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Human MTHFR gene polymorphism detection kit

A detection kit and kit technology, applied in the biological field, can solve the problems of long operation time, misjudgment of results, long detection period, etc., and achieve the effect of stable typing detection.

Pending Publication Date: 2019-09-24
江苏正大天创生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the high-resolution melting curve method has special requirements for equipment, there are certain difficulties in clinical promotion; PCR-PFLP technology relies on restriction endonuclease digestion, electrophoresis analysis and other reasons, often resulting in misjudgment of results, affecting its detection accuracy In addition, its detection cycle is long and the throughput is low, and it is not suitable for rapid screening of a large number of people.
As the gold standard of genetic testing, DNA direct sequencing method is difficult to achieve large-scale promotion due to its low cost, long operation time and low sensitivity.
DNA chip technology has been widely used in SNP detection due to the advantages of high throughput, simple and fast operation, etc., but this technology is expensive, complicated, poor in repeatability, and low in sensitivity, which is not conducive to large-scale promotion

Method used

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  • Human MTHFR gene polymorphism detection kit
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  • Human MTHFR gene polymorphism detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] 1. Primer and probe synthesis:

[0083] Design and synthesize 2 sets of specific primers SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6; 2 sets of specific probes SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQ ID NO:10, and labeled with FAM, HEX, Cy3 and Cy5 fluorescent groups at the 5' end, and NFQ-MGB labeled at the 3' end does not emit light Quenching group; 2 enrichment primers SEQ ID NO:14, SEQ ID NO:15. The primers and probes were prepared into 100 μM stock solutions for storage.

[0084] 1. Preparation of internal standard system: design and synthesize a pair of internal standard primers designed for the human genome, the primer pair sequences are SEQ ID NO: 11 and SEQ ID NO: 12; design and synthesize internal standard probes, the probes is SEQ ID NO:13. The primers and probes were prepared into 100 μM stock solutions for storage.

[0085] 2. Prepare other reagents: prepare PCR buffer, which contains 1.0mM MgCl2, dATP, dUTP, dGTP and...

Embodiment 2

[0092] Use the human MTHFR gene polymorphism detection kit prepared in Example 1 to detect the sample to be treated.

[0093] 1. Genomic DNA extraction from blood samples

[0094]Use the blood genome column extraction kit to extract the human blood genome according to the instructions. Use an ultraviolet spectrophotometer to detect the concentration of the obtained sample DNA solution, then dilute the sample DNA to 10ng / μl, take 2μl-5μl respectively and add them to the kit prepared in Example 1 and carry out the next step of PCR reaction.

[0095] 2. Take 2 μl of the DNA samples diluted in step 1 and add them to the reaction system of the kit in Example 1 of 23 μl in turn, and put them into a fluorescent quantitative PCR instrument, set the PCR reaction program as shown below and perform amplification reaction:

[0096] 50°C for 2 minutes, 95°C for 10 minutes;

[0097] 95℃ for 15s, 48℃~53℃ for 1min, 5 cycles;

[0098] 95°C for 15s, 60°C-62°C for 1min, 40 cycles; after each...

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Abstract

The present invention relates to a method for detecting gene polymorphism using labeled probes and specific primer sequences, and a kit comprising the specific probes and the specific primer sequences, and is suitable for use in the fields of biotechnology and medicines. The kit provides the specific primer sequences and the labeled probes for simultaneous detection of human MTHFR gene C677T site and A1298C site polymorphisms, and the kit contains a Taq enzyme, the specific primers, the specific probes and an internal standard system. The provided primers and kit are used for simultaneously detecting the MTHFR gene C677T site and A1298C site polymorphisms, and have advantages of strong specificity, high sensitivity, simple and rapid operation, high throughput, safety, objective result interpretation, etc.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polymorphism detection kit of C677T site and A1298C site of human MTHFR gene and its preparation method and application. Background technique [0002] Folic acid is a water-soluble B vitamin (vitamin B9). It does not exist in nature and has no biological activity, but it is a precursor of biologically active folate (folate), and its active form in the body is 5 -Methyltetrahydrofolate, which can transfer a one-carbon group (methyl or formyl) to deoxyuridine acid to make it into deoxythymidylate, and then synthesize DNA. It is an essential substance for tissue repair and an indispensable nutrient for embryonic development. In recent years, a large number of studies have confirmed that folic acid is an indispensable nutrient for fetal growth and development, and can help prevent neural tube defects, including very serious birth defects such as spina bifida and anencephaly. In addit...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2537/143C12Q2561/101C12Q2531/113C12Q2545/101C12Q2547/101C12Q2535/137C12Q2521/531C12Q2563/107
Inventor 魏赵延李思慧徐倩曹丹枫
Owner 江苏正大天创生物工程有限公司
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