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Method and oligonucleotide for detecting cyp2c19*2 mutation site

A technology of CYP2C19 and mutation sites, which is applied in biochemical equipment and methods, microbiological measurement/inspection, DNA/RNA fragments, etc., can solve the problems of strict detection conditions, expensive sequencing equipment, long detection cycle, etc., and meet the conditions The effect of less dependence, lower synthesis cost, and shorter detection cycle

Active Publication Date: 2016-08-17
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional DNA sequencing methods, such as Sanger sequencing and pyrosequencing, have a long detection cycle, usually 1 to 2 days, complex operations, easy contamination, and expensive sequencing equipment, which is difficult to configure in general laboratories
Although ARMS-PCR improves the detection specificity, the detection conditions are strict. In actual operation, it is easy to cause false positives due to primer mismatch.

Method used

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  • Method and oligonucleotide for detecting cyp2c19*2 mutation site
  • Method and oligonucleotide for detecting cyp2c19*2 mutation site
  • Method and oligonucleotide for detecting cyp2c19*2 mutation site

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Oligonucleotides and kits for detecting CYP2C19*2 mutation sites

[0043] The oligonucleotide for detecting the CYP2C19*2 mutation site is characterized in that the oligonucleotide includes a pair of amplification primers SEQ NO 1 and SEQ NO 2, and detection probes SEQ NO 3 and SEQ NO 4, which The base sequence is as follows:

[0044] SEQ NO 1: 5'-GTATAATCAGAGAGAATTACTACACATG-3';

[0045] SEQ NO 2: 5'-GTTGATGTCCATCGATTCTTGGTG-3';

[0046] SEQ NO 3: FAM-CCACTATCATTGATTATTTCCCG-MGB;

[0047] SEQ NO 4: HEX-CCACTATCATTGATTATTTCCCA-MGB.

[0048] Among them, SEQ NO 3 can only bind to the DNA template of the wild-type CYP2C19*2 sample, and SEQ NO 4 can only bind to the DNA template of the mutant CYP2C19*2 sample.

[0049] A kit for detecting a CYP2C19*2 mutation site, comprising a sample DNA extraction reagent, a detection system PCR reaction solution, a positive control substance, a negative control substance and a blank control substance.

[0050] Sample DNA ...

Embodiment 2

[0060] Example 2: Detection process of CYP2C19*2 mutation site

[0061] (1) Use blood DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract genomic DNA in blood samples:

[0062] 1) Take 500 μl of blood and add 1000 μl of erythrocyte lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed.

[0063] 2) Add 20 μl proteinase K solution and mix well.

[0064] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0065] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner ...

Embodiment 3

[0080] Embodiment 3: clinical sample detection

[0081] Select 21 cases of clinical blood samples to be tested, and use the kit of the present invention to detect. Sample DNA extraction, fluorescent real-time PCR amplification and result analysis were carried out according to the method in Example 2, and the same sample to be tested was sequenced by Sanger sequencing method, and the results are shown in Table 3:

[0082] Table 3 CYP2C19*2 mutation site detection results

[0083]

[0084]

[0085] As can be seen from Table 3, the detection results obtained by the detection method provided by the present invention are completely consistent with the detection results obtained by the Sanger sequencing method, which shows that the method has a very high detection accuracy, wherein the letter Y in Table 3 Indicates that there is a CYP2C19*2 mutation site, that is, the base G at position 681 of exon 5 of the CYP2C19 gene is mutated to A; the letter N indicates that there is no...

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Abstract

The invention provides a method and oligonucleotide for highly-sensitive detection of a mutation site of CYP2C19*2 by utilizing a real-time fluorescent PCR (Polymerase Chain Reaction) technology. The oligonucleotide comprises a pair of amplification primers SEQNO1 and SEQNO2, and detection probes SEQNO3 and SEQNO4, wherein MGB groups are modified at the 3 end of each of two detection probes so as to enhance the specificity. The method and oligonucleotide provided by the invention have the advantages of short detection period, high specificity, high accuracy, high sensitivity, low pollution risk and the like, and nearly do not depend on conditions.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, and in particular relates to a method and an oligonucleotide for detecting a CYP2C19*2 mutation site with high sensitivity using fluorescence real-time PCR technology. Background technique [0002] Clopidogrel is an antiplatelet drug widely used in cardiovascular diseases. Platelet activation and aggregation are the key factors for arterial thrombosis after acute coronary syndrome and percutaneous coronary intervention (PCI). Antiplatelet therapy combining the two drugs clopidogrel and aspirin is essential for the prevention of ischemic events in patients with acute coronary syndrome and after PCI. However, after receiving this antiplatelet therapy, about 10% of patients still suffer from stent thrombosis, death, stroke and other cardiovascular events, mainly due to the differences in the responses of different patients to clopidogrel, and the influencing factors are Many aspects, in whic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 陈奕磊周晓犊
Owner 杭州艾迪康医学检验中心有限公司
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