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PCR primer combination for adverse reaction genotype polymorphism of statin drugs and application

A technology of adverse reactions and primer combinations, which is applied in the field of biomedical molecular detection, can solve the problems of increasing the workload of probes to detect point mutations, difficulty in completely quenching fluorescence, and high requirements for instrument consumables, so as to reduce manual operations, control detection costs, Improve the effect of economic and social benefits

Inactive Publication Date: 2019-03-29
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantages are: the fluorescence quenching is difficult to complete, and the background is high; the hydrolysis of the probe depends on the exoenzyme activity of Taq, so the quantification is affected by the performance of the enzyme and the quality of the reagent; because the probe is bound to the template, it is difficult for the amplification Increased efficiency has a certain impact; longer probes need to be designed, which increases the workload of probe design; the ability to detect point mutations is relatively insufficient; the requirements for instruments and consumables are high

Method used

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  • PCR primer combination for adverse reaction genotype polymorphism of statin drugs and application
  • PCR primer combination for adverse reaction genotype polymorphism of statin drugs and application
  • PCR primer combination for adverse reaction genotype polymorphism of statin drugs and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Multiplex PCR primer design and screening

[0066] Search SNP locus SLCO1B1 gene*5c.521T>C(rs4149056), SLCO1B1 gene c.388A>G(rs2306283), ApoE gene c.388T>C(rs429358) and ApoE gene c.526C>T(rs7412) from NCBI After bioinformatics comparison and analysis, a section of highly conserved regions containing one of the above-mentioned SNP sites was found, and each highly conserved region containing the corresponding SNP site (4 in total) was designed 3 -5 specific primer pairs (4 groups in total) and 3 to 5 single base extension primers for the corresponding SNP sites (4 groups in total), the combination of primer pairs between the 4 groups through different sequence analysis software such as DNAstar Perform interference analysis and evaluation on the combination of primers and single-base extension primers respectively. Finally, from each set of primer pairs, select the best two primer pairs after combining with other primer pairs (as shown in Table 1); and Choose a s...

Embodiment 2

[0072] Example 2, Quadruple PCR primer combination optimization (sensitivity detection experiment)

[0073] Randomly combine the four sets of primer pairs shown in Table 1 (total 2 4 After a combination), perform sensitivity testing as follows:

[0074] Take a purified genomic DAN sample of an adult EDTA anticoagulated blood sample and dilute it with sterile water to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.01ng / μL, 0.005ng / μL, 0.0025ng / μL, respectively draw 10μL as a template, and use the primer pair combinations shown in Table 3 to perform quadruple PCR detection. Each amplified product is sequentially subjected to SAP reaction, single base Extension reaction (using the combination of the four single-base extension primers shown in Table 2) and mass spectrometry detection, count the detection results of each primer pair combination for each sample concentration, the experiment set water as a negative control, and a single primer pair of eight Re-PCR is a pos...

Embodiment 3

[0104] Example 3. Blind sample detection

[0105] Use the primer pair combination 1-1 / 2-1 / 3-2 / 4-1 in Table 3 and the single-base extension primer combination in Table 2, and detect 10 copies respectively according to the method of steps 1-7 in Example 2. The genotype of the unknown sample (adult EDTA anticoagulated blood) at rs4149056, rs2306283, rs429358 and rs7412, where the concentration of the template DNA solution in step 2 is 10ng / μL (excess).

[0106] Control 1: Use traditional PCR fluorescent probe method to detect the genotype of each SNP site individually.

[0107] Control 2: Use the Sanger sequencing method to detect the genotype of each SNP site with the product obtained in step 2.

[0108] Results: The test results of the above three methods are exactly the same. The test results of one of the samples are as follows:

[0109] Mass spectrometry results such as figure 1 As shown in —4, the results show that the genotype of rs4149056 locus of the tested sample is TT, the gen...

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Abstract

The invention discloses a PCR primer combination for adverse reaction genotype polymorphism of statin drugs and an application and belongs to the technical field of biomedical molecular detection. ThePCR primer combination comprises primer pairs 1-4 for amplifying SNP sites rs4149056, rs2306283, rs429358 and rs7412, with the sequences shown in SEQ ID No.1-2, 5-6, 11-12 and 13-14; the PRC primer combination also comprises a single base extension primer combination for specifically amplifying the SNP sites, with the sequence shown in SEQ ID No.17. By means of the primer combination and the detection method, the technical problem that multiple target amplification conditions are not compatible is solved, the effect that multiple primers are added to one reaction hole and not interfere one another is realized, and the lowest detection limit can reach 0.01 ng / mu L when four mutant site genotypes on different genes about safety and curative effects of statin drugs are detected.

Description

Technical field [0001] The present invention belongs to the technical field of biomedical molecular detection, and specifically relates to the detection of four SNP sites: SLCO1B1 gene *5c.521T>C (rs4149056), SLCO1B1 gene c.388A>G (rs2306283), ApoE gene c.388T> Detection primers for nucleotide polymorphisms of C (rs429358) and ApoE gene c.526C>T (rs7412) and their applications. Background technique [0002] Statins (3-hydroxy-3-methylglutarate-coenzyme A reductase inhibitors) are a class of drugs used to lower cholesterol (LDL-C) by inhibiting the key enzyme of cholesterol synthesis in the liver-hydroxymethyl HMG-CoA reductase (HMG-CoA reductase) reduces the synthesis of cholesterol by the liver and removes excess cholesterol from the blood. In addition to their lipid-lowering effects, statins also exhibit pleiotropic effects such as reducing systemic inflammation, improving endothelial function and reducing platelet hyperresponsiveness, and can significantly reduce ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113C12Q2533/101C12Q2565/627
Inventor 刘文兰叶秀峰徐迹李赟黄建林
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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