Specific primer and kit for detecting gene polymorphism of cardiovascular disease drugs by virtue of liquid-phase chip technique

A liquid phase chip, specific technology, applied in the field of biotechnology and medicine, can solve the problems of unsuitable for clinical promotion, expensive testing equipment, low flexibility, etc., achieve great clinical application value, good sensitivity and stability, avoid sample pollution effect

Active Publication Date: 2018-12-07
东莞市厚街医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In the prior art, PCR-direct sequencing method, PCR-pyrosequencing method, fluorescent quantitative PCR method, PCR-high resolution melting curve method, allele-specific PCR method, PCR-restriction fragment length polymorphism method are often used Methods and solid chip methods are used to detect polymorphisms of one or several cardiovascular drug-related genes. Most of these methods have low flexibility, high cost, low throughput, long time, expensive detection equipment, and are not suitable for clinical promotion. , the cost of reagents for one inspection per person is tens to hundreds of dollars, and the existing detection kits do not target the spectrum of Chinese genetic diseases

Method used

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  • Specific primer and kit for detecting gene polymorphism of cardiovascular disease drugs by virtue of liquid-phase chip technique
  • Specific primer and kit for detecting gene polymorphism of cardiovascular disease drugs by virtue of liquid-phase chip technique
  • Specific primer and kit for detecting gene polymorphism of cardiovascular disease drugs by virtue of liquid-phase chip technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1. Primer set for detection of cardiovascular drugs-related genes by liquid chip technology

[0071] Design of primers and probes:

[0072] According to the rs numbers of the SNPs published on NCBI, the nucleotide sequences near the 15 mutation points were searched in Genbank, and Primer 6.0 primer design software was used to design specific primers for amplifying the following common cardiovascular drug-related target gene loci: CYP2C9 C430T polymorphism rs1799853 of gene, A1075C polymorphism rs1057910 of CYP2C9 gene, G1639A polymorphism rs9923231 of VKORC1 gene, C1347T polymorphism rs2108622 of CYP4F2 gene, G681A polymorphism of CYP2C19 gene rs4244285, G636A polymorphism of CYP2 Sex rs4986893, C806T polymorphism rs12248560 of CYP2C19 gene, Q192R polymorphism rs662 of PON1 gene, C2850T polymorphism rs16947 of CYP2D6 gene, C100T polymorphism rs1065852 of CYP2D6 gene, G1165C polymorphism of ADRB1 gene rs1801253, DH2 gene Primers for the G504A polymorphism rs671 ...

Embodiment 2

[0085] Example 2. Kit for simultaneous detection of polymorphisms of multiple cardiovascular drugs-related target genes using liquid chip technology of the present invention

[0086] The kit of the present invention includes:

[0087] 1. The primers in Table 1 and Table 2 designed in Example 1.

[0088] 2. 30 MagPlex-Tag microspheres coupled with addressing probe sequences capable of complementary pairing with tag sequences in ASPE extension primers. The 30 kinds of microsphere numbers and the anti-Tag sequences coupled to the microspheres are shown in Table 3:

[0089] Table 3. 30 MagPlex-Tag microspheres coupled with addressing probe sequences (anti-Tag sequences)

[0090]

[0091]

[0092] The selected 30 kinds of MagPlex-Tag microspheres were purchased from Luminex Company in the United States at a concentration of 2.5 × 10 6 pcs / ml, diluted to 2500 pcs per microsphere when used, wherein the addressing probe sequence is coupled to the microsphere and is complement...

Embodiment 3

[0094] Example 3. Detection of common cardiovascular drug-related target gene polymorphisms using the primer set and kit of the present invention

[0095] In this example, the primer set of Example 1 and the kit of Example 2 were used to detect 15 target gene polymorphisms of common cardiovascular drugs.

[0096] The specific experimental steps are as follows:

[0097] 1. Multiplex PCR amplification reaction: A total of 14 whole blood samples (blood samples from Dongguan Houjie Hospital) were extracted. According to the experimental requirements, another negative control was set up to extract DNA. Use the specific amplification primers in Table 1 of Example 1 to prepare a multiplex PCR reaction system (50 μl):

[0098] Element

Dosage (μl)

gDNA

50ng (2μl)

10x PCR reaction buffer

5

Taq TM HotStar

0.25

dNTPs (2.5mM each)

4

Upstream and downstream primers (10μM)

0.5 each

Deionized water

30.75

[00...

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Abstract

The invention discloses a primer group for detecting the gene polymorphism of cardiovascular disease drugs by virtue of a liquid-phase chip technique. The primer group comprises 15 groups of primers,each group of the primers include a pair of specific amplification primers for detecting the polymorphism of a target gene as well as a wide ASPE primer and a mutant ASPE primer respectively aiming atthe target gene. The invention further discloses a kit including the primer group and a use method of the kit. The primer group and the kit can be used for amplifying a target sequence through one-step PCR reaction and simultaneously detecting the polymorphism of relevant target genes of 15 common cardiovascular drugs through the combination of MagPlex microspheres, the detection flux and the cost performance are high, the medication guidance can be provided for patients with cardiovascular diseases through once detection, and laboratory basis can be provided for the clinical rational drug use; and the kit has very high sensitivity, stability and accuracy in the actual use and is well matched with a gene sequencing result, and the detection cost is greatly lowered.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to specific primers for detecting polymorphisms of commonly used drugs for cardiovascular diseases using liquid-phase chip technology. The invention also relates to a kit comprising these specific primers and a method for using the kit. Background technique [0002] Cardiovascular disease is caused by disorders of the heart and blood vessels, including coronary heart disease (heart attack), cerebrovascular disease (stroke), hypertension (high blood pressure), peripheral vascular disease, rheumatic heart disease, congenital heart disease, cardiac failure and cardiomyopathy. Cardiovascular disease is a common disease that seriously threatens the health of human beings, especially the middle-aged and elderly people over 50 years old. At present, cardiovascular disease death accounts for the first cause of death among urban and rural residents, with 45.01% in rural areas and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/16C12Q2600/156C12Q2537/143C12Q2535/125C12Q2563/149
Inventor 付文金
Owner 东莞市厚街医院
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