) gene is located on chromosome 1p36.3, with 12 exons in total, encoding 656 amino acid residues MTHFR is an important enzyme involved in the metabolism of folic acid cycle, which is related to DNA synthesis, methylation, etc., and its main function is to convert 5,10-methylene tetrahydrofolate into 5-methyl folate with biological functions Tetrahydrofolate. Folic acid is a B vitamin, which is an indispensable component of cell division and synthesis of DNA. The clinical function of folic acid is not only to prevent fetal neural tube defects, but also to reduce pregnancy-induced hypertension, spontaneous abortion, fetal intrauter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2535/137C12Q2537/143C12Q2531/113
Inventor 付金玲王宁李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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