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Nucleic acid, kit and method for rapid detection of C3435T polymorphism of ABCB1 gene

A polymorphism and kit technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problems of complex operation process, complicated experimental operation and low detection sensitivity of liquid chip method. , to achieve the effect of reducing the process of diluting the sample, fast and simple synthesis, and high sensitivity

Active Publication Date: 2017-05-31
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the DNA sequencing method is the gold standard for genotyping, its detection sensitivity is low, the sample is easily contaminated, false positives occur, the experimental operation is complicated, time-consuming, and the detection efficiency is low
Although the melting curve method is fast and simple, it has a high false positive rate.
The operation process of the liquid chip method is complicated, and it is easy to be polluted, and the false positive rate is high

Method used

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  • Nucleic acid, kit and method for rapid detection of C3435T polymorphism of ABCB1 gene
  • Nucleic acid, kit and method for rapid detection of C3435T polymorphism of ABCB1 gene
  • Nucleic acid, kit and method for rapid detection of C3435T polymorphism of ABCB1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 primer, probe, verification template design

[0052] The selected primer pins and probes of the present invention are designed for the SNP site at C3435T of the ABCB1 gene. The specific principle of primers and probes is: design wild-type and mutant ARMS primers and Taqman probes for the mutation sites, combined with fluorescent quantitative PCR reaction, to detect genomic DNA extracted from human peripheral blood cells, through real-time fluorescent PCR The signal is collected on the instrument, and the △Ct value of wild type and mutant type is calculated to determine the genotype of the sample DNA.

[0053] After experimental verification, it was finally determined that the specific detection primers for detecting the C3435T polymorphism site of the ABCB1 gene included wild-type downstream primers (ABCB1-RW), mutant downstream primers (ABCB1-RM), common upstream primers (ABCB1-F) and Public detection probe (ABCB1-P), the nucleotide sequence is as follows...

Embodiment 2

[0073] Example 2 The method for detecting the C3435T polymorphic site of the ABCB1 gene by real-time fluorescent PCR

[0074] The wild-type upstream primer, mutant upstream primer, public downstream primer and public detection probe designed according to Example 1 are synthesized, and a fluorophore can be connected to the 5' end of the public detection probe, and a quencher can be connected to the 3' end. Group, wherein the fluorescent group is any one of FAM, JOE, CY3, HEX, and the quenching group is any one of MGB, BHQ1, TAMRA, BHQ2.

[0075]Apply the wild-type primer pair, mutant-type primer pair and public detection probe designed by the present invention to the DNA template of the detection sample to perform real-time fluorescent PCR amplification detection of the C3435T polymorphic site of the ABCB1 gene, using the wild-type reaction system and For the mutant reaction system, a 40 μL reaction system is used, and the 30 μL reaction system is configured as shown in Table 1...

Embodiment 3

[0090] Example 3 The kit for detecting the C3435T polymorphic site of the ABCB1 gene

[0091] The real-time fluorescent PCR kit for rapid detection of ABCB1 gene C3435T polymorphic site includes the following components:

[0092] Wild-type downstream primer: the nucleotide sequence is shown in SEQ ID No.1;

[0093] Mutant downstream primer: the nucleotide sequence is shown in SEQ ID No.2;

[0094] Public upstream primer: nucleotide sequence as shown in SEQ ID No.3

[0095] Public detection probe: the nucleotide sequence is shown in SEQ ID No.4, the 5' end of the public detection probe is connected to a fluorescent group, and the 3' end is connected to a quenching group;

[0096] Wild-type positive control: containing the nucleotide sequence shown in SEQ ID No.8;

[0097] Mutant positive control: containing the nucleotide sequence shown in SEQ ID No.9.

[0098] In order to avoid missed detection and wrong detection, it also includes: quality control primer pairs, quality co...

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Abstract

The invention discloses a nucleic acid, kit and method for rapid detection of C3435T polymorphism of an ABCB1 gene. The nucleic acid comprises detection primers and a detection probe, wherein the detection primers comprise a wild downstream primer, a mutant downstream primer and a common upstream primer; a nucleotide sequence of the wild downstream primer is as shown in SEQ ID No.1, a nucleotide sequence of the mutant downstream primer is as shown in SEQ ID No.2, a nucleotide sequence of the common upstream primer is as shown in SEQ ID No.3, and a nucleotide sequence of the detection probe is as shown in SEQ ID No.4. The kit and method for real-time fluorescent quantitative PCR rapid detection of C3435T polymorphism of the ABCB1 gene have the advantages of being convenient to use, simple to operate and high in automatic degree, greatly simplifying the operation process and reducing pollution in the operation process, and the detection effect is good; the kit has the characteristics of high sensitivity, specificity, accuracy and precision.

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 rapidly detecting the C3435T polymorphism of the ABCB1 gene by using fluorescent quantitative PCR technology. Background technique [0002] Coronary heart disease is one of the common cardiovascular diseases in my country. Percutaneous coronary intervention (PCI), as a method of minimal trauma in myocardial revascularization, can greatly improve the long-term prognosis of patients with coronary heart disease, and has become an important means of treating coronary heart disease. However, thrombus is easy to form at the stent implantation site after PCI, resulting in varying degrees of obstruction, leading to a series of acute complications such as unstable angina, acute myocardial infarction, and even sudden death. Even unstented coronary arteries are frequently associated with thrombotic adverse events due to progression of atherosc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 杨惠娟邹芳段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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