Typing kit, primer and typing method for vitamin K metabolism related genes

Abstract

The invention relates to the technical field of gene detection, in particular to a typing kit, primers and a typing method for vitamin K metabolism related genes. The typing kit at least comprises a nucleic acid amplification reagent and a single-base extension reaction reagent, the nucleic acid amplification reagent contains PCR (Polymerase Chain Reaction) amplification primers as shown in SEQ ID NO: 1 to SEQ ID NO: 22; the single base extension reaction reagent contains single base extension primers as shown in SEQ ID NO: 23 to SEQ ID NO: 43. The typing method comprises the following steps: carrying out a PCR amplification reaction on the PCR amplification primer, carrying out alkaline phosphatase digestion, carrying out a single-base extension reaction on the single-base extension primer, carrying out desalination treatment, and carrying out high performance liquid chromatography-mass spectrometry detection. The kit and the detection method provided by the invention can improve the typing detection efficiency of the vitamin K metabolism related genes, and are simple, convenient, feasible, high in accuracy and high in automation degree.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a typing kit, primers and typing method for genes related to vitamin K metabolism. Background technique [0002] Vitamin K is an essential fat-soluble vitamin for blood clot formation. Naturally available in two natural forms: phylloquinone (VK1) synthesized by green plants and menaquinone (VK2) synthesized by bacteria. VK3 and VK4 are human-made synthetic products, which are water-soluble and have no activity themselves. They can be alkylated into MK4 (VK2) in the liver in vivo and have biological activity. Vitamin K is essential for the conversion of inactive precursors into functional clotting factors and is therefore essential for clot formation. Once vitamin K is utilized, it becomes inactive and must be reactivated in order to exert its biological effects. This vitamin K recycling process is known as the vitamin K cycle. Vitamin K is also important for bone healt...

AI Technical Summary

Problems solved by technology

[0006] PCR-direct sequencing method is recognized as the gold standard for genotyping detection, but it is not easy to popularize due to long detection cycle, low detection throughput, low sensitivity, high cost, special requirements for reagents and instruments

The detection sensitivity of PCR-restriction fragment length polymorphism analysis is also not high, the throughput is low, the operation steps are cumbersome, and it is only suitable for the typing of some SNP sites. The detection results still need to be verified again by the next generation sequencing method, especially when When the sample size is large, it is easy to cause cross-contamination of PCR products, and it is prone to insufficient or excessive digestion, resulting in false negative or false positive results

The PCR-gene chip method has high throughput, but the accuracy and repeatability of the detection results are poor, the sensitivity is low, the cost is high, special equipment is required, and the operation is complicated

Although the detection method of fluorescent quantitative PCR has the advantages of high sensitivity, accurate typing, simple and fast operation, good repeatability of results, and easy popularization and promotion of the instruments used, it is a very good means for detecting SNP sites, but the throughput of this method is Limited, it cannot conveniently and quickly meet the clinical needs for the detection of dozens to hundreds of sites of multiple genes, and the cost of probes is high, so it is mainly suitable for typing a small number of sites and large samples

Although the throughput of high-throughput sequencing is extremely high, its cost, time-consuming, and personnel requirements are not suitable for the detection of gene SNP sites

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