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Kit for detecting polymorphism of human CYP2C9 and VKORC1 genes

A gene polymorphism and kit technology, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as inability to type multiple tubes, complicated amplification procedures, and complicated operations

Active Publication Date: 2020-02-07
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with domestic patents for the detection of CYP2C9 and VKORC1 gene polymorphisms by fluorescent quantitative PCR, there are still the characteristics of long detection time, complicated operation, and inability to multi-tube typing
For example, the patent applied by Wuhan Haijili Biotechnology Co., Ltd. (application publication number: CN106399560A) uses fluorescent quantitative PCR single-site multi-tube typing, which is complicated to operate, time-consuming, and liquid reagents are stored at -20°C
The patent (application publication number: CN104962609A) applied by Shenyang Youjinuo Biotechnology Co., Ltd. has incomplete detection sites, complex amplification procedures and long time-consuming

Method used

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  • Kit for detecting polymorphism of human CYP2C9 and VKORC1 genes
  • Kit for detecting polymorphism of human CYP2C9 and VKORC1 genes
  • Kit for detecting polymorphism of human CYP2C9 and VKORC1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Example 1: Composition of the kit

[0193] The composition of the kit of the present invention is as follows:

[0194] Table 1 Composition of the kit

[0195]

Embodiment 2

[0196] Example 2: Extraction of genomic DNA from human whole blood / peripheral blood samples

[0197] In this embodiment, human whole blood / peripheral blood samples are collected, and genomic DNA is extracted therefrom.

[0198] Genomic DNA was extracted from EDTA anticoagulated plasma and used as a template for PCR detection. Use Zhengzhou Antu Bioengineering Co., Ltd.'s pretreatment reagents and nucleic acid extraction and purification reagents (magnetic bead method), and proceed according to the instructions. The details are as follows:

[0199] 1. Sample pretreatment:

[0200] Frozen whole blood is pre-treated with red blood cell lysate:

[0201] ① Take 200μL of clinical freeze-thaw or hemolyzed whole blood sample, add 600μL of red blood cell lysate, gently invert and mix 10

[0202] Second, after the red blood cells are lysed, the solution should be clear and transparent. Centrifuge at 12000rpm for 3min, discard the supernatant;

[0203] ②Add 600μL 0.2mg / ml proteinase K, incubate at...

Embodiment 3

[0215] Example 3: Crude extraction of genomic DNA from human oral swab samples

[0216] In this embodiment, the human oral swab sample is simply processed, and the genomic DNA of the sample is roughly extracted.

[0217] 1. Take a clean oral swab, extend the sampling site of the swab head into the left and right sides of the sampled person’s mouth, scrape up and down 5 times, the oral epithelial exfoliated cells will adhere to the swab head, and swab it quickly Soak the seeds in 1ml of rinse solution (purified water or normal saline can also be substituted), rinse them up and down 10 times, discard the swab, and save the swab rinse solution for later use.

[0218] 2. Place the swab rinse solution on a metal bath at 90°C and heat for 5 minutes. After cooling to room temperature, centrifuge at 12000 rpm for 3 minutes. Take the supernatant to obtain the crudely extracted genomic DNA.

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Abstract

The invention relates to the technical field of in vitro diagnosis, and particularly relates to a kit for detecting polymorphism of human CYP2C9 and VKORC1 genes. The kit for detecting the polymorphism of the human CYP2C9 and VKORC1 genes comprises 2 CYP2C9 primers and probes thereof, 3 VKORC1 primers and probes thereof, a VKORC1 primer and a probe thereof, as well as an internal control primer and a probe thereof. According to the invention, amplification reagents with relatively high amplification specificity and relatively low cost are prepared by using the specific primers and probes, so that the kit disclosed by the invention is easy to operate, strong in specificity, high in sensitivity, and feasible for detection in cooperation with an automatic instrument.

Description

Technical field [0001] The present invention relates to the technical field of in vitro diagnosis, in particular to a kit for detecting human CYP2C9 and VKORC1 gene polymorphisms. Background technique [0002] Warfarin is a first-line oral anticoagulant commonly used in clinical treatment of blood embolic diseases (such as deep vein thrombosis, atrial fibrillation, heart valve replacement, pulmonary embolism, etc.), and its annual prescription accounts for 0.5% to 1.5% of the total population. Since its advent in the 1950s, it has been used as an anticoagulant in the market for more than half a century. Because of its low cost and significant anticoagulant effect, it is still the first choice for patients with thromboembolism as an anticoagulant. However, its treatment window is narrow, and it has been the most frequently reported drug in the FDA's adverse drug reaction monitoring system. [0003] CYP2C9 (cytochrome P450 superfamily 2C9) is the most important metabolizing enzyme o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 崔奇新李江浩张肖洁李振红李静静杜美付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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