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Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene

A polymorphism and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of easy false negative, high detection cost, low detection sensitivity, etc., to avoid non-specific Difficulties in heterosexual amplification, overcoming easy contamination, and good specificity

Inactive Publication Date: 2017-03-22
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 direct sequencing method results in more accurate sequencing results, its detection sensitivity is low, false negatives are prone to occur, the operation process is complicated, the sample is easily contaminated, it takes a long time, and the detection cost is high
The gene chip method and the liquid phase chip method are also complicated in operation process, high in detection cost, long in detection cycle, and the accuracy of the result is not high, and sample contamination is prone to occur
The above shortcomings limit the development and application of these three methods in actual clinical work, and cannot meet the needs of rapid clinical evaluation

Method used

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  • Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene
  • Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene
  • Nucleic acid, kit and method for detecting G1165C polymorphism of human ADRB1 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 primer, probe, verification template design

[0058] The invention is designed for the G1165C SNP site of the human ADRB1 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 tissues such as human peripheral blood cells or oral swabs. For detection, the genotype of the sample DNA is determined by collecting signals on a real-time fluorescent PCR instrument and calculating the △Ct value of the wild type and mutant type. And through the screening of mutant ARMS primers and Taqman probes and the optimization of the mutation detection system, a real-time fluorescent PCR mutation detection system was established to achieve high sensitivity and high specificity detection of ADRB1 gene polymorphism (G1165C). Through numerous experiments, optimization, and finally obtain the sequences of wild...

Embodiment 2

[0075]Example 2 The method for detecting the G1165C polymorphism of the human ADRB1 gene by real-time fluorescent PCR The wild-type downstream primers, mutant downstream primers, public upstream primers and public detection probes designed in Example 1 are synthesized, and the detection probes are synthesized. The 5' end is connected with a fluorescent group, and the 3' end is connected with a quenching group, wherein the fluorescent group can be any of FAM, JOE, VIC, CY3, JUN, ROX, HEX, and the quenching group can be MGB, Any one of BHQ1, TAMRA, BHQ2.

[0076] The method for detecting the G1165C polymorphism of the human ADRB1 gene by real-time fluorescent PCR is as follows:

[0077] (1) Extract DNA from the sample or obtain sample DNA;

[0078] (2) Real-time fluorescent PCR amplification detection of human ADRB1 gene G1165C site polymorphism is carried out on the extracted sample DNA, adopting wild-type reaction system and mutant reaction system, wherein, in the wild-type s...

Embodiment 3

[0099] Embodiment 3 is used for detecting the kit of human ADRB1 gene G1165C polymorphism

[0100] The real-time fluorescent PCR kit for detecting the G1165C polymorphism of the human ADRB1 gene includes the following components:

[0101] Wild-type downstream primer: the nucleotide sequence is shown in SEQ ID No.1;

[0102] Mutant downstream primer: the nucleotide sequence is shown in SEQ ID No.2;

[0103] Public upstream primer: nucleotide sequence as shown in SEQ ID No.3

[0104] 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;

[0105] Wild-type positive control: containing the nucleotide sequence shown in SEQ ID No.8;

[0106] Mutant positive control: containing the nucleotide sequence shown in SEQ ID No.9.

[0107] In order to avoid missed detection and wrong detection, it also includes: quality control primer pairs, q...

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Abstract

The invention discloses nucleic acid and a kit for detecting G1165C polymorphism of the human ADRB1 gene, and also establishes a method for detecting the G1165C polymorphism of the human ADRB1 gene, which is strong in specificity, high in sensitivity, high in degree of accuracy, and easy to operate. The kit for detecting the G1165C polymorphism of the human ADRB1 gene is applicable to the selection for various clinical samples, and has the remarkable advantages of strong specificity, high sensitivity, short experimental period, operation simplicity, safety and non-toxicity, low cost, and the like. The detection method provided by the invention adopts the completely closed tube operation, and is easy, convenient and rapid to operate, the detection result is obtained by directly directing fluorescence signal values during the PCR process, the PCR aftertreatment or electrophoresis detection is not needed, the large possibility of pollution and false positive property during the conventional PCR technology are overcome, the difficulty of non-specific amplification can be effectively avoided, and the detection method is suitable for detection samples in large batches.

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 the G1165C polymorphism of human ADRB1 gene. Background technique [0002] Cardiovascular and cerebrovascular diseases are collectively referred to as cardiovascular and cerebrovascular diseases. Diseases are characterized by high morbidity, disability and mortality. Epidemiological statistics show that the number of people who die from cardiovascular and cerebrovascular diseases in the world is as high as 15 million every year, ranking first among all causes of death in the world. Hypertension is an important cause and risk factor of cardiovascular and cerebrovascular diseases. Long-term hypertension can affect the structure and function of the heart, brain, kidney and other organs, and eventually lead to the failure of these organs, which is one of the main causes of death in patients with cardiovascular and cerebrovascu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2561/101
Inventor 李惠邹芳杨惠娟段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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