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Kit for simultaneously detecting multisite mutation of genes CYP2C19 and CYP2D6

A CYP2C19, multi-site mutation technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of not being able to obtain multiple site information at the same time, and achieve the effect of high sensitivity

Inactive Publication Date: 2016-08-17
钟诗龙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional fluorescent PCR technology and products are often only designed for a single site, and cannot obtain information on multiple sites at the same time

Method used

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  • Kit for simultaneously detecting multisite mutation of genes CYP2C19 and CYP2D6
  • Kit for simultaneously detecting multisite mutation of genes CYP2C19 and CYP2D6
  • Kit for simultaneously detecting multisite mutation of genes CYP2C19 and CYP2D6

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The design of embodiment 1 primer and probe

[0043] Because CYP2C19 and CYP2D6 genes have highly homologous sequences in the genome, it is generally difficult to design specific primers and probes for the Taqman allele discrimination assay for detecting CYP2D6 gene variation. The present inventors carried out careful design, repeated verification, screening and optimization, and finally obtained specific primers and probes based on the Taqman allele discrimination analysis method, as shown in Table 1. Table 2 and Table 3 are the primers and probes that cannot successfully specifically detect the mutations of CYP2C19 and CYP2D6 genes.

[0044] Table 1

[0045]

[0046]

[0047]

[0048] Table 2

[0049]

[0050]

[0051]

[0052] table 3

[0053]

[0054]

Embodiment 2

[0055] Embodiment 2 fluorescence quantitative PCR detection process

[0056] 1. A 96-well plate design with 8 rows x 12 columns is adopted, in which 6 clinical samples are detected in columns 1 to 6, and corresponding wild-type, mutant, and heterozygous quality controls are installed in columns 7 to 9. Template, columns 10 to 12 were filled with 6 different ratios of copy number standards (Table 4).

[0057] Table 4 shows an example of a suggested layout for a 96-well PCR reaction plate

[0058]

[0059]

[0060] Note: S stands for sample, S1 to S6 stands for 6 samples; W stands for wild-type quality control template; M stands for mutant quality control template; H stands for heterozygous quality control template; NTC stands for wells without DNA samples; STD stands for Copy number gradient standard; where STD1 to STD6 represent 10-fold serial dilutions of the plasmid standard containing CYP2D6 and ALB gene fragments (1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1 :10000...

Embodiment 3

[0073] The assembly of embodiment 3 detection kits

[0074] A kit for simultaneously detecting multi-site mutations in CYP2C19 and CYP2D6 genes, comprising the following components: each primer described in Table 1 with a concentration of 0.25-0.75 μM and a volume of 0.5-1.25 μL (including primers designed for each gene locus) Forward primer and reverse primer, for example, for CYP2D6*2 site, add primers for detecting CYP2D6*2 site), the concentration of each probe described in Table 1 is 0.25-0.75 μM, and the volume is 0.125-0.625 μL Needle (including wild allele probes and mutant allele probes designed for each gene locus, for example, for CYP2D6*2 loci, add probes for detecting CYP2D6*2 loci), 5-15 μL 2 ×Taqman Master Mix, make up the total volume with double distilled water to 10-25 μL; wild-type quality control template; mutant quality control template; heterozygous quality control template.

[0075] In order to more concisely illustrate the composition of the above kit,...

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Abstract

The invention discloses a kit for simultaneously detecting multisite mutation of genes CYP2C19 and CYP2D6. A group of Taqman allele resolution analysis method-based specific primers and probes is obtained by meticulous designing, multiple verification, screening and optimization, and 8 functional variations of 4 types, i.e. single base displacement, deletion mutation, gene deletion and duplication mutation, of genes CYP2C19 and CYP2D6 can be detected; from DNA extraction to fluorescent PCR and then to result acquisition, 4 hours is required only, and the manual operation time is shorter than 2 hours. The kit containing the primers has the advantages of time saving, convenience, high sensitivity, capability of ensuring that the positive coincidence rate and negative coincidence rate of a sample are both over 99 percent, and the like.

Description

technical field [0001] The invention relates to the technical field of gene mutation detection, more specifically, to a kit for simultaneously detecting multiple site mutations of CYP2C19 and CYP2D6 genes. Background technique [0002] CYP2C19 isoforms play a critical role in drug response because there are significant interindividual differences in their activity, manifested as genetic polymorphisms, which generate interindividual differences in blood drug concentrations. There are multiple gene mutations in CYP2C19, among which, *1 is the normal function allele, *2~*8 is the loss of function or reduced allele, and *17 is the function enhancement allele. CYP2C19*2 and CYP2C19*3 alleles are the main poor metabolic phenotype (PM) of yellow race, accounting for more than 99%. The enzyme encoded by the *2, *3 allele is inactive. Drugs metabolized by this enzyme (such as antiplatelet drug clopidogrel, proton pump inhibitor omeprazole, antidepressant drug venlafaxine, etc.) hav...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 钟诗龙
Owner 钟诗龙
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