Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method

A technology of CYP2C19 and fluorescent probes, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., and can solve the problems of narrow analysis pan, difficult to meet, expensive reagents and instruments, etc.

Active Publication Date: 2016-04-20
智海生物工程(北京)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are many methods for the detection of CYP2C19 genotyping. The conventional methods include: (1) direct sequencing method, by extracting human peripheral venous blood, extracting genomic DNA, and performing DNA direct sequencing after amplification to determine the genotype ; This method can directly display the position and type of known mutation sites, however, its operation steps are cumbersome, reagents and instruments are expensive, the detection period is long and the cost is high
(2) Restriction sequence analysis, based on the principle of disappearance or increase of the restriction...

Method used

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  • Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method
  • Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method
  • Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method

Examples

Experimental program
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Embodiment 1

[0076] Example 1 CYP2C19 Genotyping Detection

[0077] 1. Genomic DNA Extraction

[0078] ①Put 10 μL whole blood into a 2.0 mL centrifuge tube, add 500 μL sterilized double-distilled water to the centrifuge tube, shake vigorously, and place at room temperature for 15 min.

[0079] ②Centrifuge at 13000rpm for 3min, remove the supernatant, collect the precipitate, and wash the precipitate until it is colorless or has very little hemoglobin.

[0080] ③ Add 100 μL of well-mixed nucleic acid extraction solution to the precipitation, shake repeatedly on a shaker, and incubate at 56°C for more than 30 minutes.

[0081] ④ Take it out and oscillate, incubate at 100°C for 8 minutes. After oscillating, centrifuge at 13,000 for 3 minutes, and the supernatant is used for PCR amplification.

[0082] The samples applicable to the present invention may also include one of body fluids, tissue samples or exfoliated cells, such as blood filter paper, hair with hair follicles, oral mucosal scrapi...

Embodiment 2

[0112] Example 2 CYP2C19 Genotyping Detection Verification

[0113] Using the primer pair and fluorescent probe of Example 1 of the present invention to detect 477 gene samples for verification test, all samples were sequenced and verified by the gold standard sequencing method. 231 cases of CYP2C19*1 / *1 genotype, 164 cases of CYP2C19*1 / *2 genotype, 41 cases of CYP2C19*2 / *2 genotype, 26 cases of CYP2C19*1 / *3 genotype, and 26 cases of CYP2C19*2 / *2 genotype were detected. There were 13 cases of *3 genotype and 2 cases of CYP2C19*3 / *3 genotype. The kit of the present invention detects 477 cases of gene samples compared with the gold standard sequencing results. The detection results are consistent, 0 cases are missed, and the accuracy is 100%. The test results were all negative, the sensitivity was 100%, and the specificity was 100%.

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Abstract

The invention discloses a reagent kit used for detecting CYP2C19 genotypes. Allelic genotypes of CYP2C19*2(681G/A) and CYP2C19*3(636G/A) of a CYP2C19 gene are detected by designing a specific primer pair and a fluorescent probe. Asymmetric PCR and the fluorescent probe melting curve analysis technology are combined, different genotypes are judged through melting peaks of specific temperatures, detection of six genotypes related to CYP2C19*2 and CYP2C19*3 can be achieved in one-tube PCR, subsequent processing is not needed, the possibility of reactant pollution is avoided to the largest extent, operation is easy, consumed time is short, specificity is good, accuracy is high, and results are easy to interpret.

Description

technical field [0001] The invention relates to the field of allele typing, in particular to a primer pair, fluorescent probe, kit and method for detecting CYP2C19 gene CYP2C19*2 and CYP2C19*3 genotyping. Background technique [0002] With the development of medical technology, new drugs emerge in endlessly, but there are more and more medication problems. Many drugs used clinically, such as inhibitors-omeprazole, antidepressants-fluoxetine, antiepileptic drugs-phenytoin Tumor drug-cyclophosphamide, etc., there are individual differences in drug use and drug metabolism. For example, the same drug with the same dosage is used in different patients, but the curative effect may be completely different. Some are cured, while others may have serious adverse drug reactions. Even death, according to the statistics of the World Health Organization, about 19 million people die from adverse drug reactions in the world every year. [0003] Studies have shown that individual difference...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2527/107C12Q2545/113C12Q2563/107
Inventor 杨海艳冯东沈东艳戈斌
Owner 智海生物工程(北京)股份有限公司
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