Fluorogenic quantitative PCR detection system for gallbladder carcinoma gene screening and application thereof

Abstract

The invention relates to the technical field of biological detection, and in particular relates to a fluorogenic quantitative PCR detection kit and further discloses an application of the kit for detecting gallbladder carcinoma. The fluorogenic quantitative PCR detection system comprises a detection kit for genes at special loci, calculates the score of risk of gallbladder carcinoma according to the risk value of each locus on the risk of gallbladder carcinoma, grades crowds according to an existing database, and obtains the individual gallbladder carcinoma risk level through individual score,so as to estimate the risk of illness of a subject and guide people with high-risk to improve the living style and avoid inducing factors and increase physical examination frequency. At the same time, a Chinese crowd database is established for the two selected gene polymorphic sites, thereby laying a foundation for estimating gallbladder carcinoma special for Chinese crowds.

Description

technical field

[0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR detection system for gallbladder cancer gene screening, and further discloses a method for gallbladder cancer gene screening based on the fluorescent quantitative PCR detection system. Background technique

[0002] With the continuous development of scientific research, after the completion of the Human Genome Project and the full development of the Post-Genome Project, the application of genetic engineering-led biotechnology in the field of cancer research has attracted widespread attention. At present, there has been preliminary progress in the study of tumor susceptibility using the theory and methods of genetics. Commonly used gene detection methods include: direct sequencing (DS), ligase detection reaction (LDR), restriction fragment length polymorphism (RFLP), denaturing high performance liquid chromatography Analysis (denaturing...

Examples

Embodiment 1

[0049] In this example, gallbladder cancer fluorescent quantitative PCR detection kit was used to detect gallbladder cancer-related genes, wherein two polymorphic sites on two genes related to gallbladder cancer were detected, including rs7504990 and rs693 sites, using Specifically designed primers and probes, optimized amplification system and conditions, and simultaneously complete the detection of two polymorphic sites on two genes in the same system.

[0050] In this embodiment, a single fluorescently labeled probe is used to implement 480 platform, and finally detect the rs7504990 and rs693 sites through the peak pattern of the melting curve.

[0051] Specifically, in the dual fluorescent quantitative PCR detection method for rs7504990 and rs693 sites, the design of primers and probes for rs7504990 and rs693 sites is shown in Table 1 below.

[0052] Table 1 Primer and probe design

[0053]

[0054] For the above rs7504990 and rs693 sites, respectively design the rea...

Embodiment 2

[0069] The two loci in Example 1 are from the articles published by Cha PC et al. and Gong Y et al. The article obtained two susceptibility loci directly related to gallbladder cancer through GWAS research, and calculated the two loci The risk value of the risk allele type, that is, the OR value.

[0070] Cha PC et al conducted a GWAS study on 41 gallbladder cancer patients and 866 control samples, and further verified the SNPs that may be associated with gallbladder cancer in 30 patients and 898 control samples. It was found that the SNP site rs7504990 on the DDC gene showed a genome-wide association with the susceptibility to gallbladder cancer in the Japanese population (PCombined=7.46×10 -8 ; OR=6.95; 95%CI=3.43-14.08), DCC was shown to induce apoptosis in the absence of spindlein-1 ligand; in addition, enhanced expression of spindlein-1 in the mouse digestive tract induced proliferative neoplastic lesions The spontaneous formation of , suggesting a potential role of DCC ...

Embodiment 3

[0086] The method of the present invention extracts DNA by collecting the gene sample of subject then, detects with Roche LightCycler480 real-time quantitative instrument, the result obtained is as follows: figure 1 , 3.

technical field

[0001] The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR detection system for gallbladder cancer gene screening, and further discloses a method for gallbladder cancer gene screening based on the fluorescent quantitative PCR detection system. Background technique

[0002] With the continuous development of scientific research, after the completion of the Human Genome Project and the full development of the Post-Genome Project, the application of genetic engineering-led biotechnology in the field of cancer research has attracted widespread attention. At present, there has been preliminary progress in the study of tumor susceptibility using the theory and methods of genetics. Commonly used gene detection methods include: direct sequencing (DS), ligase detection reaction (LDR), restriction fragment length polymorphism (RFLP), denaturing high performance liquid chromatography Analysis (denaturing...

Examples

Embodiment 1

[0049] In this example, gallbladder cancer fluorescent quantitative PCR detection kit was used to detect gallbladder cancer-related genes, wherein two polymorphic sites on two genes related to gallbladder cancer were detected, including rs7504990 and rs693 sites, using Specifically designed primers and probes, optimized amplification system and conditions, and simultaneously complete the detection of two polymorphic sites on two genes in the same system.

[0050] In this embodiment, a single fluorescently labeled probe is used to implement 480 platform, and finally detect the rs7504990 and rs693 sites through the peak pattern of the melting curve.

[0051] Specifically, in the dual fluorescent quantitative PCR detection method for rs7504990 and rs693 sites, the design of primers and probes for rs7504990 and rs693 sites is shown in Table 1 below.

[0052] Table 1 Primer and probe design

[0053]

[0054] For the above rs7504990 and rs693 sites, respectively design the rea...

Embodiment 2

[0069] The two loci in Example 1 are from the articles published by Cha PC et al. and Gong Y et al. The article obtained two susceptibility loci directly related to gallbladder cancer through GWAS research, and calculated the two loci The risk value of the risk allele type, that is, the OR value.

[0070] Cha PC et al conducted a GWAS study on 41 gallbladder cancer patients and 866 control samples, and further verified the SNPs that may be associated with gallbladder cancer in 30 patients and 898 control samples. It was found that the SNP site rs7504990 on the DDC gene showed a genome-wide association with the susceptibility to gallbladder cancer in the Japanese population (PCombined=7.46×10 -8 ; OR=6.95; 95%CI=3.43-14.08), DCC was shown to induce apoptosis in the absence of spindlein-1 ligand; in addition, enhanced expression of spindlein-1 in the mouse digestive tract induced proliferative neoplastic lesions The spontaneous formation of , suggesting a potential role of DCC ...

Embodiment 3

[0086] The method of the present invention extracts DNA by collecting the gene sample of subject then, detects with Roche LightCycler480 real-time quantitative instrument, the result obtained is as follows: figure 1 , 3.

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