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Real-time fluorescence PCR method of detecting rs762551 site of CYP1A2 gene, and primer-and-probe combination of real-time fluorescence PCR method

A technology of CYP1A2 and primer probes, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of specificity and sensitivity, and achieve high sensitivity, simple operation, and simple result analysis Effect

Pending Publication Date: 2020-03-27
SHANXI LIFEGEN
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Problems solved by technology

For those skilled in the field of gene detection, the current application of real-time fluorescent PCR based on TaqMan probes is mainly due to difficulties in specificity and sensitivity; because it is not just a simple combination of primers and probes, but the need to target CYP1A2 A set of specific primers and TaqMan probes were designed for the rs762551 site to efficiently, quickly and accurately detect the genotype of the sample to be tested

Method used

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  • Real-time fluorescence PCR method of detecting rs762551 site of CYP1A2 gene, and primer-and-probe combination of real-time fluorescence PCR method
  • Real-time fluorescence PCR method of detecting rs762551 site of CYP1A2 gene, and primer-and-probe combination of real-time fluorescence PCR method
  • Real-time fluorescence PCR method of detecting rs762551 site of CYP1A2 gene, and primer-and-probe combination of real-time fluorescence PCR method

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specific Embodiment

[0044] Specific embodiment: TaqMan probe real-time fluorescent PCR method detects the allele of rs762551 site.

[0045] 1. Extraction and dilution of DNA samples

[0046] After collecting venous blood in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Mini Blood Kit (Qiagen, Germany) kit; the extracted DNA was concentrated using NanoDrop 2000 Determination (A260 / 280=1.95~2.15). Using the above method, respectively measure the concentration of 104 cases of Bouyei DNA samples, and then use PCR grade H 2 O Dilute the sample to 10 ng / μl.

[0047] 2. Design primers and probes

[0048] Specific upstream primers: C allele: 5’-GGTGAGCTCTGTGGGGC-3’; A allele: 5’-GGTGAGCTCTGTGGGGA-3’;

[0049] Universal downstream primer: 5'-GCTGAGGGTTGAGATGGAGAC-3';

[0050] Probe: 5'-Cy5-ACGCATGGTAGATGGAGCTTAG-BHQ2-3'. ;

[0051] Among them, Cy5 is Cyaine5; BHQ2 is Black Hole Quenc...

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Abstract

The invention belongs to the field of gene diagnosis, and discloses a real-time fluorescence PCR method of detecting a rs762551 site of a CYP1A2 gene, and a primer-and-probe combination of the real-time fluorescence PCR method. A sequence of one forward primer FpG is 5'-GGTGAGCTCTGTGGGGC-3', a sequence of the other forward primer FpG is 5'-GGTGAGCTCTGTGGGGA-3', a sequence of a reverse primer Rp is5'-GCTGAGGGTTGAGATGGAGAC-3', and a sequence of a probe Probe is 5'-Cy5-ACGCATGGTAGATGGAGCTTAG-BHQ2-3'. After a PCR is completed, results can be analyzed through whether or not an amplification curveexists and in combination with a Ct value. The real-time fluorescence PCR method of detecting the rs762551 site of the CYP1A2 gene is applied to polymorphism detection of the rs762551 site, and has the advantages of being easy to operate, high in resolution, free of pollution, and high in throughput.

Description

technical field [0001] The invention belongs to the field of pharmacogenomics and gene detection, and specifically relates to a design of specific primers and probes and a detection method for the CYP1A2 gene rs762551 site (CYP1A2*1F, -163C>A). Background technique [0002] Mental diseases refer to serious psychological disorders, in which the patient's cognition, emotion, volition, behavior and other psychological activities may appear persistent and obvious abnormalities, and they cannot live normally. The number of mentally ill patients in my country cannot be underestimated. By the end of 2017, there were about 240 million mentally ill patients nationwide, with a total prevalence rate as high as 17.5%; there were more than 16 million patients with severe mental disorders, and the incidence rate exceeded 1%. And this number is still increasing year by year. At present, drug treatment is the main treatment for mental illness in our country, but it is still in the stage...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C12Q1/686
CPCC12Q1/6883C12Q1/686C12Q2600/106C12Q2600/166C12Q2561/101C12Q2561/113
Inventor 王会娟康星韩敏
Owner SHANXI LIFEGEN
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