Complete-set primer for SNP (Single Nucleotide Polymorphism) sites of genes of drug-metabolizing enzyme and application thereof

A technology of metabolic enzymes and loci, which is applied in the field of primer sets for detecting SNP loci of drug metabolizing enzyme genes, which can solve problems such as time-consuming, complicated operation, and failure to cover key enzyme genes and single nucleotide polymorphism loci , to achieve the effect of simple operation, saving cost and time

Active Publication Date: 2018-06-22
北京天平永达生物科技发展有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] In the research related to gene detection of drug-metabolizing enzymes, the patent (CN200810208219.5) discloses a method for detecting drug-metabolizing-related sites, and the patent (CN201610214241.5) discloses the amplification of a drug-metabolizing enzyme-related gene SNP fluorescent labeling complex For the kit, these two patents selected 5 SNP sites of 4 related genes respectively, and the detection of genes was not comprehensive, and did not cover all key enzyme genes and single nucleotide polymorphism sites related to drug metabolism; in addition, The method generally used in the above two patents and previous patents is the fluorescent quantitative PCR method. This method is suitable for detecting about 5 known SNP sites. When the number of sites increases, the operation is complicated and time-consuming.

Method used

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  • Complete-set primer for SNP (Single Nucleotide Polymorphism) sites of genes of drug-metabolizing enzyme and application thereof
  • Complete-set primer for SNP (Single Nucleotide Polymorphism) sites of genes of drug-metabolizing enzyme and application thereof
  • Complete-set primer for SNP (Single Nucleotide Polymorphism) sites of genes of drug-metabolizing enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The establishment of embodiment 1 technical scheme

[0034] S1 design technical route: determine the main components of children’s common drugs (Table 1), clarify the results of SNP typing of genes and sites related to drug metabolism and the interpretation of drug metabolism rate (Table 2);

[0035] S2 establishes the reaction system: design multiple PCR amplification primers (Table 3) and extension primers (Table 4), adjust the concentration of reaction components and reaction conditions;

[0036] S2 Analyzing samples: DNA extraction, multiplex PCR amplification, time-of-flight mass spectrometry detection, result analysis and medication advice.

[0037] Table 1 Common drugs and their main ingredients for children

[0038]

[0039] Table 2 Types of related SNP sites and drug metabolism rate

[0040]

[0041] Table 3 multiplex PCR amplification primer sequence

[0042]

[0043] Table 4 Single base extension primer sequence

[0044] serial number

Embodiment 2

[0045] Example 2 sample extraction and analysis

[0046] In order to verify the detection accuracy and effectiveness of the set of primers used to detect drug-metabolizing enzyme genes of the present invention, 10 samples were selected for gene detection, and the process was as follows:

[0047] A. Saliva DNA extraction: use Kangwei Century Oral Swab Genomic DNA Extraction Kit for saliva DNA extraction. Add a certain amount of absolute ethanol to the GW1 and GW2 reagents. Take 500 μL saliva sample from the sampling tube, add 300 μL GR, 20 μL Proteinase K and 300 μL GL, shake and mix, incubate with shaking at 56°C for 15 minutes, add 300 μL absolute ethanol, shake and mix; add 750 μL of this solution to the adsorption tube of the collection tube Add 400 μL GW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the liquid; add 400 μL GW2 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the liquid; centrifuge at 12,000 rpm for 2 min, and let...

Embodiment 3

[0071] Example 3 Analysis of genetic testing results

[0072] The 9 SNP site gene detection results (Table 5) of the 10 samples in Example 2 were analyzed, and the rational drug use suggestions were given in conjunction with the drug-metabolizing enzyme gene-related SNP site typing and drug metabolism rate (Table 2) .

[0073] Table 5 Genotypes of 9 SNP loci in drug metabolism-related genes

[0074] site

[0075] It can be seen from Table 5 that the genotypes of the 9 loci can be detected correctly, and the detection rate is 100%. The amplification primer and the extension primer designed by the invention can detect 9 SNP sites in the same reaction, and are used for developing a drug metabolizing enzyme gene detection kit and guiding safe medication. In terms of safe drug use for children, the gene detection of the present invention can understand children's ability to metabolize different types of drugs, and provide scientific reference for rational drug administr...

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Abstract

The invention discloses a complete-set primer for SNP (Single Nucleotide Polymorphism) sites of drug-metabolizing enzyme genes and application thereof. The complete-set primer comprises amplificationprimers and extension primers of 9 SNP sites of 7 drug-metabolizing enzyme associated genes of human genome DNA, wherein a pair of multiple PCR amplification primers and a single-base extension primerare respectively designed for each site, and an MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry) method is adopted to detect typing of the SNP sites of a sample to be detected. The complete-set primer disclosed by the invention has the beneficial effects that a detection method for the drug-metabolizing enzyme genes based on gene molecule typing is established, and the typing of multiple SNPs can be finished at the same time in the same reaction, so that the accurate and efficient effects are achieved and the cost is low; the covered genes and sitesare related to the metabolizing enzyme genes related to 8 major types of common drugs for children, and the coverage is most complete in gene detection products of safe drugs used for children so far.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a set of primers for detecting SNP sites of drug metabolizing enzyme genes and applications thereof. Background technique [0002] There are more than 3,500 kinds of pharmaceutical preparations in my country, of which only more than 60 are specially used for children. Among the only more than 60 special medicines for children, there are also many cases of wrong use. Because of improper medication, about 30,000 children in our country fall into a silent world every year, and it is even more difficult to count the damage caused to liver, kidney function and nervous system. In China, drug poisoning incidents among children are increasing year by year, reaching 73% in 2014. One out of every eight children has an adverse reaction due to improper medication, resulting in damage to liver, kidney function, and nervous system. [0003] The safety of medication is mainly related ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 张影
Owner 北京天平永达生物科技发展有限公司
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