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Detection kit for predicting curative effect of statins as well as detection method and application thereof

A detection kit and the technology of the kit, which are applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low specificity, easy contamination of amplified products, and difficult primer design.

Inactive Publication Date: 2021-09-17
湖南菲思特精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sequencing method and the chip method have cumbersome operation steps, long detection cycle, and the amplification product is prone to contamination; the high-resolution melting curve method has simple steps, low specificity, and high requirements for equipment; The specific amplification method uses ARMS primers for specific amplification, and the design of the primers is difficult to optimize, and the detection conditions are strict.
Taqman fluorescent probe method has high test cost, and the amplification throughput of multiple genes is not high

Method used

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  • Detection kit for predicting curative effect of statins as well as detection method and application thereof
  • Detection kit for predicting curative effect of statins as well as detection method and application thereof
  • Detection kit for predicting curative effect of statins as well as detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the preparation of kit

[0040] The detection kit of the present invention designs specific amplification primers and sequencing primers for ApoE (T388C) and ApoE (C526T), and is used for constant temperature amplification and pyrosequencing detection. The design of primers based on the recombinant enzyme polymerase amplification technology is one of the keys of the present invention. The primer design of this technology cannot be carried out by auxiliary software, and can only rely on manual design. In order to ensure the amplification speed and detection sensitivity, the primer length should be controlled at 30-35 bp. If the primer design is too short, non-specific amplification will easily increase and cause false positives. If the primer design is too long, it will easily lead to failure of amplification. Gene polymorphism sequence is subject to the public sequence in Genebank.

[0041] (1) The primer sequences of this embodiment are as follows:

[0...

Embodiment 2

[0069] Embodiment 2, pyrophosphate detection

[0070] The instrument that adopts among the present invention is as follows: thermostat;

[0071] Pyrosequencer: Wuhan First Biotechnology Co., Ltd.

[0072] (1) Reagent preparation (reagent preparation room)

[0073] Take out the reagent in advance, vortex reagent 1 for 15 seconds, and centrifuge at low speed for later use. Add 440ul of Reagent 1 directly to Reagent 2 (lyophilized), and vortex for 15 seconds to mix well. Determine the number of reactions N, N = number of samples to be tested (n) + number of quality control products (1) + blank control. It is recommended to conduct positive control and blank control analysis for each PCR experiment at the same time. Then the reaction solution was dispensed into PCR reaction tubes at 20 μL / tube.

[0074] (2) Sample testing (sample preparation room)

[0075] Add the sample DNA, positive control and blank control into the PCR reaction tube according to the amount of 5 μL, close...

Embodiment 3

[0099] Example 3. Correlation between genetic testing results and drug efficacy and cardiovascular disease

[0100] Apolipoprotein E is a very important protein in the human body, which is closely related to lipoprotein metabolism. For example, it is involved in the activation of enzymes that hydrolyze fat, immune regulation and regeneration of nerve tissue, thus affecting the occurrence, development and prognosis of many diseases.

[0101] Correspondence between phenotype and gene locus

[0102] genotype ApoE type Corresponding locus genotype E2 ε2 / ε2 388T / T+526T / T type E2 ε2 / ε3 388T / T+526T / C type E3 ε2 / ε4 388T / C+526T / C type E3 ε3 / ε3 388T / T+526C / C type E4 ε3 / ε4 388T / C+526C / C type E4 ε4 / ε4 388C / C+526C / C type

[0103]

[0104]

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Abstract

The invention discloses a detection kit for predicting the curative effect of statins as well as a detection method and application thereof, the kit is used for detecting the polymorphism of two genes, namely, ApoE T388C and ApoE C526T, related to the curative effect of statins, and the kit comprises the following components: an amplification reaction solution, ApoE T388C amplification primers, APOE T388C sequencing primers, ApoE C526T amplification primers, ApoE C526T sequencing primers and a positive control. According to the present invention, multiple RPA amplification and an optimized pyrosequencing technology are adopted as the combination to detect the gene polymorphism related to the curative effect of statins, and the kit can simultaneously detect the ApoE (T388C) and ApoE (C526T) gene polymorphism statin drugs so as to provide the gene perspective suggestion for the clinical personalized medication.

Description

technical field [0001] The invention relates to a detection kit for predicting the curative effect of statins, a detection method and application thereof, and belongs to the field of gene detection. Background technique [0002] According to my country's epidemiological survey, the overall prevalence of dyslipidemia in Chinese adults was as high as 40.40% in 2012, which was a sharp increase compared with 10 years ago. The increase in serum total cholesterol (TC) levels will lead to an increase in cardiovascular disease events in China in the future About 9.2 million. Statins can significantly reduce the risk of cardiovascular events in the primary and secondary prevention of ASCVD, and are the most important drugs for the prevention and treatment of such diseases. However, although different statins have similarities in chemical structure, there are inter-individual differences in their pharmacokinetics, interactions with other drugs, drug efficacy, and adverse reactions cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/16C12Q2600/172C12Q2531/119C12Q2537/1376C12Q2522/101C12Q2521/507C12Q2537/143C12Q2535/122C12Q2565/301
Inventor 刘丹李丽
Owner 湖南菲思特精准医疗科技有限公司