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CYP2C19 gene polymorphism detection kit and application thereof

A technology of CYP2C19 and gene polymorphism, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inapplicability of real-time fluorescent quantitative PCR instrument, complicated operation process of liquid phase chip method, and instrument requirements High-level problems, to achieve accurate and easy detection results, low instrument requirements, and improve detection efficiency

Pending Publication Date: 2020-03-31
武汉光谷联合医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although direct DNA sequencing is the most routine detection method and is the gold standard, the steps are cumbersome and time-consuming
The high-resolution melting curve method has high requirements for instruments, and most real-time fluorescent quantitative PCR instruments are not applicable
The operation process of the liquid chip method is complicated, and it is easy to be polluted, and the false positive rate is high

Method used

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  • CYP2C19 gene polymorphism detection kit and application thereof
  • CYP2C19 gene polymorphism detection kit and application thereof
  • CYP2C19 gene polymorphism detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, CYP2C19 gene polymorphism detection kit

[0042] S1. Design and synthesis of primers and probes

[0043] Primer probes for detection of CYP2C19*2(G681A):

[0044] Forward primer 1: 5'-AAATTTCCCCATCAAGATAT-3',

[0045] Reverse primer 1: 5'-TACGCAAGCAGTCACATAAC-3',

[0046] Wild-type detection probe 1: 5'-FAM-ATGACGTGATCTTGATACTATCATTGATTATTTCCCGG-3', the 37th base of its sequence is modified by LAN,

[0047] Mutant detection probe 1: 5'-HEX-TAGGTCAAGCGTAGCTGTAATTTGTTATGGGTTCCAA-3', the 36th base of its sequence is modified by LAN;

[0048] Primer probes for detection of CYP2C19*3(G636A):

[0049] Forward primer 2: 5'-ACTTTCATCCTGGGCTGTGC-3',

[0050] Reverse primer 2: 5'-TTGGGATATTCATTTCCTGTGC-3',

[0051] Wild type detection probe 2: 5'-FAM-ATGACGTGATCTTGATATTGTAAGCACCCCCTGG-3', the 34th base of its sequence is modified by LAN,

[0052] Mutant detection probe 2: 5'-HEX-TAGGTCAAGCGTAGCTCTTGGCCTTACCTGGATT-3', the 34th base of its sequence is modified ...

Embodiment 2

[0058] Embodiment 2, performance verification of the kit of the present invention

[0059] S1. Artificial plasmid synthesis and extraction

[0060] Plasmids containing two polymorphic regions of the CYP2C19 gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and polymorphic variations (CYP2C19*2 and CYP2C19*3, G>A) were introduced into the plasmids, and finally Two wild-type plasmids and two mutant plasmids of CYP2C19 gene were obtained. Four kinds of artificial plasmids were purified with a plasmid extraction kit (Shanghai Sangong) according to its operating instructions. The purified plasmid was quantified with a NanoDrop 2000 ultra-micro spectrophotometer, and diluted with deionized water to 1000 copies / μL for subsequent real-time fluorescent quantitative PCR detection. At the same time, the mutant plasmids and wild-type plasmids were mixed at a ratio of 1:100, and the mutant plasmid content in the finally obtained mixed plasmid was 1%.

[0061] S2. Real...

Embodiment 3

[0067] Example 3, using the CYP2C19 polymorphism detection kit of the present invention to detect blood samples

[0068] S1. Genomic DNA extraction

[0069] In this example, peripheral blood samples from 50 healthy individuals were collected, and genomic DNA was extracted using the Thermo Fisher Plasma Sample Genomic DNA Extraction Kit, and the DNA was eluted with TE buffer after the extraction was completed. The extracted and purified genomic DNA was quantified with a NanoDrop 2000 ultramicro spectrophotometer, and diluted with deionized water to a concentration of 10 ng / μL.

[0070] S2, fluorescent quantitative PCR

[0071] Take 2 μL of genomic DNA of the sample to be tested, and perform fluorescence quantitative PCR detection on the sample according to the same operation method as described in step S2 of Example 2. CYP2C19*2 mixed plasmid and CYP2C19*3 mixed plasmid are used as positive controls, and the TE buffer without nucleic acid is Negative control; at the same time...

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Abstract

The invention discloses a CYP2C19 gene polymorphism detection kit and an application of thereof. The kit comprises forward and reverse primers for detecting CYP2C19 * 2 (G681A), a wild type detectionprobe and a mutant type detection probe, wherein the forward and reverse primers, a wild-type detection probe and a mutant-type detection probe are used for detecting CYP2C19*3 (G681A), and a wild-type quenching probe and a mutant-type quenching probe shared by two sites, wherein the wild-type detection probe and the mutant-type detection probe are respectively used for marking different fluorescent groups. The specificity of probe binding is improved by utilizing a locked nucleic acid technology, the polymorphism of the CYP2C19 gene is detected by combining a multiple fluorescent PCR technology, the operation is convenient and rapid, the detection result is easy to interpret, the specificity is strong, the accuracy and the sensitivity are high, and an effective basis is provided for clinical medication of clopidogrel.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a CYP2C19 gene polymorphism detection kit and application thereof. Background technique [0002] Clopidogrel, the drug name Plavix, is a platelet aggregation inhibitor mediated by adenosine diphosphate receptors, which can be used to prevent and treat myocardial infarction, ischemic cerebral thrombosis, vasculitis obliterans, atherosclerosis and Complications caused by thromboembolism can significantly reduce the incidence of stent thrombosis in the application of percutaneous coronary intervention. At present, it has been used as one of the important drugs for standard antiplatelet therapy after percutaneous coronary intervention. . However, patients on long-term treatment with clopidogrel sometimes experience recurrent acute coronary syndromes, which can be severe or even life-threatening. A large number of studies have proved that this is due to the individual differences in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2563/107C12Q2547/101
Inventor 赵平锋
Owner 武汉光谷联合医学检验所股份有限公司
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