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Primer, kit and method for detecting human CYP2C9 genetic typing

A technology of genotyping and kits, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc. It can solve the problems that are difficult to meet clinical tests, achieve low cost, simplify the operation process, save cost effect

Inactive Publication Date: 2017-01-25
SHENZHEN UNI MEDICA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current detection methods for CYP2C9 genotyping are limited to the above-mentioned gene chip method and traditional fluorescent quantitative PCR probe method, and its shortcomings are increasingly difficult to meet the requirements of clinical testing
In the study of detecting CYP2C9 genotyping using a novel SNP detection technology with dual fluorescently labeled probes, no one has reported

Method used

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  • Primer, kit and method for detecting human CYP2C9 genetic typing
  • Primer, kit and method for detecting human CYP2C9 genetic typing
  • Primer, kit and method for detecting human CYP2C9 genetic typing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 detects the kit of human CYP2C9 genotyping

[0046] Primer and Probe Design

[0047] According to the NCBI human CYP2C9 gene sequence, the inventors designed primers and probe nucleotide sequences capable of detecting the polymorphism of the 1075-site gene as follows:

[0048] Upstream primer SEQ ID NO: 1: 5'-CAGCTAAAGTCCAGGAAGAGAT-3';

[0049] Downstream primer SEQ ID NO:2: 5'-TTCTGAATTTAATGTCACAGGT-3';

[0050] Probe SEQ ID NO:3: 5'-CAGAGATAC ATTGACCTTTCTCCCCACC -3'.

[0051] Among them, the sequence between the 10th nucleotide and the 3' end of the probe is complementary to the corresponding sequence on the target nucleic acid, the probe is labeled with a dual fluorescent reporter group, its 5' end is labeled with FAM, and its 3' end is labeled with VIC , the 11th position of the probe marks the quenching group ZEN TM Internal Quencher.

[0052] Preparation of CYP2C9 Fluorescence Quantitative PCR Reaction Solution

[0053] The concentration o...

Embodiment 2

[0060] Fluorescent quantitative PCR detection and analysis of embodiment 2 CYP2C9 genotyping

[0061] (1) Extraction of genomic DNA from human blood samples

[0062] In this example, the blood genome extraction and purification kit produced by Bao'an Branch of Shenzhen Huayinkang Gene Technology Co., Ltd. was used to extract sample DNA. The specific operations are as follows:

[0063] (1) Take out several 1.5ml centrifuge tubes from the extraction kit, and add 200 μl of anticoagulated blood to each tube.

[0064] (2) Add 500 μl buffer CLG solution to the anticoagulant blood, close the cap of the centrifuge tube tightly, vortex for more than 20 seconds, and fully invert and mix. It must be fully mixed or vortexed to ensure the release of genomic DNA.

[0065] (3) Add 100 μl buffer PP solution, vortex or invert back and forth for 10 seconds.

[0066] (4) Centrifuge at 12,000 g for 5 min.

[0067] (5) Take out the DNA purification column from the extraction kit, transfer the s...

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Abstract

The invention belongs to the technical field of genetic typing, and particularly relates to a primer, a probe, a kit and a method for detecting human CYP2C9 genetic typing. The primer and the probe comprise the following nucleotide sequences: an upstream primer SEQ ID NO.1: 5'-CAGCTAAAGTCCAGGAAGAGAT-3', a downstream primer SEQ ID NO.2: 5'-TTCTGAATTTAATGTCACAGGT-3', and a probe SEQ ID NO.3: 5'-CAGAGATACATTGACCTTCTCCCCACC-3'. The kit comprises the primer and the probe, dNTPs (Deoxyribonucleoside Triphosphate), MgCl2, mixed enzyme prepared from UNG (Uracil-N-Glycosylase) and DNA (Deoxyribose Nucleic Acid) polymerase having the activity of flap endonuclease, and positive and negative reference substances. The method for detecting the human CYP2C9 genetic typing by using the kit is capable of detecting different types on a target nucleic acid SNP (Single Nucleotide Polymorphism) site, so that the operation process is simplified, the cost is reduced, the resolution ratio of a detection result is high, and quickness and accuracy are realized.

Description

technical field [0001] The invention belongs to the technical field of genotyping, and in particular relates to a primer, a probe, a kit and a method for detecting human CYP2C9 genotyping. Background technique [0002] In the cytochrome P450 superfamily, the metabolic enzymes related to drug metabolism are mainly CYP1, CYP2, and CYP3 families. CYP2C9 is an important member of the CYP2C subfamily, metabolizing about 10% of clinical drugs, including warfarin, phenytoin, tolbutamide, fluoxetine, etc. The enzyme has genetic polymorphism, and there are individual and racial differences, which lead to differences in the drug metabolism ability of different individuals and races. The most common allele in the Chinese Han population is CYP2C9*3, which occurs at the 1075th nucleotide A>C mutation, and the 359th amino acid sequence is changed from Ile to Leu, resulting in the destruction of a β sheet for substrate recognition, and then The catalytic ability of the enzyme is reduc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2563/107C12Q2545/113C12Q2521/301
Inventor 谭爱女罗晓腾郭永超周代志
Owner SHENZHEN UNI MEDICA TECH
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