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Nucleic acid, kit and method for detecting polymorphism of human CYP2D6 gene

A gene polymorphism and kit technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection. The complex operation process of the phase chip method can avoid the problems of non-specific amplification, high accuracy and easy interpretation.

Active Publication Date: 2017-05-31
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The operation process of this method is cumbersome, it cannot be batched and automated, and the results are not easy to interpret, and the probability of false positives is high
For the melting curve method, neither the dye melting curve nor the probe melting curve method can effectively distinguish multiple mutation types at the same site
The operation process of the liquid chip method is complicated, and it is easy to be polluted, and the false positive rate is high

Method used

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  • Nucleic acid, kit and method for detecting polymorphism of human CYP2D6 gene
  • Nucleic acid, kit and method for detecting polymorphism of human CYP2D6 gene
  • Nucleic acid, kit and method for detecting polymorphism of human CYP2D6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design of primers, probes, and verification templates

[0042] According to the sequence information of CYP2D6*10 and CYP2D6*14 published in the NCBI dbSNP database (https: / / www.ncbi.nlm.nih.gov / SNP / ), the two alleles of the CYP2D6 gene were designed as shown in Table 1. The detection primers and probes of polymorphism sites, after screening, optimization, and a large number of experimental verifications, the primer and probe sequences are as follows:

[0043] (1) Primers and probes for CYP2D6*10 SNP gene polymorphism detection:

[0044] C100T wild-type allele upstream primer (1W-F):

[0045] 5'-CGCCAACGCTGGGCTGCACGCTAC-3' (SEQ ID NO. 1),

[0046] C100T mutant allele upstream primer (1M-F):

[0047] 5'-CGCCAACGCTGGGCTGCACGCTAT-3' (SEQ ID NO. 2),

[0048] Common downstream primer for C100T allele (1C-R):

[0049] 5'-CCTCAGGACCTCTGCCGCCCTCC-3' (SEQ ID NO.3),

[0050] C100T allele-specific probe (1P):

[0051] 5'-TGTTCTGGAAGTCCACATGCAGCA-3' (SEQ ID NO. 4);...

Embodiment 2

[0094] Example 2 Kit for detecting CYP2D6 gene polymorphism sites

[0095] The real-time fluorescent PCR kit for rapid detection of CYP2D6 gene polymorphism sites includes the following components: primers for each gene as described in Example 1, detection probes, wild-type positive controls, mutant positive controls, The 5' end of the detection probe of each gene is connected with a fluorescent group, and the 3' end is connected with a quenching group; wherein, the fluorescent group is any one of FAM, JOE, CY3, and HEX, and the quenching group is MGB , any of BHQ1, TAMRA, BHQ2.

[0096] In order to avoid missed detection and wrong detection, it also includes: quality control primer pair, quality control probe and quality control positive control as internal quality control, the sequence is as described in Example 1, and the fluorophore of the quality control probe Fluorophores other than detection probes.

[0097] In order to prevent the reaction system from volatilizing du...

Embodiment 3

[0099] Example 3 Preparation of detection kit

[0100] The kit in this example is based on Example 2, and is prepared as a kit that can be directly added to the test sample for detection. It is composed of a CYP2D6 6-linked PCR reaction strip and a CYP2D6 positive control. The components in each tube in the kit are See Table 2.

[0101] Table 2 Kit composition

[0102]

[0103]

[0104] The full name of Taqman Master Mix is ​​TaqMan Real-Time PCR Master Mixes, purchased from ThermoFisher Scientific (Thermo Fisher Scientific); 10×PCR buffer (Mg 2+ Plus) uses TaKaRa Bioengineering (Dalian) Co., Ltd., and can also apply buffers and DNA polymerases from other companies on the market.

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Abstract

The invention discloses nucleic acid, a kit and a method for quickly detecting polymorphism of a human CYP2D6 gene, which are particularly used for detecting site polymorphism of CYP2D6*10 genes C100T and G4268C as well as a CYP2D6*14 gene G1758A. The detection kit for quickly detecting the polymorphism of the CYP2D6 gene by using real-time fluorescent quantitative PCR, has remarkable advantages of high specificity, high sensitivity, short experiment cycle, simplicity in operation, safety, no toxicity, low cost and the like; the detection method is simple and convenient to operate and high in automation degree; the operation process is greatly simplified, pollution in the operation process is reduced, and the detection result is accurate and reliable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid, a kit and a method for detecting human CYP2D6 gene polymorphism. Background technique [0002] CYP2D6 is one of the important members of the CYP enzyme family. The gene is located at 22q13.1 and contains 9 exons and 8 introns, with a total length of about 7kb. It is a complete functional gene. Studies have shown that CYP2D6 in the liver only accounts for 2-9%, but it is involved in the metabolism of 20% to 30% of drugs, including antidepressants, antiarrhythmic drugs, antipsychotics, and analgesics. CYP2D6 has a wide range of gene polymorphisms, and more than 100 gene variants have been reported, such as single base mutation, deletion or insertion and deletion of large fragments. These genetic variations can cause differences in enzyme activity and quantity, leading to individual differences in drug metabolism. The metabolic phenotypes of CYP2D6 can be d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 胡金吴志伟段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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