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MTHFR and MTRR gene polymorphism detection primer, probe, kit and application

A technology for gene polymorphism and detection primers, which is applied in the field of molecular biology, can solve the problems of inability to accurately type polymorphisms in samples, nucleic acid loss, and high equipment requirements, so as to avoid the risk of template cross-contamination and inhibit nuclease activity , the effect of high sensitivity

Inactive Publication Date: 2019-11-08
北京协和洛克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the DNA sequencing method is the gold standard for mutation detection, but this method has disadvantages such as complicated operation, long detection cycle, easy pollution, and high cost; the gene chip method is difficult to prepare chips, cumbersome to operate, and expensive to detect; the HRM method requires high equipment requirements , special analysis software needs to be configured, which limits the popularization of this method; the traditional fluorescent quantitative PCR method is widely used in clinical practice, but this method generally uses the Genotyping method for genotyping, which has certain requirements for the number of samples and the distribution of polymorphisms. When the sample size is small, the polymorphism of the sample cannot be accurately typed
In addition, the template DNA used in the above detection methods needs to go through cumbersome extraction and purification steps, which is not only time-consuming and costly, but also easily leads to nucleic acid loss and cross-contamination between samples

Method used

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  • MTHFR and MTRR gene polymorphism detection primer, probe, kit and application
  • MTHFR and MTRR gene polymorphism detection primer, probe, kit and application
  • MTHFR and MTRR gene polymorphism detection primer, probe, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The invention provides a detection kit for MTHFR and MTRR gene polymorphisms, the kit includes the following components: nucleic acid release agent, 2 × PCR reaction mixture (comprising hot start Taq DNA polymerase, UDG enzyme), 3 Group specific primers, 3 group specific probes and internal standard system.

[0089] The 3 groups of specific primers are MTHFR C677T primers, MTHFR A1298C primers and MTRR A66G primers, and the specific primer sequences are listed as follows:

[0090] MTHFR C677T upstream primer 5′-CTTCACAAAGCGGAAGAATGTG-3′SEQ ID NO:1

[0091] MTHFR C677T downstream primer 5′-CCTGAAGCACTTGAAGGAGAA-3′SEQ ID NO:2

[0092] MTHFR A1298C upstream primer 5′-TGGTTCTCCCGAGAGGTAAA-3′SEQ ID NO:3

[0093] MTHFR A1298C downstream primer 5′-GGACTACTACCTTCTTCTACCTGAA-3′SEQ ID NO:4

[0094] MTRR A66G upstream primer 5′-GTGATGAGGAGGTTTCTGTTACT-3′SEQ ID NO:5

[0095] MTRR A66G downstream primer 5′-CGGCTCTAACCTTATCGGATTC-3′SEQ ID NO:6

[0096] The 3 groups of specific p...

Embodiment 2

[0130] This example is used to illustrate the compatibility of the kit of the present invention with respect to sample types.

[0131] In this example, taking the homozygous wild type (C / C) at the C677T site of the MTHFR gene as an example, the fingertip blood, EDTA anticoagulated fresh whole blood and EDTA anticoagulated frozen whole blood samples were collected from the same person for fluorescent quantitative PCR experiments .

[0132] The kit used in this example is the same as the primer and probe for detecting the C677T site of the MTHFR gene in Example 1, and other components are also the same, and the detection method used is also the same as in Example 1.

[0133] The result is as Figure 9 As shown, from the homozygous wild-type (C / C) amplification curve at the C677T site of the MTHFR gene, it can be seen that the amplification results of different sample types of the same person basically overlap, and there is no significant difference.

Embodiment 3

[0135] This example is used to illustrate the selection of the sample addition amount of the kit of the present invention.

[0136] In this example, taking the homozygous wild type (C / C) at the C677T site of the MTHFR gene as an example, 2 μl, 5 μl and 10 μl of fingertip blood were respectively taken for fluorescent quantitative PCR experiments.

[0137] The kit used in this example is the same as the primer and probe for detecting the C677T site of the MTHFR gene in Example 1, and other components are also the same, and the detection method used is also the same as in Example 1, the difference is only in the amount of sample added .

[0138] For the homozygous wild-type (C / C) amplification curves of the MTHFR gene C677T locus of samples with different addition amounts, see Figure 10 , the results showed that the amplification curve decreased gradually with the increase of the sample addition amount, and the amplification curve decreased significantly when the sample additio...

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Abstract

The invention provides an MTHFR and MTRR gene polymorphism detection primer and probe combination, which includes an MTHFR C677T, MTHFR A1298C and MTRR A66G gene polymorphism detection primer and probe combination. The invention further provides a detection kit for the MTHFR and MTRR gene polymorphism. The kit is prepared from the following components of a nucleic acid releasing agent, 2*PCR reaction mixture (including a thermally activated Taq DNA polymerase and a UDG enzyme), three sets of specific primers, three sets of specific probes and an internal standard system. The kit only needs tobriefly treat a 2 [mu]l blood sample at the room temperature to produce high-quality DNA for repeated detection, the detection specificity is good, the coincidence rate with a gold standard sequencingmethod is 100%, the sensitivity is high, accurate typing of 0.2ng genomic DNA can be achieved, the detection process is short in consumed time, the process from sample processing to detection resultscan be completed within 1 hour.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer, a probe, a kit for rapid detection of MTHFR and MTRR gene polymorphisms and applications thereof. Background technique [0002] Folic acid is a water-soluble B vitamin, an element necessary for the synthesis of nucleic acid, a substance necessary for cell growth and tissue repair, and an indispensable nutrient for embryonic development. Reduced activity of key enzymes in the folic acid metabolic pathway can lead to hypofolate and hyperhomocysteinemia, which is a major cause of birth defects in newborns, pregnancy-induced hypertension in pregnant women, spontaneous abortion, decreased sperm quality in men, H-type hypertension, One of the important risk factors for atherosclerosis and coronary heart disease. [0003] 5,10-methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are key enzymes that affect folic acid metabolism, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/166C12Q2600/16
Inventor 刘永吴建榕孙淼
Owner 北京协和洛克生物技术有限责任公司
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