Composition for detecting polymorphism of MTHFR and MTRR genes and application of composition

A gene polymorphism, MTHFR677-TFP technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve problems such as low accuracy and specificity, increase probes, etc., to achieve results Accurate, avoid cross-contamination, accurate and effective detection effect

Active Publication Date: 2017-11-24
北京鑫诺美迪基因检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And on this basis, the probe is modified by locking nucleic acid and secondary structure, increasing the Tm value of the probe, and further improving the specificity of the probe binding. Finally, through the adjustment of the above system, the accuracy and specificity are evenly balanced. It can reach 100%, and the minimum detection concentration can reach 1ng / μL, which solves the problem of low accuracy and specificity in the detection of MTHFR and MTRR gene polymorphisms in the past

Method used

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  • Composition for detecting polymorphism of MTHFR and MTRR genes and application of composition
  • Composition for detecting polymorphism of MTHFR and MTRR genes and application of composition
  • Composition for detecting polymorphism of MTHFR and MTRR genes and application of composition

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Embodiment 1

[0085] Embodiment 1: On the basis of multiplex fluorescent PCR, the present invention is formulated into a premixed primer composition for detection sites. The extracted sample can be tested directly, which makes the detection step easier.

[0086] Specific steps are as follows:

[0087] 1) Genomic DNA was extracted from the whole blood sample (using a commercial peripheral blood genomic DNA extraction kit).

[0088] 2) Measure the concentration of the extracted genomic DNA, and dilute the concentration to 10-20 ng / μL for subsequent PCR reaction process.

[0089] 3) Composition of the reaction system: dNTPs, Mg 2+ , DNA polymerase buffer, reverse transcription buffer, gene primer pair, DNA polymerase, etc.

[0090] 4) PCR reaction conditions: UDG enzyme reaction at 37°C for 2min; pre-denaturation at 95°C for 3min; denaturation at 94°C for 15s, annealing and extension at 60°C for 35s, 40 cycles.

[0091] 5) Preparation of PCR reaction system:

[0092] PCR amplification MIX...

specific Embodiment approach 2

[0116]Collect 200 peripheral blood control samples from Wuhan Tongji Hospital, and use the above-mentioned finished kit for detection. The specific operation steps are as described in Example 1.

[0117]

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Abstract

The invention discloses a composition for detecting polymorphism of MTHFR and MTRR gene and an application of the composition. Single nucleotide polymorphism sites in MTHFR and MTRR genes are detected through multi-PCR reaction of FAM, HEX and ROX (modified) channels. In order to improve the detection simplicity and specificity, one-tube typing detection is achieved through technical means of probe typing, and the whole operation and reaction processes are simplified; locked nucleic acid modification and secondary structure modification are carried out on a probe on the basis, the Tm value of the probe is increased, the probe combining specificity is further improved and finally the lowest detection limit, accuracy and specificity of the whole kit are improved through adjusting the ratio of a primer pair, the dosage of Taq enzyme and the dosage of magnesium ions; and the minimum detection concentration of the kit can reach 1ng / muL and the accuracy and the specificity can reach 100%.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a technology for detecting MTHFR and MTRR gene polymorphisms. Background technique [0002] Folic acid is a water-soluble B vitamin that was originally extracted from spinach leaves, so it is called folic acid. Folic acid is an essential element for the synthesis of nucleic acid, an essential substance for cell growth and tissue repair, and an indispensable nutrient for embryonic development. Numerous studies in recent years have confirmed that folic acid deficiency is the main cause of birth defects. In addition to preventing fetal neural tube defects, the clinical function of folic acid can also reduce the incidence of pregnancy-induced hypertension, spontaneous abortion, intrauterine growth retardation, premature delivery, and low birth weight in newborns. [0003] There are two reasons for the lack of folic acid in the body: one is insufficient folic acid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 刘沛王林海
Owner 北京鑫诺美迪基因检测技术有限公司
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