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Kit and method for detecting MTHFR (methylene tetrahydrofolate reductase) and MTRR (methionine synthase reductase) gene polymorphism simultaneously with molecular beacon probes and melting curve

A technology of molecular beacon probes and gene polymorphism, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc. Complicated problems, to achieve stable polymorphism detection, reduce false positives, and high sensitivity

Inactive Publication Date: 2018-03-23
沈阳迪安医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is complicated to operate, and it is easy to cause cross-contamination of PCR products when there are many samples, and it is easy to cause false negative or false positive results due to insufficient or excessive enzyme digestion, and the reliability is low

Method used

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  • Kit and method for detecting MTHFR (methylene tetrahydrofolate reductase) and MTRR (methionine synthase reductase) gene polymorphism simultaneously with molecular beacon probes and melting curve
  • Kit and method for detecting MTHFR (methylene tetrahydrofolate reductase) and MTRR (methionine synthase reductase) gene polymorphism simultaneously with molecular beacon probes and melting curve
  • Kit and method for detecting MTHFR (methylene tetrahydrofolate reductase) and MTRR (methionine synthase reductase) gene polymorphism simultaneously with molecular beacon probes and melting curve

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Prepare MTHFR and MTRR gene polymorphism detection kit of the present invention, comprise the following steps:

[0065] 1. Primer and probe synthesis:

[0066] Design and synthesize 3 pairs of specific primers SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:8; 3 molecular beacon probes SEQ ID NO:3, SEQ ID NO:6; SEQ ID NO:9, and label the CY5 fluorescent group at the 5' end of SEQ ID NO:3, and label the DABSYL non-luminescence quenching group at the 3' end, SEQ ID NO: The 5' end of 6 is labeled with a HEX fluorescent group, the 3' end is labeled with a DABSYL non-luminescent quencher, the 5' end of SEQ ID NO: 9 is labeled with a FAM fluorescent group, and the 3' end is labeled with a DABSYL non-luminescent quencher. The primers and probes were prepared into 100 μM mother solution for storage.

[0067] 2. Prepare a positive control, which contains 6 kinds of plasmid DNAs, which respectively contain 6 different polymorphisms of MTHFR 677C, MTHF...

Embodiment 2

[0072] The human MTHFR and MTRR gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested.

[0073] In this embodiment, 20 cases of anticoagulated whole blood samples of folic acid metabolism disorders were collected, and genomic DNA was extracted therefrom, and the MTHFR and MTRR of the samples to be tested were detected with the human MTHFR and MTRR gene polymorphism detection kit obtained in Example 1. genetic polymorphism.

[0074] 1: Extraction of genomic DNA from human whole blood (self-prepared reagents)

[0075] 1) Take 200 μl of whole blood, add 20 μL of Proteinase K (20 mg / ml) solution, mix well, then add 300 μl of lysate BL, shake and mix well, and bathe in water at 56°C for 10 minutes.

[0076] 2) Add 260 μl of absolute ethanol, and mix well by inverting, at this time flocculent precipitation may appear.

[0077] 3) Add the solution and flocculent precipitate obtained in the previous step into an adsorption column (the adso...

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Abstract

The invention relates to an application of molecular beacon probes and a melting curve technology in detection of gene polymorphism, in particular to a method for detecting MTHFR (methylene tetrahydrofolate reductase) and MTRR (methionine synthase reductase) gene polymorphism simultaneously, and further relates to amplification primers of MTHFR and MTRR genes, the molecular beacon probes and a kitcontaining the amplification primers and the molecular beacon probes. The method is simple and fast to operate, good in specificity, high in sensitivity, accuracy and flux and low in detection cost and has broad application prospect. According to the method, PCR amplification and detection are performed synchronously, the overall detection process does not require uncovering operation, so that detection period is shortened and detection efficiency is improved while detection cost is saved, and besides, risk of false positive caused by PCR product pollution is reduced.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a kit and a method for simultaneously detecting MTHFR and MTRR gene polymorphisms by using a molecular beacon probe melting curve method. Background technique [0002] Folic acid is one of the B vitamins. It participates in the synthesis of DNA, RNA, protein and other important compounds. It plays an important role in the body's metabolic process. It is often used in nutritional supplements during pregnancy to prevent homocysteinemia and neural tube disease. Defects and other diseases. At present, it has been confirmed that the genes closely related to folic acid metabolism are methylenetetrahydrofolate reductase (MTHFR) and methionine synthesis reductase (MTRR) genes. MTHFR and MTRR are the key enzymes in the folic acid metabolism system. Changes in their gene structure can lead to a decrease in the activity of key enzymes in folic acid metabolism and affect ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/16C12Q2600/156C12Q2531/107C12Q2537/143C12Q2563/107
Inventor 任绪义罗英张锋
Owner 沈阳迪安医学检验所有限公司
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