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Method for detecting CYP2D6 gene polymorphism through fluorescent quantitative PCR

A technology for gene polymorphism and fluorescence quantification, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc. Reduced cost, high specificity, high sensitivity, and improved specificity

Pending Publication Date: 2020-02-21
吉林和合医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the most commonly used sequencing method can directly detect the position and type of the mutation site, but this method has cumbersome operation steps, long detection cycle, low sensitivity, and the amplification product is prone to contamination

Method used

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  • Method for detecting CYP2D6 gene polymorphism through fluorescent quantitative PCR
  • Method for detecting CYP2D6 gene polymorphism through fluorescent quantitative PCR
  • Method for detecting CYP2D6 gene polymorphism through fluorescent quantitative PCR

Examples

Experimental program
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Embodiment 1

[0030] A method for detecting CYP2D6 gene polymorphism The preparation method of the DNA sample to be tested comprises the following steps: using the blood / cell / tissue genomic DNA extraction kit (DP304) to extract genomic DNA, and using NP80-touch (Germany IMPLEN) to measure DNA concentration and purity, and then save genomic DNA.

Embodiment 2

[0032] 1. Primer and Probe Design

[0033]The present invention designs specific primers and specific fluorescent probes respectively according to the base sequence of the CYP2D6*10 gene locus. The specific primers are designed at both ends of the SNP locus by Primer Premier 5.0, and the most suitable one is selected by software analysis. Primers; specific probes are located in the region between a pair of primers, where the Tm value should be between 60-70°C, usually 5-10°C higher than that of the primers. The 5' end of the probe for detecting the wild type is labeled with a fluorescent reporter group (FAM), the 5' end of the probe for detecting the mutant type is labeled with a fluorescent reporter group (VIC), and the 3' end is labeled with a non-fluorescent quencher group ( NFQ) label, and the MGB modification group is also linked to the probe; in actual operation, the fluorescent reporter group can be replaced according to the actual situation, as long as the fluorescent ...

Embodiment 3

[0049] Embodiment 3: the preparation of kit

[0050] According to the above experimental results, this embodiment provides a preferred kit for detecting CYP2D6 gene polymorphisms by fluorescent quantitative PCR, which includes the following reagents:

[0051] 1. 2×TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (including dUTP);

[0052] 2. Probe Mix: the final concentration of fluorescent probes is 0.6 μM;

[0053] 3. Primer Mix: Primer pair, the final concentration of F upstream primer and R downstream primer is 0.6 μM;

[0054] 4. Sample DNA extraction reagents;

[0055] 5. Sample collection storage box (such as containing oral swabs and swab storage boxes / tubes);

[0056] 6. Ultrapure water.

[0057] The reagents are properly placed in the kit, and then put into the instructions for use of the kit (PCR test tubes, orifice plates or pipette guns can be optionally reconfigured). The instructions for use include the collection steps of the samples to...

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Abstract

The invention provides a primer probe combination for detecting CYP2D6 gene polymorphism through fluorescent quantitative PCR. The combination comprises primers and double probes as shown in a sequence table. Meanwhile, the invention provides a method for detecting the polymorphism of the CYP2D6 gene through the fluorescent quantitative PCR. The CYP2D6 gene polymorphism is detected by adopting a real-time fluorescent quantitative PCR Taqman-MGB probe method, and a wild type and mutant type double-probe detection method is adopted, so that the specificity is improved, and the cost is reduced. The Taqman-MGB probe is superior to common probes in the aspects of accuracy, repeatability, specificity, sensitivity and the like of experimental results. Therefore, the method for detecting the CYP2D6 gene polymorphism established by the invention has the advantages of accurate result, high specific sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a combination of primers and probes and a detection method for detecting CYP2D6 gene polymorphisms by fluorescent quantitative PCR. Background technique [0002] CYP2D6 is an important oxidative metabolic enzyme in the cytochrome P450 enzyme system, which plays an important role in the metabolism of endogenous and exogenous substances in organisms. It is mainly expressed in the small intestine, liver and lung, located on human chromosome 22q13.1, with a total length of about 7Kb, 9 exons and 8 introns, encoding 497 amino acids. At present, more than 70 genetic variations of CYP2D6 gene have been found, and different mutation types of these gene polymorphisms have different effects on enzyme activity and drug metabolism. According to the activity of each genotype, CYP2D6 is divided into: poor metabolizers (PMs), intermediate metabolizers (IMs), intensive metabolizers (EMs)...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2561/101C12Q2563/107
Inventor 李晓娇智慧芳倪君君
Owner 吉林和合医学检验有限公司
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