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Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation

A multi-site mutation and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of not being able to obtain multiple site information at the same time, and achieve the effect of high sensitivity

Inactive Publication Date: 2016-07-27
钟诗龙
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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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  • Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation
  • Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation
  • Kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Design of primers and probes

[0049] The specific primers and probes based on the Taqman allele resolution analysis method are shown in Table 1. At the same time, Tables 2 and 3 are the other two sets of primers and probes based on the Taqman allele resolution analysis method, as shown in Table 2 and Table 3. The primers and probes can not specifically detect multi-site mutations in SLCO1B1, APOE and LDLR genes.

[0050] Table 1 Specific primer and probe sequence

[0051]

[0052]

[0053]

[0054] Table 2 Primers and probes that failed to detect multiple mutations in SLCO1B1, APOE and LDLR genes

[0055]

[0056]

Embodiment 2

[0057] Example 2 Fluorescence quantitative PCR detection process

[0058] 1. A 96-well plate design with 8 rows×12 columns is adopted, in which 8 clinical samples are tested in columns 1 to 8, and corresponding negative no-DNA control templates, wild type, and mutant types are installed in columns 9 to 12 , Hybrid quality control template, its specific design is shown in Table 4.

[0059] Table 496-well PCR reaction plate recommended layout example

[0060]

[0061] Note: S stands for samples, S1 to S8 stands for 8 clinical samples; NTC stands for negative control wells without DNA samples; W stands for wild-type quality control template; M stands for mutant quality control template; H stands for heterozygous quality control template .

[0062] Among them, the preparation of wild-type quality control template, mutant-type quality control template, and hybrid-type quality control template is as follows: the wild-type and mutant-type DNA fragments containing the above sites are synthes...

Embodiment 3

[0072] Example 3 Assembly of detection kit

[0073] A kit for simultaneous detection of SLCO1B1, APOE and LDLR gene multi-site mutations, including the following components: the concentration of 0.5μM, the volume of 0.5 ~ 1.25μL of each primer described in Table 1 (including design for each gene site Forward primer and reverse primer), each probe described in Table 1 (including wild allele probes and mutant allele probes designed for each gene locus) with a concentration of 0.25μM and a volume of 0.125~0.625μL Needle), 5-15μL 2×TaqmanMasterMix, with double distilled water to make up the total volume to 10-25μL, the total volume of the reaction system is 10-25μL; wild-type quality control template; mutant quality control template; heterozygous quality control template.

[0074] In order to explain the composition of the above kit more concisely, here is an example of the components. The components of the kit are as follows: sample genomic DNA at an appropriate concentration and volu...

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Abstract

The invention belongs to the technical field of gene mutation detection, and concretely discloses a kit for simultaneously detecting SLCO1B1, APOE and LDLR gene multisite mutation. Through meticulous design, multi-time verification, screening and optimization, specific primers and probes based on a Taqman allelic gene resolution analysis method are obtained; eight functional variation of the SLCO1B1, APOE and LDLR genes can be detected; the time from DNA (Deoxyribonucleic Acid) extraction to fluorescent PCR (Polymerase Chain Reaction) to result obtaining is less than four hours; and the manual operation time is less than two hours. The kit comprising the primer pairs and the probe pairs has the advantages that the time is saved; convenience is realized; the sensitivity is high; and both the positive conformity rate and the negative conformity rate of a sample are higher than 99 percent, and the like. A detection method provided by the invention is mainly used for the personalized medication auxiliary diagnosis of statins such as simvastatin, atorvastatin, fluvastatin and rosuvastatin.

Description

Technical field [0001] The present invention relates to the technical field of gene mutation detection, and more specifically, to a kit for simultaneously detecting multiple site mutations of SLCO1B1, APOE and LDLR genes. Background technique [0002] Statins are widely used lipid-lowering drugs that can effectively prevent cardiovascular and cerebrovascular diseases. In the United States, more than 25% of people over 45 years old take statins. In the secondary prevention of coronary heart disease after percutaneous stent implantation (PCI), the use of active statin lipid-lowering therapy is of great significance to the prevention and treatment of coronary heart disease. [0003] As the dose of statins increases, the effect of lowering LDL increases, but at the same time the incidence of adverse reactions is also high. One of the main side effects of statins is myopathy, and 5 to 29% of patients will develop statin-induced myopathy (statin-induced myopathy, SIM), mainly manifested...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2545/113C12Q2561/101C12Q2537/143
Inventor 钟诗龙
Owner 钟诗龙
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