CN107312837A - A kind of primer sets of detection rs2066853 loci gene types and its detection kit and application - Google Patents

Abstract

The invention discloses a primer set for detecting the genotype of the AHR gene rs2066853 site, a detection kit and application thereof. The primer set includes a pair of specific primers and two wild-type and mutant fluorescent probes, the sequences of which are shown in SEQ ID NO: 1-4; the specific detection method is: S1. Extract the DNA of the sample to be tested ; S2. Use the DNA described in step S1 as a template, and use the above primers to perform fluorescent quantitative PCR detection to determine the genotype of the rs2066853 locus; the susceptibility assessment of lung cancer can be performed through the genotype of the locus, if the genotype of the rs2066853 locus If it is A, the sample is susceptible to lung cancer. The rs2066853 site detection primer designed by the invention has strong specificity and high amplification efficiency; the detection method adopted is simple and efficient, and realizes the automaticity of judging results, and has great application prospects in disease prevention detection.

Description

A primer set for detecting the genotype of rs2066853 site and its detection kit and application

technical field

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

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.

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.

Population epidemiology and basic etiology studies have shown that tumors are the result of a combination of environmental and genetic factors. Among them, tobacco exposure is considered to be one of the main risk factors for lung cancer. It is estimated that by 2030, the number of lung cancer deaths caused by smoking will reach more than 10 million cases per year. Hundreds of carcinogens and carcinogens can be produced during the burning of tobacco, including polycyclic aromatic hydrocarbons (PAHs), benzene, nicotine, and tobacco tar, which are closely related to lung cancer. Benzo(a)pyrene (BaP), a representative polycyclic aromatic hydrocarbon in smoke, is metabolized in the body to form an active product, diol epoxybenzo(a)pyrene (BPDE), which forms adducts with guanine to cause DNA damage , induced mutations, can increase cell genome instability and the risk of lung cancer. After entering the cell, PAHs first bind to the aromatic hydrocarbon receptor AHR. AHR is a ligand-activated transcription factor. When combined with PAHs, it immediately activates the expression of a series of metabolic enzyme genes, and then participates in the metabolic activation process of PAHs. AHR is a key hub for regulating the expression of metabolic enzymes and downstream regulatory genes induced by smoking. Studies have confirmed that the abnormality of AHR signaling pathway is closely related to the occurrence and development of tumors. Many studies have shown that there are abnormal changes in the expression of genes related to AHR signaling pathway in various human malignant tumors. Therefore, in-depth research on AHR signaling pathway genes will provide a theoretical basis for gene prevention and treatment of lung cancer.

SNP (Single nucleotide polymorphism), that is, single nucleotide polymorphism, is the third-generation genetic marker of human genome mapping, mainly referring to a type of DNA sequence polymorphism caused by single base variation, including single base Transitions, transversions, and single-base insertions/deletions, etc. SNP is the most widely-existing type of polymorphic marker in the genome. Its high density, fast and large-scale screening and easy typing characteristics suitable for dimorphism determine that it is more suitable for the identification of polymorphic markers than other polymorphic markers. The study of complex diseases has become a tool for studying genomic polymorphisms and identifying and locating diseases. Currently, the main detection methods routinely used for disease susceptibility gene variation include PCR-restriction fragment length polymorphism (RFLP) and real-time fluorescent quantitative PCR. The principle of the RFLP method is that when a mutation affects the cleavage site of a certain restriction endonuclease, the PCR product of the patient can be processed by enzyme digestion and then electrophoresis to detect whether there is a mutation. However, this method has disadvantages such as time-consuming and cumbersome operation. In 1996, the real-time fluorescent quantitative PCR technology for SNP detection was successfully developed, which realized the leap from qualitative to quantitative PCR and also realized the automaticity of judging the results. Fluorescent quantitative PCR technology can be used to develop kits for rapid detection of disease susceptibility.

The human AHR gene is located at position 7p21.1 of chromosome 7, with a total length of about 47 kb, a full length of mRNA of 43,800 bp, a total of 11 exons, and a protein encoding 848 amino acids. The protein encoded by the AHR gene is a key regulatory molecule in the AHR signaling pathway, which can inhibit the p53 and BRCA1 signaling pathways, thereby regulating the cell cycle, cell proliferation, promoting angiogenesis, and inflammatory responses. A large number of studies have shown that the abnormal overexpression of AHR gene is common in the process of tumorigenesis. Abnormal expression of AHR can also promote tumor invasion and metastasis. In addition, studies have shown that SNPs located in the AHR gene sequence can affect the expression or function of the AHR gene and be associated with the occurrence of diseases.

rs2066853 is a G>A polymorphism located in the coding region of the AHR gene on chromosome 7. In the frequency distribution of the world population, G accounts for 0.73 and A accounts for about 0.27. The distribution of Chinese population G accounts for 0.62, and A accounts for about 0.37. Biological experimental studies have shown that the substitution of G>A can change the combination of AHR gene and transcription factor TBP, thereby affecting the expression of AHR downstream target genes and regulating certain diseases.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned prior art, and provide a primer set for detecting the genotype of the rs2066853 locus in the coding region of the AHR gene; the primer set has high specificity, high amplification efficiency, and can accurately identify The genotype of the rs2066853 SNP locus.

The purpose of the present invention is to provide a primer set for detecting the genotype of the polymorphic site rs2066853 in the coding region of the AHR gene.

Another object of the present invention is to provide a method for detecting the genotype of the rs2066853 locus.

Another object of the present invention is to provide a lung cancer susceptibility detection kit.

Above-mentioned purpose of the present invention is given to realize by following technical scheme:

The present invention firstly provides the application of the polymorphic site rs2066853 in the coding region of the AHR gene in the assessment of the individual's susceptibility to lung cancer or as a target for the assessment of the individual's susceptibility to lung cancer.

The present invention finds through a large-scale population lung cancer case-control study that the G>A polymorphism in the coding region of the AHR gene is associated with the occurrence of lung cancer, and has a gene dose-response relationship. This correlation persisted even after confounding factors.

Specifically, the application is to first extract the genomic DNA of the sample, and then determine the genotype of the rs2066853 site by SNP genotyping technology. If the rs2066853 site is A, the sample is susceptible to lung cancer.

Preferably, the SNP genotyping technique is Taqman-MGB probe method.

A primer set for detecting the genotype of the polymorphic site rs2066853 in the coding region of the AHR gene, the primer set includes a pair of specific primers and two wild-type and mutant fluorescent probes, and its sequence is as shown in SEQ ID NO: 1 ~4 shown.

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

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

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

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

The rs2066853 site G>A polymorphism of the present invention is related to the occurrence of lung cancer, so the application of the detection primers of the rs2066853 site genotype in the detection of lung cancer susceptibility and/or in the preparation of lung cancer susceptibility detection kits is also included in the present invention. within the scope of invention protection.

A method for detecting the genotype of the polymorphic site rs2066853 in the coding region of the AHR gene, comprising the steps of:

S1. Extract the DNA of the sample to be tested;

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 the genotype of the rs2066853 site.

Preferably, the fluorescent quantitative PCR reaction system described in step S2 is: 2 μL of DNA template, 0.25 μL of 10 μM specific primers, 0.1 μL of 5 μM fluorescent probes, 0.03 μL of 5 U/μL Taq DNA polymerase, 0.1 μL of 20 mM dNTP mixture, 25mMMgCl ₂ 0.5μL, 5× fluorescent quantitative PCR reaction buffer 2.37μL, deionized water 4.3μL, a total of 10μL.

Preferably, the fluorescent quantitative PCR reaction system described in step S2 is: 50°C for 2 minutes, 95°C for 10 minutes; 92°C for 15 seconds, 60°C for 1 minute, a total of 45 cycles.

Preferably, the fluorescent quantitative PCR detection instrument is an ABI 7900HT fluorescent quantitative PCR instrument, which comes with software SDS (Sequence Detection Systems) V2.3 for genotype interpretation and automatic analysis of results.

In addition, the application of the detection method in the detection of lung cancer susceptibility and/or in the preparation of a lung cancer susceptibility detection kit is also within the protection scope of the present invention.

Specifically, the application is to use the above-mentioned rs2066853 genotype detection method to detect the genotype of rs2066853 in the sample. If the rs2066853 locus is A, the sample is susceptible to lung cancer.

At the same time, the present invention also provides a lung cancer susceptibility detection kit, which includes the above-mentioned primer set for detecting the genotype of the rs20668531 locus.

Preferably, the kit also contains reagents required for fluorescent quantitative PCR.

More preferably, the reagents required for the fluorescent quantitative PCR are Taq DNA polymerase, dNTP mixed solution, MgCl ₂ solution, fluorescent quantitative PCR reaction buffer and deionized water.

Preferably, the kit also contains reagents required for sample genomic DNA extraction.

More preferably, the kit also contains consumables required for the extraction of genomic DNA by silica gel adsorption.

As a preferred implementation mode, the method of using the lung cancer susceptibility detection kit of the present invention is as follows:

S1. Extract the DNA of the sample to be tested;

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 the genotype of the rs2066853 site.

S3. If the genotype of the rs2066853 locus is A, the sample is susceptible to lung cancer.

The fluorescent quantitative PCR reaction system is: 2 μL of DNA template, 0.25 μL of 10 μM specific primers, 0.1 μL of 5 μM fluorescent probes, 0.03 μL of 5U/μL Taq DNA polymerase, 0.1 μL of 20mM dNTP mixture, 0.5 μL of 25mM MgCl ₂ , 2.37 μL of 5× fluorescence quantitative PCR reaction buffer, 4.3 μL of deionized water, a total of 10 μL.

The fluorescence quantitative PCR reaction system is as follows: 50°C for 2 minutes, 95°C for 10 minutes; 92°C for 15 seconds, 60°C for 1 minute, a total of 45 cycles.

Compared with the prior art, the present invention has the following beneficial effects:

The rs2066853 locus genotype detection primer of the present invention has strong specificity and high amplification efficiency. The detection method can detect the genotype of the rs2066853 locus of the population, and evaluate the susceptibility of lung cancer occurrence of the population carrying different genotypes; the detection method is simple, easy, and efficient, and can be used for disease prevention guidance for high-risk groups of lung cancer.

Description of drawings

Fig. 1 is a flow chart of the detection of lung cancer susceptibility in the present invention.

Fig. 2 is a graph of PCR amplification curve and fluorescence signal of each primer set in the embodiment of the present invention. A is the PCR amplification curve and fluorescence signal graph of the primer set of group 1; B is the PCR amplification curve and fluorescence signal graph of the primer set of group 2; C is the PCR amplification curve and fluorescence signal graph of the primer set of group 3 .

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

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

1. Primer design

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.

Table 1

2. Fluorescent quantitative PCR detection

(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 0.5μL, 5× fluorescent quantitative PCR reaction buffer 2.37μL, deionized water 4.3μL.

(2) The reaction was carried out in an ABI 7900HT fluorescent quantitative PCR instrument, and the reaction conditions were 50°C for 2 minutes, 95°C for 10 minutes; 92°C for 15 seconds, and 60°C for 1 minute, a total of 45 cycles. After the reaction, the fluorescence quantity was read on the ABI 7900HT fluorescent quantitative PCR instrument.

(3) The ABI 7900HT fluorescent quantitative PCR instrument was used to interpret the genotype with the software SDS (Sequence Detection Systems) V2.3.

3. Results

The results are shown in Figure 2. The primers and probes of group 1 were well designed, and the PCR target product could be amplified, and the fluorescence signal value could be read. However, the primer probes of groups 2 and 3 could not amplify the target fragment and had no fluorescent signal.

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

1. Extraction of DNA

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.

2. Genotyping detection

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.

3. Guide people to actively prevent lung cancer

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 the level of susceptibility to lung cancer and the probability of developing the disease. The individualized health guidance report is based on the genotyping test results of the tested subject, combined with the results of the questionnaire survey on their living and eating habits, to assess the relative risk of the tested subject's genetic susceptibility to lung cancer. At the same time, a personalized health action plan for the subjects was formulated, including suggestions for improvement in eating habits and lifestyle, and the health knowledge of lung cancer prevention was popularized for the subjects.

4. Results

The present invention analyzes the relationship between the AHR gene rs2066853G>A site and the susceptibility to lung cancer through a case-control study, and finds that the rs2066853G>A site is closely related to the incidence of lung cancer. Table 2 shows the basic information of the lung cancer and normal control population in this study. The results show that the comparison of smoking, smoking amount and alcohol consumption between the case group and the control group has statistical significance, while other factors such as age, gender, and tumor family There were no significant differences in history and other variables between the two groups. The results in Table 3 show that compared with the GG wild-type genotype, individuals carrying the AG or AA variant genotype have a significantly increased risk of developing lung cancer. It indicated that the rs2066853G>A locus is a genetic susceptibility marker for the onset of lung cancer.

Table 2 Demographic characteristics and distribution of main factors for lung cancer and controls

^Pa values are ^two -sided χ2 tests.

Table 3 The genotype frequency distribution of rs2066853G>A locus and its relationship with the risk of lung cancer

^a In the control group, the genotype frequencies of the selected loci were in Hardy-Weinberg equilibrium (both P>0.05); ^b The correction factors in the logistic regression model included age, gender, smoking, drinking, and family cancer history.

Example 3 Lung Cancer Susceptibility Detection Kit

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;

The primer sequences are as follows:

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

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

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

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

2. The method of use of the kit

S1. Extract the DNA of the sample to be tested;

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 the genotype of the rs2066853 site.

S3. If the genotype of the rs2066853 locus is A, the sample is susceptible to lung cancer.

The fluorescent quantitative PCR reaction system is: 2 μL of DNA template, 0.25 μL of 10 μM specific primers, 0.1 μL of 5 μM fluorescent probes, 0.03 μL of 5U/μL Taq DNA polymerase, 0.1 μL of 20mM dNTP mixture, 0.5 μL of 25mM MgCl ₂ , 2.37 μL of 5× fluorescence quantitative PCR reaction buffer, 4.3 μL of deionized water, a total of 10 μL.

The fluorescence quantitative PCR reaction system is as follows: 50°C for 2 minutes, 95°C for 10 minutes; 92°C for 15 seconds, 60°C for 1 minute, a total of 45 cycles.

SEQUENCE LISTING

<110> Guangzhou Medical University

<120> A primer set for detecting rs2066853 locus genotype, its detection kit and application

<130> 2017

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Claims (10)

1. The application of the polymorphic site rs2066853 in the coding region of the AHR gene in the assessment of individual lung cancer susceptibility or as a target for the assessment of individual lung cancer susceptibility. 2. The application according to claim 1, characterized in that, the application is to first extract the genomic DNA of the sample, and then measure the genotype of the rs2066853 site by SNP genotyping technology, if the rs2066853 site is A, then the sample is Susceptibility to lung cancer. 3. A primer set for detecting the polymorphic site rs2066853 genotype in the coding region of the AHR gene, characterized in that, the primer set includes a pair of specific primers and two wild-type and mutant fluorescent probes, and its sequences are sequentially As shown in SEQ ID NO: 1-4. 4. The use of the primer set according to claim 1 in the detection of lung cancer susceptibility and/or in the preparation of a lung cancer susceptibility detection kit. 5. A detection method for the polymorphic site rs2066853 genotype in the coding region of the AHR gene, characterized in that it comprises the following steps: S1. Extracting the genomic DNA of the sample to be tested; S2. Using the DNA described in step S1 as a template, and using the primer set described in claim 1 to perform fluorescent quantitative PCR detection to determine the genotype of the rs2066853 site. 6. The detection method according to claim 4, wherein the fluorescent quantitative PCR reaction system in step S2 is: 2 μL of DNA template, 0.25 μL of 10 μM specific primers, 0.1 μL of 5 μM fluorescent probes, 5 U/μL Taq DNA polymerase 0.03 μL, 20 mM dNTP mixture 0.1 μL, 25 mM MgCl ₂ 0.5 μL, 5× fluorescent quantitative PCR reaction buffer 2.37 μL, deionized water 4.3 μL, 10 μL in total. 7. The detection method according to claim 4, characterized in that the fluorescent quantitative PCR reaction system in step S2 is: 50°C for 2 minutes, 95°C for 10 minutes; 92°C for 15 s, 60°C for 1 min, a total of 45 cycles. 8. Application of the detection method according to any one of claims 5 to 7 in the detection of lung cancer susceptibility and/or in the preparation of a lung cancer susceptibility detection kit. 9. The application according to claim 8, characterized in that, specifically, the genotype detection of the rs2066853 locus of the sample is performed using the detection method described in any one of claims 5 to 7, and if the rs2066853 locus is A, the sample susceptibility to lung cancer. 10. A lung cancer susceptibility detection kit, characterized in that it comprises the primer set according to claim 3.