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Human aspirin resistance gene polymorphism detection kit as well as preparation method and application thereof

A technology for detecting kits and genes, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of long time-consuming experiments, low detection sensitivity, poor specificity, etc., and prevent false positives Negative and false positive results, effect of guaranteeing accuracy

Inactive Publication Date: 2018-12-21
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The PCR-sequencing method has low sensitivity and takes a long time for the experiment, so it is not suitable for clinical promotion; the chip hybridization method has low detection sensitivity and poor specificity, and is prone to false positive results; the high-resolution melting curve method has special requirements for equipment and is not suitable for clinical promotion. Detection sensitivity is not high
The Taqman-qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and it is genotyped by the Genotyping method, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting mutation frequency too low position

Method used

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  • Human aspirin resistance gene polymorphism detection kit as well as preparation method and application thereof
  • Human aspirin resistance gene polymorphism detection kit as well as preparation method and application thereof
  • Human aspirin resistance gene polymorphism detection kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 prepares aspirin resistance gene detection kit of the present invention

[0066] 1. Primer design and synthesis:

[0067] Each system of the 3 sites of PEAR1, GP1BA and GPIIIa contains 6 primers; two sense chain upstream primers F1 and F2, two antisense chain downstream primers R1 and R2, F1 and R1 are common primers, F2 and R2 Specific ARMs primers with fluorophore and tag sequence, screened by primers and PCR reaction conditions to ensure that the primer pair F1R1 enriches the template, while F2 and R1, F1 and R2 can normally amplify specific genes with fluorescence Type PCR products; at the same time, internal reference primers were added to each gene locus.

[0068] The specific sequence is shown in Table 1:

[0069] Table 1 Primer Sequence

[0070] name

sequence

serial number

note

PEAR1-F1

5'-TTGTGGACTTCTCTTCCTTC-3'

SEQ ID NO.1

PEAR1-F2

5'-VIC-ATCCCTTGTCATCGTCAGATACG-3'

SEQ ID NO.2

Specific ...

Embodiment 2

[0099] The human aspirin resistance gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested. In this example, 100 cases of normal human peripheral blood samples were collected, genomic DNA was extracted, and the human aspirin resistance gene polymorphism detection kit was used to detect the polymorphism of the aspirin resistance gene. The specific operation process is as follows:

[0100] (1) Genomic DNA extraction from blood samples: Use a commercial extraction kit to extract genomic DNA. After the extraction is complete, use TE buffer to elute the DNA and measure the DNA concentration; dilute the genomic DNA to 20ng / μl;

[0101] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C f...

Embodiment 3

[0118] Use the kit prepared in Example 1 to detect and verify the minimum detection line of this kit, specifically including the following steps:

[0119] (1) Using samples of the genotypes of the corresponding loci of PEAR1, GP1BA, and GPIIIa genes known in Example 2, select one case each of the wild-type genome, mutant genome, and heterozygous genome samples for each locus, and dilute the above-mentioned samples Up to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.001ng / μL;

[0120] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, collect fluorescence; each concentration gradient of each sample is repeated 20 times;

[0121] (3) After the PCR reaction is com...

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Abstract

The invention belongs o the technical field of biology and specifically relates to a human aspirin resistance gene polymorphism detection kit as well as a preparation method and application thereof. The kit is prepared from PCR premixed reaction liquid, a positive control product and a negative control product, wherein the PCR premixed reaction liquid, the positive control product and the negativecontrol product are respectively used for amplifying polymorphism of 3 genes of GPIIIa, GP1BA and PEAR1 which are related to human aspirin resistance; the PRC premixed reaction liquid is prepared from a specificity primer sequence set, a probe set and PCR reaction liquid; the specificity primer sequence set is used for amplifying all the sites; the specificity primer set is prepared from an ordinary outer primer and an ARMs primer with fluorescent label specificity. The kit disclosed by the invention is used for detecting the human aspirin resistance gene polymorphism and has the advantages of high flexibility, high specificity, convenience in operation, reliable results and the like; furthermore, detection can be finished within 1 hour, and result reading is simple and objective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human aspirin resistance gene polymorphism detection kit and its preparation method and application. Background technique [0002] Aspirin is the basic antithrombotic drug in the treatment of acute coronary syndrome and percutaneous coronary intervention, and is widely used in the primary and secondary prevention of cardiovascular and cerebrovascular diseases. It is clinically found that some patients cannot effectively inhibit platelet activity despite long-term low-dose aspirin use, that is, aspirin resistance. The incidence rate is about 50% to 60%, and there are obvious racial differences. Studies have shown that polymorphisms of GPIIIa, GP1BA and PEAR1 genes play an important role in aspirin resistance; [0003] The platelet membrane glycoprotein IIIa gene is encoded by the ITGB3 gene, and the mutation (rs5918) of the 1565th amino acid in exon 2, namely T1565C (Le...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 晏星李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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