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A primer set for detecting p.pro189ala locus genotype and its detection kit and application

A detection kit and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of increasing the risk of oral cancer and reducing the efficiency of AHRR gene inhibition, etc., to achieve the detection method Easy to use, strong specificity, and high amplification efficiency

Active Publication Date: 2020-12-01
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Research reports have shown that the genetic variation site p.Pro189Ala (rs2292596; 565C>G) in the coding region of the AHRR gene can reduce the inhibition efficiency of the AHRR gene and increase the risk of oral cancer. Related, we need to study further

Method used

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  • A primer set for detecting p.pro189ala locus genotype and its detection kit and application
  • A primer set for detecting p.pro189ala locus genotype and its detection kit and application
  • A primer set for detecting p.pro189ala locus genotype and its detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 AHR gene coding region p.Pro189Ala site primer design

[0048] 1. Primer design

[0049] In this implementation, the amplification primers and Taqman-MGB probes shown in Table 1 were designed by comparing the sequences of the coding regions of the AHRR gene, and according to the upstream and downstream sequences of the p.Pro189Ala SNP site on the AHR gene, through extensive research.

[0050] Table 1

[0051]

[0052] 2. Fluorescent quantitative PCR detection

[0053] (1) The 3 pairs of primers designed in step 1 were reacted according to the following fluorescent quantitative PCR reaction system. The total volume is 10 μL, including 2 μL of DNA template (20ng / μL), 0.25 μL each of 10 μM specific primer pair (two pairs), 0.1 μL each of 5 μM specific probe pair (two pairs), (5 unit / μL) Taq DNA polymerization Enzyme 0.03μL, 20mM dNTP Mixture 0.1μL, 25mM MgCl 2 Solution 0.5 μL, 5× fluorescence quantitative PCR reaction buffer 2.37 μL, deionized water 4.3 μL...

Embodiment 2p

[0058] Example 2p. Application of Pro189Ala site detection method in lung cancer susceptibility detection

[0059] 1. Extraction of DNA

[0060] The subjects were consulted before the service, and the subjects signed the informed consent and filled out the questionnaire on living and eating habits. 5ml of EDTA anticoagulated blood was extracted by conventional method. Genomic DNA of blood samples was extracted by silica gel adsorption method.

[0061] 2. Genotyping detection

[0062] Using the detection method of Example 1, the p.Pro189Ala polymorphic site in the AHRR gene coding region of the DNA of the subject to be tested was detected by fluorescent quantitative PCR to determine the genotype of the site.

[0063] 3. Guide people to actively prevent lung cancer

[0064] Through the analysis of the genotype of the tested person, the genotyping test report and the individualized health guidance report for the tested person are issued. The genotyping test report details th...

Embodiment 3

[0074] Example 3 Lung Cancer Susceptibility Detection Kit

[0075] 1. A lung cancer susceptibility detection kit, said kit comprising the following components: p.Pro189Ala site-specific amplification primers, mutant and wild-type fluorescent probes, Taq DNA polymerase, dNTP mixed solution, MgCl2 Solution, fluorescent quantitative PCR reaction buffer and deionized water;

[0076] The primer sequences are as follows:

[0077] Specific primer F: 5'-ACACCCACCTGTCTCCAAGG-3' (SEQ ID NO: 1)

[0078] Specific primer R: 5'-GCACCAGAACATTTATGACTAC-3' (SEQ ID NO: 2)

[0079] Wild-type fluorescent probe: FAM-CGGGGCCTGCCCAAACA-MGB (SEQ ID NO: 3)

[0080] Mutant fluorescent probe: HEX-CGGGGGCTGCCCAAAACACC-MGB (SEQ ID NO: 4)

[0081] 2. The method of use of the kit

[0082] S1. Extract the DNA of the sample to be tested;

[0083] S2. Using the DNA described in step S1 as a template, and using the primers described in claim 1 to perform fluorescent quantitative PCR detection to determine...

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Abstract

The invention discloses a primer group for detecting a p.Pro189Ala site genotype as well as a detection kit and application thereof. The primer group comprises a pair of specific primers and two wild and mutant fluorescent probes, and a sequence thereof is successively shown as SEQ ID NO. 1 to SEQ ID NO. 4. The detection method specifically comprises the following steps: S1, extracting DNA of a sample to be detected; S2, performing fluorescent quantitative PCR detection by using the primer with the DNA of step S1 as a template, and determining a genotype of a p.Pro189Ala site; and evaluating the lung cancer susceptibility by virtue of the genotype of the site, wherein if an amino acid for coding the p.Pro189Ala site is Ala, the sample is susceptible to the lung cancer. The p.Pro189Ala site detection primer designed by the present invention is high in specificity and high in amplification efficiency; and the adopted detection method is simple and efficient, the automation of a determination result is realized, and the application prospect in the disease prevention and detection is relatively great.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a primer set for detecting the genotype of the p.Pro189Ala site, a detection kit and applications thereof. Background technique [0002] Lung cancer is one of the most common malignant tumors in humans, and its morbidity and mortality rate ranks first among all kinds of tumors, seriously threatening the health and life safety of residents. The latest research data shows that there are approximately 1.8 million new cases of lung cancer each year worldwide; while in my country, the number of new lung cancer patients reached 733,000 in 2015. Because lung cancer is often accompanied by early metastasis, 70% of lung cancer patients are found at advanced stage, and the average 5-year survival rate is lower than 20%. Studies have shown that lung cancer is a multifactorial and polygenic disease, which is the result of the joint action of environmental and genetic factors. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156C12Q2600/172
Inventor 吕嘉春丘福满杨磊
Owner GUANGZHOU MEDICAL UNIV
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