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Nucleic acid, kit and method used for detecting polymorphism of COX-1, COX-2 and GPIIIa genes

A gene polymorphism, COX-1 technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of cross-contamination of PCR products, insufficient enzyme digestion, and complicated steps. , to avoid the problem of non-specific amplification, the detection results are reliable, and the effect of overcoming easy contamination

Active Publication Date: 2017-06-13
武汉海吉力生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are many methods for gene polymorphism detection. The simplest method is PCR-RFLP, but this method is complicated to operate, and it is easy to cause cross-contamination of PCR products when there are many samples, and it is prone to insufficient or excessive digestion. And false negative or false positive results, low reliability
Although the DNA sequencing method is the gold standard, the steps are cumbersome, the process is complicated, and cross-contamination between samples is prone to occur, resulting in sequencing failure. In addition, the price of the sequencer is beyond the tolerance of general clinical testing laboratories.
High-resolution melting curve method is fast, simple, economical and practical, but it is controlled by the temperature of the instrument, and the false positive is high

Method used

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  • Nucleic acid, kit and method used for detecting polymorphism of COX-1, COX-2 and GPIIIa genes
  • Nucleic acid, kit and method used for detecting polymorphism of COX-1, COX-2 and GPIIIa genes
  • Nucleic acid, kit and method used for detecting polymorphism of COX-1, COX-2 and GPIIIa genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 primer, probe, verification template design

[0073] The specific principle of the present invention is: design wild-type and mutant ARMS primers and Taqman-MGB probes respectively for different gene loci, and combine with fluorescent quantitative PCR reaction to detect genomic DNA extracted from human peripheral blood cells or oral swabs , which can realize the detection of cyclooxygenase 1 (COX-1) gene A842G, cyclooxygenase 2 (COX-2) gene G-765C and platelet membrane glycoprotein (GPIIIa) gene in one 6-way PCR reaction strip at one time The gene polymorphism of T1565C is detected, and the genotype of the sample DNA is determined by collecting signals on a real-time fluorescent PCR instrument and calculating the ΔCt values ​​of the wild type and the mutant type.

[0074] Design wild-type upstream primers, mutant upstream primers, public downstream primers and public detection probes for the A842G site of the human COX-1 gene, the G-765C site of the COX-2 g...

Embodiment 2

[0092] Example 2 Method for detecting COX-1, COX-2 and GPIIIa gene polymorphisms by real-time fluorescent PCR

[0093] The wild-type upstream primers, mutant upstream primers, public downstream primers and public detection probes of each gene screened and designed in Example 1 were synthesized, and a fluorescent group was connected to the 5' end of the detection probe, and a quencher was connected to the 3' end. Group, wherein, the fluorescent group can be any one of FAM, JOE, CY3, HEX, and the quenching group can be any one of MGB, BHQ1, TAMRA, BHQ2.

[0094] A method for detecting polymorphisms of COX-1, COX-2 and GPIIIa genes by real-time fluorescent PCR. Specific steps are as follows:

[0095] (1) Obtain the DNA of the sample to be tested;

[0096] (2) Apply the designed detection primers and detection probes to perform real-time fluorescent PCR amplification detection on the A842G, G-765C, and T1565C polymorphisms of the COX-1, COX-2, and GPIIIa genes in the DNA of the ...

Embodiment 3

[0122] Embodiment 3 is used for detecting the kit of COX-1, COX-2 and GPIIIa gene polymorphism

[0123] The real-time fluorescent PCR kit for detecting polymorphisms of COX-1, COX-2 and GPIIIa genes includes the following components:

[0124] COX-1 gene A842G wild-type primer pair: the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3;

[0125] COX-1 gene A842G mutant primer pair: the nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3;

[0126] COX-1 gene A842G detection probe: the nucleotide sequence is shown in SEQ ID NO.4;

[0127] COX-2 gene G-765C wild-type primer pair: the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.7;

[0128] COX-2 gene G-765C mutant primer pair: the nucleotide sequence is shown in SEQ ID NO.6, and the downstream primer is shown in SEQ ID NO.7;

[0129] COX-2 gene G-765C detection probe: the nucleotide sequence is shown in SEQ ID NO.8;

[0130] GPIIIa gene T1565C wild-type primer pair: the nucleotide sequences...

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Abstract

The invention discloses nucleic acid and a kit used for detecting polymorphism of COX-1, COX-2 and GPIIIa genes as well as detecting method for detecting polymorphism of the COX-1, COX-2 and GPIIIa genes, and the method is strong in specificity, is high in sensitivity, is high in accuracy and is simple to operate. The detected results can provide guidance meaning for aspirin resistance. The detecting method provided by the invention adopts completely closed-tube operation, is simple to operate, is convenient and quick, obtains the detected results by directly detecting a fluorescence signal value in a PCR process, PCR post-treatment or electrophoresis detection is not needed, the problems such as easy pollution and false positive results in the conventional PCR technology are overcome, the nonspecific amplification problem can be effectively avoided, and therefore, the nucleic acid, the kit and the method are suitable for large-scale sample detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid, a kit and a method for detecting COX-1, COX-2 and GPIIIa gene polymorphisms. Background technique [0002] Single Nucleotide Polymorphisms (Single Nucleotide Polymorphisms, SNP) refers to the genetic markers formed by the variation of a single nucleotide on the genome (including substitutions, transversions, deletions and insertions). Compared with microsatellite molecular markers, its number Many, polymorphic rich. Generally speaking, a SNP refers to a single nucleotide variation with a variation frequency greater than 1%. There is approximately one SNP for every 1000 bases in the human genome, and the total number of SNPs on the human genome is approximately 3×10 6 indivual. Therefore, SNP has become the third-generation genetic marker, and many phenotypic differences in the human body, sensitivity to drugs or susceptibility to diseases, etc. may be r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2561/113C12Q2535/137C12Q2563/107
Inventor 李存耀史学晖段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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