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Probe, primer and kit for detecting ACE2 gene polymorphism

A gene polymorphism and kit technology, applied in the field of molecular biology, can solve the problems of high cost, time-consuming and laborious, unsuitable detection, etc., and achieve the effect of simple operation, improved curative effect, and rapid screening

Pending Publication Date: 2022-04-12
上海利康精准医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe, primer and kit for detecting ACE2 gene polymorphism
  • Probe, primer and kit for detecting ACE2 gene polymorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Probe and Primer Design

[0026] The present invention designs probe and primer sequences for the ACE2 SNP site. The specific principle is to use the conformational change of the fluorescent probe and the target sequence to release the fluorescent dye, and to judge the genotyping result according to the peak maps and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher When the quenching groups are separated from each other, the fluorescent signal can be detected.

[0027] Design probes and primers so that when the template does not exist at the annealing temperature, the probe is in a stem-loop state, including the loop sequence and its reverse complementary stem sequences on both sides, with a total length o...

Embodiment 2

[0034] Example 2 Detection of different genotype standards

[0035] 1. Use the plasmid ACE2 to construct and prepare the wild-type standard plasmid and mutant standard plasmid containing the rs2285666 site of the target gene (the source of the plasmid and the synthesis of the plasmid containing the target gene are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) . The accuracy of the sequence was confirmed by sanger sequencing. The genotype of the wild-type standard plasmid rs2285666 was CC; the genotype of the mutant standard plasmid rs2285666 was TT. The standard plasmid DNA concentration was normalized to 10 ng / ul.

[0036] 2. Use the probes and primers in Example 1.

[0037] 3. PCR reaction system:

[0038] 1) Add 7.5 ul of PCR Mix, 0.5 uM of primer 1 solution, 0.5 uM of primer 2 solution, and 0.1 uM of probe to each PCR reaction well in turn, and then add wild-type standard plasmid to 3 different PCR reaction wells respectively DNA, mutant standard plasmid D...

Embodiment 3

[0041] Example 3 Detection method performance analysis experiment

[0042] 1. Precision test

[0043]The wild-type standard plasmid DNA and mixed-type DNA (rs2285666 plasmid source, and the synthesis of the plasmid containing the target gene were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The wild-type standard plasmid and mutant standard plasmid were according to 1: 1 ratio of mixing), each of which was tested 3 times a day for 5 consecutive days. The amplification reaction procedure adopted the method in Example 2, and the results were shown in figure 2 . It shows that the fluorescence PCR amplification reaction of the present invention has good repeatability (the coincidence rate is greater than 95%, and the coefficient of variation CV of the Ct value of the detection result is less than 5%).

[0044] 2. Coincidence rate experiment

[0045] Twenty DNA samples from healthy volunteers in Shanghai were selected, and the method of Example 2 was used to detect...

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Abstract

The invention provides a probe for detecting ACE2 gene polymorphism. The sequence of the probe is as shown in SEQ ID NO.: 1, or two ends of the sequence of SEQ ID NO.: 1 are modified by groups. The invention also provides a primer for detecting the polymorphism of the ACE2 gene, and the sequence of the primer is shown as SEQ ID NO.: 2 and SEQ ID NO.: 3. The probe and the primer for detecting the polymorphism of the ACE2 gene have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when being used for detecting the polymorphism of the ACE2 gene.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a probe, primer and kit for detecting ACE2 gene polymorphism. Background technique [0002] The protein encoded by the ACE2 (angiotensin I converting enzyme 2) gene belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidase, and has considerable homology with human angiotensin-1 converting enzyme. This secreted protein catalyzes the cleavage of angiotensin I to angiotensin 1-9 and angiotensin II to the vasodilator angiotensin 1-7. Organ- and cell-specific expression of this gene suggests that it may play a role in regulating cardiovascular and renal function and fertility. Furthermore, the encoded protein is a functional receptor for the spike glycoprotein of human coronaviruses SARS and HCoV-NL63. [0003] At present, the commonly used mutation detection method is the direct DNA sequencing method. The PCR product is directly analyzed by DNA sequence, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
Inventor 张奕毛丹丹傅咏南王校黄芬芬
Owner 上海利康精准医疗技术有限公司
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