CN101608217A - Liver cancer genetic test kit - Google Patents

Abstract

The invention discloses a kind of test kit that detects the liver cancer genetic risk.This test kit comprises that the genotypic Auele Specific Primer of mononucleotide polymorphism site that detects Cytochrome P450 1A1 gene (CYP1A1), microsome epoxide enzyme gene (EPHX1), glutathione S-transferase M1 gene (GSTM1) and glutathione S-transferase T1 gene (GSTT1) is to reaching the specificity fluorescent probe to, quantification PCR normal assembly, PCR reaction component etc.Test kit of the present invention is assessed individual liver cancer genetic risk by detection simultaneously and the pleomorphism site genotype on the closely-related gene of liver cancer genetic risk.

Description

Liver cancer genetic test kit

Technical field

The present invention relates to molecular biology and medical field, more specifically, the present invention relates to a kind of test kit that detects individual liver cancer genetic susceptibility, assess individual liver cancer genetic susceptibility by detection simultaneously and the mononucleotide polymorphism site genotype on the closely-related Cytochrome P450 1A1 of liver cancer gene (CYP1A1), microsome epoxide enzyme gene (EPHX1), glutathione S-transferase M1 gene (GSTM1) and the glutathione S-transferase T1 gene (GSTT1).

Background technology

Liver cancer is because liver is subjected to various adverse factors (mainly being chemical carcinogen) and interior some the carcinogenic long term of body in the external environment, make liver cell or bile duct epithelial cell etc. that hyperplasia take place, cause normal configuration to wreck and a kind of malignant tumour of forming.Liver cancer is one of China's common malignancy, is to occupy second cancer " killer ", is common in middle-aged male, and the age occurred frequently is 40～49 years old, and the ratio of men and women's morbidity is 6: 1.The average course of disease of liver cancer is two-and-a-half years, but its early symptom is not obvious, and generally patient's survival time only is 6 months after symptom occurring.Because of its grade of malignancy height, disease progression fast, person " king in the cancer ".

A large amount of association studies both at home and abroad show that liver cancer and inherited genetic factors are in close relations, and the inheritance susceptible Journal of Sex Research of this disease mainly concentrates on I phase metabolic enzyme and the mutually metabolic genes involved of II.

The reactivation process that foreign compound metabolism in vivo comprises I phase metabolic enzyme mediation and II be the detoxification processes of metabolic enzyme mediation mutually.Cytochrome P450 is the more I phase metabolic enzyme of research, and there be more than 100 kind of isozyme in this enzyme system.Many chemical substances are by after the oxygenizement of cytochrome P 450 enzymes, and toxicity does not weaken, and strengthen on the contrary, as just having carcinogenic activity after the oxidative metabolism activation of benzopyrene by cytochrome P 450 enzymes.CYP1A1 gene Ile462Val is the A/G polymorphic site that is positioned at No. 7 exon place of this gene, and this site is positioned at the haem bonding pad of this enzyme.This polymorphic site has three kinds of genotype: Ile/Ile, Ile/Val and Val/Val, and wherein Ile/Val and Val/Val are the tumor susceptibility gene types of liver cancer.Studies show that CYP1A1 zymoprotein 462 amino acids become Val by Ile, have increased the activity of enzyme, the epoxide concentration that causes having carciongenic potency increases, and causes damage and the chromosome aberration of DNA, causes the onset risk of liver cancer.

Epoxide hydrolase is an important II phase metabolic enzyme in the body, can express in various organs of body and tissue, mainly is present in endocytoplasmic reticulum and the cytosol.To be that catalysis is various have a various unsettled epoxide hydrolysis that is produced in active exogenous epoxide and the I phase metabolic process to the major function of this enzyme, forms the material of solubility and excrete.Epoxide is highly activation owing to have strong polar carbon-oxygen bond, is easy to and DNA, protein and other covalent attachment formation adducts and cause cytotoxicity or genetic material mutation.EPHX1 gene Tyr113His site is a polymorphic site at No. 3 exon place of this gene, and the genotype of this polymorphic site has three kinds: Tyr/Tyr, Tyr/His, His/His, wherein His/His is confirmed as the risk genes type, and is relevant with the morbidity of liver cancer.Studies show that the activity of the EPHX1 enzyme of His113His homozygote coding can reduce by 40%, thereby the epoxide hydrolysis rate is slowed down, too much epoxide is accumulated in vivo, the latter can with the N7 site covalent attachment of DNA guanine, cause the dna single chain break.Therefore, risk genes type carrier is because slow to the metabolism of epoxide with carcinogenesis, and increased the susceptibility of liver cancer.

GSTM1 is mutually important transferring enzyme during bio-transformation II in the body, and it is by directly being incorporated into chemical carcinogen or bringing into play function of detoxification by combining with gsh.Null/Present is the common polymorphism of GSTM1, and this is polymorphic three kinds of genotype ,+/+,+/-and-/-, the no allelotrope of representative lacks respectively, lack an allelotrope, lack two allelotrope (absence type fully), wherein absence type (Null) is relevant with the morbidity of liver cancer fully.Lot of domestic and foreign studies show that, absence type person's liver can not be expressed glutathione S-transferase M1 fully, the function of detoxification of body reduces, therefore homozygous disappearance person is when a large amount of carcinogens of contact, can not make carcinogens be converted into low, the water-soluble strong meta-bolites of toxicity and excretes timely and effectively.These do not get rid of external carcinogens can form dna adduct with important tumour cancer suppressor gene in the body, easily causes variation of tumour cancer suppressor gene and afunction, thereby has increased the onset risk of liver cancer.

GSTT1 is an important II phase metabolic enzyme in the body, and it is by directly being incorporated into chemical carcinogen or bringing into play function of detoxification by combining with gsh.Null/Present is the common polymorphism of GSTT1, this is polymorphic three kinds of genotype, + /+,+/-and-/-, the no allelotrope of representative lacks respectively, lack an allelotrope, lack two allelotrope (absence type fully), wherein complete absence type (/-) relevant with the morbidity of liver cancer, be the risk genes type of liver cancer.The disappearance of GSTT1 gene is polymorphic, makes the forfeiture of this gene function, and the function of detoxification of body reduces, and make carcinogens can not be converted into low, the water-soluble strong meta-bolites of toxicity and excrete timely and effectively, the carcinogen in vivo accumulation, concentration increases.The carcinogens of accumulation can form dna adduct with important tumour cancer suppressor gene in the body, easily causes variation of tumour cancer suppressor gene and afunction, thereby has increased the onset risk of liver cancer.

Summary of the invention

Can be used to assess on the basis of individual liver cancer genetic susceptibility based on 4 SNP loci polymorphisms on Cytochrome P450 1A1 gene (CYP1A1), microsome epoxide enzyme gene (EPHX1), glutathione S-transferase M1 gene (GSTM1), the glutathione S-transferase T1 gene (GSTT1), the invention provides a kind of test kit that detects individual liver cancer genetic susceptibility.

This test kit comprises:

The genotypic Auele Specific Primer of Ile462Val SNP polymorphism is right to reaching the specificity fluorescent probe on the detection CYP1A1 gene;

The genotypic Auele Specific Primer of Tyr113His SNP polymorphism is right to reaching the specificity fluorescent probe on the detection EPHX1 gene;

The genotypic electrophoresis detection primer of SNP polymorphism is right on the detection GSTM1 gene;

The genotypic electrophoresis detection primer of SNP polymorphism is right on the detection GSTT1 gene;

The PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, PCR reaction buffer, deionized water etc.);

The quantitative fluorescent PCR reaction component (comprises Taq enzyme, dNTP mixed solution, MgCl ₂Solution, reaction buffer, deionized water etc.).

Primer described in this test kit is to being that those skilled in the art can finish design easily, and the synthetic technology of available routine is synthesized.

Specificity fluorescent probe described in this test kit is to finishing design easily, and the specificity fluorescent probe synthesizes the probe synthetic technology of available routine.

The component and the content of test kit of the present invention comprise:

1. quantitative fluorescent PCR reacts suit

10X quantitative fluorescent PCR reaction buffer 2 μ l,

25mM dNTP mixed solution 0.2 μ l,

25mM MgCl2 solution 1.2 μ l,

5units/ μ l Taq archaeal dna polymerase 0.05 μ l,

20 μ M Auele Specific Primers are to each 0.225 μ l of every primer,

10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe,

Deionized water 10.65 μ l.

2. electrophoresis detection suit

10X PCR reaction buffer 2.5 μ l,

25mM dNTP mixed solution 0.2 μ l,

25mM MgCl ₂Solution 1.5 μ l,

5units/ μ l Taq archaeal dna polymerase 0.125 μ l,

20 μ M electrophoresis detection primers are to each 0.25 μ l of every primer,

Deionized water 18.675 μ l,

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The use of embodiment 1. detection kit

The extraction of step 1:DNA template

Genomic dna with silica gel adsorption extracting mouth epithelial cells.

Step 2: electrophoresis detection somatotype

Use the electrophoresis detection suit in the detection kit, the system of reaction is cumulative volume 25 μ l, comprising concentration is dna profiling 1 μ l, the 10X PCR reaction buffer 2.5 μ l of 12.5ng/ μ l, 25mM dNTP mixed solution 0.2 μ l, 25mM MgCl2 solution 1.5 μ l, 5units/ μ l Taq archaeal dna polymerase 0.125 μ l, 20 μ M electrophoresis detection primers are to each 0.25 μ l of every primer, deionized water 18.675 μ l.

React on the pcr amplification instrument, reaction conditions is 94 ℃, 12 minutes, carries out 94 ℃ of 30 round-robin, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds, carries out then 72 ℃, 10 minutes.

Utilize electrophoretic technique then, carry out agarose gel electrophoresis, observe band quantity.

Step 3: quantitative fluorescent PCR reaction

Use the quantitative fluorescent PCR suit in the detection kit, carry out 2 independently quantitative fluorescent PCR reactions respectively, the system of each reaction is cumulative volume 10 μ l, and comprising concentration is dna profiling 2 μ l, 1 μ l, 10 X quantitative fluorescent PCR reaction buffers, 0.1 μ l 25mM dNTP mixed solution, the 0.6 μ l 25mM MgCl of 20ng/ μ l ₂The band VIC fluorescent probe that adopted primer and antisense primer each 0.225 μ l, 10 μ M are arranged of solution, 0.025 μ l (5units/ μ l) Taq archaeal dna polymerase, 20 μ M and each 0.25 μ l of band FAM fluorescent probe, deionized water 5.325 μ l.

React on the pcr amplification instrument, reaction conditions is 50 ℃, 2 minutes, 95 ℃, 10 minutes, carries out 95 ℃ of 60 round-robin, 30 seconds, 60 ℃, 1 minute.Reaction finishes the back and read the fluorescent amount on quantitative real time PCR Instrument.

Step 4: gene type assay

According to the gene type diagram that the test kit working instructions indicate gene type assay is carried out in the SNP site.

Be familiar with position that those skilled in the art of electrophoresis detection technology can be by DNA band on the identification electrophorogram and quantity and determine the genotype in the SNP site detected.

The those skilled in the art that are familiar with fluorescent quantitative PCR technique can be by the final sample fluorescence volume that shows on the identification quantitative real time PCR Instrument, can determine the genotype in the SNP site detected according to the power of different sequence probe VIC and FAM fluorescent signal.

Embodiment 2. applying detection test kits carry out the service that individual liver cancer genetic susceptibility detects

Step 1:DNA extracts

Instructing the examinee to use the oral cavity sampling to wipe away by the hospital laboratory doctor carries out the mouth epithelial cells sampling, adopts silica gel adsorption to carry out the DNA extracting of mouth epithelial cells.

Step 2: genotype tests

Use test kit provided by the invention, the GSTM1 gene of detected person's genomic dna and the SNP site on the GSTT1 gene are carried out pcr amplification and electrophoresis detection, Tyr113His SNP on Ile462Val SNP site, the EPHX1 gene on the CYP1A1 gene is carried out fluorescence quantitative PCR detection in the site, determine the genotype in these 4 SNP sites.

Step 3: the analysis of individual liver cancer genetic susceptibility

By to the genotypic analysis of detected person SNP, provide the analysis report list of individual liver cancer genetic susceptibility.Describe the height of detected person's liver cancer genetic risk in the report in detail, and describe and understand individual liver cancer genetic susceptibility analysis report list in detail to the examinee by the doctor.

Claims (2)

1. test kit that detects individual liver cancer genetic susceptibility is characterized in that: comprise detect simultaneously Cytochrome P450 1A1 gene (CYP1A1) go up Ile462Val SNP site, microsome epoxide enzyme gene (EPHX1) go up Tyr113His SNP site, glutathione S-transferase M1 gene (GSTM1) go up the Auele Specific Primer of SNP site, glutathione S-transferase T1 gene (GSTT1) going up the SNP site to and the specificity fluorescent probe to, Taq enzyme, dNTP mixed solution, MgCl ₂Solution, fluorescent quantitation PGR reaction buffer, deionized water.

2. test kit according to claim 1 is characterized in that: the component and the content of test kit comprise

1) quantitative fluorescent PCR reaction suit: 10X quantitative fluorescent PCR reaction buffer 2 μ l, 25mM dNTP mixed solution 0.2 μ l, 25mM MgCl2 solution 1.2 μ l, 5units/ μ l Taq archaeal dna polymerase 0.005 μ l, 20 μ M Auele Specific Primers are to each 0.225 μ l of every primer, 10 μ M specificity fluorescent probes are to each 0.25 μ l of every probe, deionized water 10.65 μ l.

2) PCR reaction suit: 10X PCR reaction buffer 2.5 μ l, 25mM dNTP mixed solution 0.2 μ l, 25mM MgCl2 solution 1.5 μ l, 5units/ μ l Taq archaeal dna polymerase 0.125 μ l, 20 μ M Auele Specific Primers are to each 0.25 μ l of every primer, deionized water 18.675 μ l.

This test kit detects for a person-portion and uses, and the storage temperature of test kit is-20 ℃.