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Nucleic acid for detecting A1166C polymorphic site of AGTR1 gene, kit and method

An A1166C, polymorphic site technology, applied in the field of nucleic acid and kits of A1166C polymorphic sites, can solve the problems of inability to meet the requirements of rapid clinical evaluation, unsuitable mutation site detection, restricted development and application, etc. , to avoid the problem of non-specific amplification, the detection results are reliable, and the detection effect is good.

Active Publication Date: 2017-05-10
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

The Sanger sequencing method is the gold standard for mutation analysis, which can find known and unknown mutation sites, but the instrument is expensive, the sensitivity is low, the operation is complicated, the cycle is long, and it is easy to be polluted; the ordinary PCR-RFLP method is simple in technology, cheap in price, and suitable for a small amount of samples laboratory testing, but can only detect mutations with enzyme cleavage sites, and cannot be detected without enzyme cleavage sites, and there is a risk of false positives caused by PCR product contamination; gene chip technology has the characteristics of high throughput, miniaturization, and automation , suitable for genome-wide mutation scanning, not suitable for mutation site detection of a single gene, and has low precision and high price
These shortcomings limit the development and application of these three methods in actual clinical work, and cannot meet the requirements of rapid clinical evaluation

Method used

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  • Nucleic acid for detecting A1166C polymorphic site of AGTR1 gene, kit and method
  • Nucleic acid for detecting A1166C polymorphic site of AGTR1 gene, kit and method
  • Nucleic acid for detecting A1166C polymorphic site of AGTR1 gene, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 primer, probe, verification template design

[0057] The selected ARMS primers of the present invention are designed for the A1166C SNP site. The selected ARMS primers are selected from a set of primer combinations that artificially add mismatched mutation sites at the 3' end and its vicinity. By screening primers that can distinguish single site alleles, a high degree of specificity.

[0058] After experimental verification, the specific detection primers for detecting the A1166C polymorphism site of the AGTR1 gene were finally determined to include wild-type upstream primers (AGRT1-FW), mutant upstream primers (AGRT1-FM), and common downstream primers (AGTR1-R). And public detection probe (AGTR1-P), nucleotide sequence is as follows:

[0059] AGRT1-FW (SEQ ID No. 1):

[0060] 5'-GCAGCACTTCACTACCAAATGAGTA-3';

[0061] AGTR1-FM (SEQ ID No. 2):

[0062] 5'-GCAGCACTTCACTACCAAATGAGTC-3';

[0063] AGTR1-R (SEQ ID No. 3):

[0064] 5'-AAAATGTGGCTTTGCTTTGTC...

Embodiment 2

[0079] Example 2 The method for detecting the A1166C polymorphic site of the AGTR1 gene by real-time fluorescent PCR

[0080] 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.

[0081] 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, and perform real-time fluorescent PCR amplification detection of the A1166C polymorphic site of the AGTR1 gene, using a wild-type reaction system and mutant reaction system, both use 40 μL reaction system as shown in Table 1;

[0082] Table 1 PCR reaction solution configurati...

Embodiment 3

[0090] Example 3 Kit for detecting the A1166C polymorphic site of the AGTR1 gene

[0091] The real-time fluorescent PCR kit for rapid detection of the A1166C polymorphic site of the AGTR1 gene includes the following components:

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

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

[0094] Common downstream primers: the nucleotide sequence is 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, ...

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Abstract

The invention discloses a nucleic acid for quickly detecting an A1166C polymorphic site of an AGTR1 gene and establishes a detection kit and a quick detection method for efficiently and sensitively detecting the A1166C polymorphic site of the AGTR1 gene. The detection kit has the characteristics of convenience in use, simple and convenient operation, high degree of automation, capability of greatly simplifying the operation process and reducing the pollution in the operation process, good detection effect, high sensitivity, high specificity, high accuracy and high precision. According to the detection method provided by the invention, the fully closed tube operation is adopted, the operation is simple, convenient and quick, the detection result is acquired in the manner of directly detecting a fluorescence signal value in a PCR process, the PCR post-treatment or electrophoresis detection is not required, the easiness in pollution and false positive of the conventional PCR technology can be overcome, the non-specificity amplification problem can be effectively avoided and the method is suitable for the detection of large-batch samples.

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 A1166C polymorphic site of the AGTR1 gene. Background technique [0002] Essential hypertension (Essential Hypertension, EH) is one of the most common diseases that endanger human health. It is a polygenic disease that interacts with genetic and environmental factors. A large number of studies have found that the renin-angiotensin system (Rennin-Angiotensin System, RAS) plays an important role in the occurrence and development of EH. Angiotensin Ⅱ (Angiotenssin Ⅱ, Ang Ⅱ) is the most important vasoconstrictor active substance in the RAS system, it can act on peripheral blood vessels, make the veins contract, increase the blood volume returned to the heart, and finally lead to the occurrence of hypertension. Ang II has two main receptor subtypes, angiotensin II receptor subtype 1 and subtype 2 (Angiotensin II Type 1 / 2 Re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2563/107
Inventor 周艳琳邹芳段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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