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Primer set for detecting genotype of rs2066853 site and detection kit and application thereof

A technology for detecting kits and genotypes, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as time-consuming and cumbersome operations, and achieve strong specificity and high amplification efficiency , The detection method is simple and easy to implement

Inactive Publication Date: 2017-11-03
GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has disadvantages such as time-consuming and cumbersome operation.

Method used

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  • Primer set for detecting genotype of rs2066853 site and detection kit and application thereof
  • Primer set for detecting genotype of rs2066853 site and detection kit and application thereof
  • Primer set for detecting genotype of rs2066853 site and detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Design of primers for the rs2066853 site in the coding region of the AHR gene

[0049] 1. Primer design

[0050] In this implementation, the amplification primers and Taqman-MGB probes shown in Table 1 below were designed according to the upstream and downstream sequences of the rs2066853 SNP site on the AHR gene by comparing the sequences of the coding regions of the AHR gene.

[0051] Table 1

[0052]

[0053] 2. Fluorescent quantitative PCR detection

[0054] (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 2

[0059] Example 2 Application of rs2066853 site detection method in the detection of lung cancer susceptibility

[0060] 1. Extraction of DNA

[0061] 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.

[0062] 2. Genotyping detection

[0063] Using the detection method of Example 1, the rs2066853 polymorphic site in the AHR 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.

[0064] 3. Guide people to actively prevent lung cancer

[0065] 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 ...

Embodiment 3

[0075] Example 3 Lung Cancer Susceptibility Detection Kit

[0076] 1. A lung cancer susceptibility detection kit, said kit comprising the following components: rs2066853 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;

[0077] The primer sequences are as follows:

[0078] Specific primer F: 5'-CCCAGGGCTCTTTCAAGATAGTAA-3' (SEQ ID NO: 1)

[0079] Specific primer R: 5'-TCAACCTC ACCAGAAAAATCATTTC-3' (SEQ ID NO: 2)

[0080] Wild-type fluorescent probe: FAM-TTGAAGACATCAGACAC-MGB (SEQ ID NO: 3)

[0081] Mutant fluorescent probe: HEX-ATTTTGAAGACATCAAAC-MGB (SEQ ID NO: 4)

[0082] 2. The method of use of the kit

[0083] S1. Extract the DNA of the sample to be tested;

[0084] 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 deter...

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Abstract

The invention discloses a primer set for detecting the genotype of an rs2066853 site and a detection kit and application thereof. The primer set comprises a pair of specific primers, a wild-type fluorescent probe and a mutant fluorescent probe, and the sequences thereof are sequentially shown as SEQ ID NOs: 1-4; a detection method comprises the following specific steps: S1, extracting DNA from a sample to be detected; S2, with the DNA in the step S1 as a template, performing fluorescent quantitative PCR detection by using the primers to determine the genotype of the rs2066853 site; through the genotype of the site, lung cancer susceptibility assessment can be performed, and if the genotype of the rs2066853 site is A, the sample is susceptible to lung cancer. The rs2066853 site detecting primers designed by the invention are strong in specificity and high in amplification efficiency; the adopted detection method is simple, convenient and efficient; automatic result judgment is achieved; the primer set has a relatively good application prospect in disease prevention and detection.

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 rs2066853 site, a detection kit and applications thereof. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world, which seriously threatens human health and life safety. [0003] 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 in advanced stage, and the current average 5-year survival rate is only 16%. In recent years, the incidence and mortality of lung cancer have been increasing year by year, and it has become a serious social public health problem. [0004] Population epidemiology and basic eti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 吕嘉春杨磊丘福满
Owner GUANGZHOU MEDICAL UNIV
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