Full premix MTHFR and MTRR multiple PCR gene polymorphism detection kit and method thereof

A gene polymorphism and polymorphism technology, applied in the field of biomedical clinical molecular detection, can solve the problems of cumbersome data analysis, high price, low efficiency, etc., and achieve the effect of wide promotion value, simple operation and fast response

Inactive Publication Date: 2018-03-27
SUZHOU KUANGYUAN MOLECULAR BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention aims at the deficiencies such as high price, long period, low efficiency, cumbersome data analysis and the like when detecting MTHFR and MTRR gene polymorphisms in the prior art, and provides a fully premixed folic acid metabolic pathway MTHFR and MTHFR based on a real-time fluorescent quantitative PCR technology platform. MTRR multiple primer-specific PCR detection kit and method

Method used

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  • Full premix MTHFR and MTRR multiple PCR gene polymorphism detection kit and method thereof
  • Full premix MTHFR and MTRR multiple PCR gene polymorphism detection kit and method thereof
  • Full premix MTHFR and MTRR multiple PCR gene polymorphism detection kit and method thereof

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Effect test

Embodiment 1

[0125] Example 1. Design of primers and probes:

[0126] According to the reference sequence (NG_013351.1) of the MTHFR gene published by GeneBank, the nucleic acid sequence database of the National Center for Biotechnology Information NCBI, and the c.677 site (C / T) polymorphism (SNP ID: rs1801133) published by the dbSNP database and c.1298 site (A / C) polymorphism (SNP ID: rs1801131), reference sequence of MTRR gene (NG_008856.1) and c.66 site (A / G) polymorphism published in dbSNP database (SNP ID: rs1801394), ABI's Primer Express 3.0 software was used to design and detect the c.677 site (C / T) polymorphism and c.1298 site (A / C) polymorphism of the MTHFR gene and the MTRR gene c. Primers and probes for polymorphism at position 66 (A / G). In order to monitor the effectiveness of the reaction system, internal control primers and probes are added to the detection system. The present invention selects a sequence of the human conserved gene GAPDH (its GeneBank reference sequence num...

Embodiment 2

[0157] Example 2. Preparation of primers

[0158] The designed primers and probe sequences are handed over to the synthesis company for synthesis, generally using automatic chemical synthesis with instruments, and a synthesis inspection report is required.

Embodiment 3

[0159] Example 3: Preparation of the complete premixed folic acid metabolic pathway MTHFR and MTRR multiple primer-specific PCR gene polymorphism detection kit

[0160] Preparation of specific reaction solutions for detecting the c.677 site C allele of the human MTHFR gene, the c.1298 site A allele of the human MTHFR gene and the c.66 site A allele of the human MTRR gene: MTHFR and MTRR A PCR reaction solution; specific reaction solution for detection of human MTHFR gene c.677 site T allele, human MTHFR gene c.1298 site C allele and human MTRR gene c.66 site G allele: MTHFR and MTRR B PCR reaction solution; wherein the PCR reaction solution contains not only the buffer, magnesium ions, dNTP and other substances necessary for the PCR reaction, but also includes detection primers and probes, internal control primers and probes, and antibody-modified TaqDNA polymerase and PCR protectant trehalose. The concentration of the PCR reaction solution components and the sequence informa...

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Abstract

The invention provides a complete premixed multiple primer-specific PCR detection kit and method for detecting nucleotide polymorphisms at three sites of c.677 and c.1298 of the folic acid metabolic pathway and c.1298 of the MTRR gene. "Allele-specific PCR" principle, design multiple primer-specific PCR, realize two-tube reaction on the real-time fluorescent quantitative PCR technology platform, and simultaneously detect three folic acid metabolic pathway MTHFR genes c.677 and c.1298 and MTRR gene c.66 The nucleotide type of the site. In addition, modified Taq DNA polymerase and disaccharide PCR protective agent are added to the reaction system to increase the stability of the reaction system and realize the long-term stability of the pre-mixed detection system.

Description

technical field [0001] The invention belongs to the field of biomedical clinical molecular detection, and specifically relates to the specific PCR detection of full premixed multiple primers for detection of nucleotide polymorphisms at three sites of MTHFR gene c.677 and c.1298 and MTRR gene c.66 Kits and assay methods. Background technique [0002] Folic acid (Folic Acid) refers to a class of water-soluble B vitamins with related biological activities. Its active form in the body is 5-methyltetrahydrofolate, which is an essential substance for cell growth and reproduction, and participates in many important metabolic reactions. As a carrier of one-carbon units in the body, folic acid participates in the synthesis and transformation of purines, thymine, nucleotides and other substances, and at the same time affects the production of phospholipids, creatine, and nerve mediators, and promotes the development of nerve cells and brain cells. Folic acid and its metabolic inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2563/107C12Q2535/137
Inventor 王进吴梦华赵小龙何晓辉吴子侠
Owner SUZHOU KUANGYUAN MOLECULAR BIOTECH
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