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A method for detecting multi-site mutation of cyp2d6 gene

A multi-site mutation, gene technology

Active Publication Date: 2016-08-24
广州海思医疗科技有限公司
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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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  • A method for detecting multi-site mutation of cyp2d6 gene
  • A method for detecting multi-site mutation of cyp2d6 gene
  • A method for detecting multi-site mutation of cyp2d6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Use a 96-well plate design with 8 rows x 12 columns, or a combination of several 8-tube tubes. Each row of 8 wells detects one sample, each well detects a mutation site, and the 8th well detects the copy number of CYP2D6. The corresponding NTC, wild-type, heterozygous, and mutant quality control templates are installed in columns 8 to 11, and 6 standard solutions with different ratios of copy numbers are installed in column 12.

[0063] (1) Genomic DNA extraction from blood: DNA extraction kit from Qiagen was used to extract genomic DNA as a template. The extracted genomic DNA was dissolved in TE buffer solution, that is, 10mmol / L Tris-HCl (PH8.0) and 1mmol / L EDTA (PH8.0), and the concentration was measured by an ultraviolet spectrophotometer to prepare a 50ng / μL solution. as template for PCR amplification.

[0064] (2) Design specific primers and fluorescent probes, the sequences of which are shown in SEQ ID NO: 1-34.

[0065] (3) Fluorescent quantitative PCR reacti...

Embodiment 2

[0080] Comparison between the CYP2D6 gene polymorphism detection kit currently on the market and the detection method of Example 1 of the present invention (Table 2).

[0081] Table 2

[0082]

[0083]

Embodiment 3

[0085] Since patients with coronary heart disease often use beta-receptor blockers, 148 patients with coronary heart disease were selected, and the method described in Example 1 of the present invention was used to detect 8 mutations of CYP2D6 gene in these patients. The Sanger sequencing method was used for verification, and the positive coincidence rate (clinical sensitivity) and negative coincidence rate (clinical specificity) were calculated (Table 3).

[0086] table 3

[0087]

[0088]

[0089]

[0090]

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Abstract

The invention discloses a multi-site mutation detection method of a CYP2D6 gene. The detection method disclosed by the invention is an allele differential analysis method based on Taqman; and detection of eight functional haplotype variations in four types (single-base substitution, deletion mutantion, gene deletion and duplication) of the CYP2D6 gene is provided. The multi-site mutation detection method of the CYP2D6 gene has the advantages that 98% of functional sites of the CYP2D6 gene can be covered; all operation processes can be finished within only 4 hours; and the manual operation time is less than 2 hours; thus time and labor are saved. The detection method disclosed by the invention is high in detection sensitivity and suitable for popularization and application, and the positive coincidence rate and the negative coincidence rate of samples are both over 99%.

Description

technical field [0001] The invention relates to the field of gene mutation detection, in particular to a method for detecting multiple sites of CYP2D6 gene. The method can detect eight functional haplotype variations of CYP2D6 gene quickly, efficiently and accurately. Background technique [0002] According to the World Health Organization (WHO), the proportion of adverse drug reactions in hospitalized patients in various countries in the world is 10%-20%, and 5% of patients die of severe adverse drug reactions. In my country, patients with adverse drug reactions account for 10%-30% of hospitalized patients. Every year, 5 million patients are admitted to hospital due to adverse drug reactions, and about 190,000 people die of adverse drug reactions every year. Therefore, pharmacogenomics research has become a global hotspot, and individualized medicine and individualized treatment are the focus of discussions at various international medical and biological conferences. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2561/101C12Q2545/113
Inventor 钟诗龙
Owner 广州海思医疗科技有限公司