Primer pair and kit for detecting folate metabolism-related gene polymorphism in hypertensive patients

Abstract

The invention relates to a primer pair and a kit for detecting folate metabolism-related gene polymorphism in hypertensive patients, and belongs to the technical field of in vitro nucleic acid detection. The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein the 5' end of the reverse amplification primer is labeled with biotin. Thekit comprises a PCR (Polymerase Chain Reaction) solution containing a forward amplification primer and a reverse amplification primer, a sequencing primer, uracil DNA (Deoxyribonucleic Acid) glycosylase, Taq polymerase and a positive control product. The kit provided by the invention has the advantages of accurate detection result, high specificity, short detection period, and simple operation, and the clinical inspection requirements can be effectively met; in addition, the kit also has the advantages of real-time monitoring of reaction progress, and short reaction time, the PCR product canbe used for sequencing on a pyrosequencing instrument after being simply treated, the sensitivity is higher than that of a gold standard method, i.e., a capillary electrophoresis sequencing method, and the kit is more applicable to mutation analysis.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer pair and a kit for detecting gene polymorphisms related to folic acid metabolism in hypertensive patients. Background technique [0002] Folic acid is a compound composed of pterinidine, p-aminobenzoic acid and glutamic acid, and is a water-soluble B vitamin. It exists in many plants and animal tissues, the human body cannot synthesize it, and must be taken from the outside world. [0003] Folic acid is a cofactor for enzymes that play an important role in DNA and RNA synthesis. It provides a one-carbon unit for the de novo synthesis of nucleotides and participates in methyl transport in the methylation cycle of amino acids. Its active unit in the body is 5-methyl base tetrahydrofolate. On the other hand, folic acid is the most important coenzyme in the metabolism of homocysteine ​​(Hcy), and its deficiency will lead to metabolic disorders in th...

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Problems solved by technology

[0009] At present, among the existing publicly reported literature, only the Chinese patent CN201510675462.8 applied by our research team in 2016 has carried out the "gold standard" (PCR-direct sequencing method) for MTHFR genotyping detection in hospitals. However, the technical solution involved in this patent CN201510675462.8, after the two products were put into the clinical market, especially in the process of large-scale application in hospitals, it was found that the specificity of the designed primers and kits was not yet high. It is extremely high, and the accuracy of the test results will occasionally fluctuate. It is speculated that there may be an adaptability problem between the design of the primers and the environment of the actual molecular diagnostic laboratory in the hospital. The need for two genotyping of metabolism MTHFR and MTRR has caused the problem that the clinical practicability cannot be fully matched; therefore, the kit containing the two genes of folic acid metabolism MTHFR and MTRR in patients with hypertension was redesigned and optimized and the corresponding PCR system was stable After investigating the environment of clinical molecular diagnosis laboratories in various hospitals and statistically analyzing the results of problematic test reports, we comprehensively re-optimized the design of the primers for the research and development projects of the two gene kits, MTHFR and MTRR. Optimized the PCR reaction system of the new kit, and significantly improved the product performance and clinical practicability of the new kit

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