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Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene

A fluorescent probe and primer pair technology, which is applied in the fields of probes, primer pairs, and fluorescent PCR kits, can solve the problems of increased cost and complicated detection procedures, and achieve the effects of short cycle, low detection cost, and low price

Inactive Publication Date: 2016-02-03
智海生物工程(北京)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method does not need to open the tube and is easy to interpret. However, the above method needs to design two different FRET probes for detection, the detection procedure is complicated, and the cost is increased.

Method used

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  • Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene
  • Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene
  • Primer pair, fluorescence probe and kit for detecting polymorphism of MTHFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Detection of C677T polymorphism of methylenetetrahydrofolate reductase gene

[0069] 1. DNA extraction

[0070] DNA was extracted from human whole blood samples using the Chelex-100 method. The specific steps are as follows:

[0071] A. Take 10 μL of whole blood and add it to a 2.0 mL centrifuge tube, add 500 μL of sterilized double-distilled water to it, shake vigorously, let it stand at room temperature for 15 minutes, centrifuge at 13,000 rpm for 3 minutes, discard the supernatant, and collect the precipitate (use double-distilled water repeatedly if necessary) Wash the precipitate until it is colorless or has little hemoglobin);

[0072] B. Add 100 μL of nucleic acid extraction solution (contains particles, shake well before each suction to ensure that the particles are sucked out together) into the precipitation, shake repeatedly on the shaker, and keep warm at 56°C for more than 30 minutes;

[0073] C. Fully shake after taking out, keep warm at 100°C ...

Embodiment 2

[0092] Example 2. Detection of C677T polymorphism of methylenetetrahydrofolate reductase gene

[0093] 1. DNA extraction

[0094] DNA extraction was the same as Example 1 of the present invention.

[0095] 2. Design of primers and fluorescent probes

[0096] According to the MTHFRC677T polymorphism (rs1801133) information published in the dbSNP database of the National Center for Biotechnology Information NCBI, the Primer5 software was used to design primers and probes, as follows:

[0097] Forward primer SEQ ID NO: 3: 5'-GAAGCACTTGAAGGAGAA-3',

[0098] Reverse primer SEQ ID NO: 4: 5'-GTGTCAGCCTCAAAAGAAA-3'.

[0099] Fluorescent probe SEQ ID NO: 7:

[0100] 5'-FAM-TGTCTGCGGGAGCCGATTTCATCATC-BHQ-3';

[0101] Primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. After probe quantification, store at -20°C in the dark. In this embodiment, the fluorescent probe can also be selected as the probe shown in SEQ ID NO: 8 in the sequenc...

Embodiment 3

[0107] Example 3 Detection and Verification of C677T Polymorphism of Methylenetetrahydrofolate Reductase Gene

[0108] Using the primer pair and fluorescent probe of Example 1 of the present invention to detect 477 gene samples for verification test, all samples were sequenced and verified by the gold standard sequencing method. 148 cases of 677TT homozygous mutant genotype, 92 cases of 677CC homozygous wild genotype, and 237 cases of 677CT heterozygous mutant genotype were detected. The kit of the present invention detected 477 cases of gene samples compared with the gold standard sequencing results, and the detection results were consistent. 0 cases were detected, and the accuracy was 100%; for other genotype positive and negative samples not within the detection range of this kit, the detection results of this kit were all negative, the sensitivity was 100%, and the specificity was 100%.

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Abstract

The invention provides a primer pair, specific oligonucleotide fluorescence probe and kit for detecting the nucleotide polymorphism of a C677T locus of an MTHFR gene. The new specific primer pair and the corresponding fluorescence probe are designed, PCR amplification is performed by adopting an asymmetric PCR technology, melting curve analysis is performed after PCR amplification is finished, and a genetype is judged through the melting peak at specific temperature. According to the kit, the MTHFR gene can be amplified in a highly specific mode, detection of three kinds of genetypes of the C677T locus in a single-pipe PCR system can be completed, the detection specificity is high, the result is easy to judge and read, the operating steps are simple, the detection cost is low, the cycle is short, and the efficiency is high.

Description

technical field [0001] The invention relates to the field of single nucleotide polymorphism detection, in particular to a primer pair set, a probe and a fluorescent PCR kit for detection of the nucleotide polymorphism at the C677T site of the MTHFR gene. Background technique [0002] 5,10-methylenetetrahydrofolate-reductase (MTHFR) in human genomic DNA is one of the key enzymes in the metabolic pathway of folic acid and methionine in the human body, which can convert 5,10-methylene 4-methyltetrahydrofolate is reduced to 5-methyltetrahydrofolate. On the one hand, it participates in the synthesis of purines and pyrimidines and the methylation of DNA, RNA, and proteins as a methyl carrier; on the other hand, under the catalysis of methionine synthase, vitamin B12 is used as a coenzyme to make the The remethylation of homocysteine ​​to methionine maintains normal homocysteine ​​levels in the body. [0003] Studies have found that the C677T polymorphism of the MTHFR gene can le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2563/107C12Q2561/113
Inventor 杨海艳冯东沈东艳
Owner 智海生物工程(北京)股份有限公司
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