CN102108411A - Kit for detecting gastric cancer susceptibility - Google Patents

Abstract

The invention discloses a kit for detecting the gastric cancer susceptibility, which is used for detecting four genes nearly concerned with gastric cancer as follows: MTHFR gene, CYP2E1 metabolic enzyme gene, XRCC1 and ADPRT repairase gene. The kit can simultaneously detect the following SNP (single nucleotide polymorphism) sites: the rs1801133 site of the MTHFR gene, the rs2031920 site of the CYP2E1, the rs25478 site of the XRCC1 gene and the rs1136410 site of the ADPRT gene. By using the kit disclosed by the invention, a group of genes and sites related to the gastric cancer susceptibility are detected, and whether the subject population carry gastric cancer susceptible genes can be judged clearly by using a specific primer and a specific probe by a mononucleotide extension technology combined with a microarray chip technology, thereby screening out the gastric cancer susceptible population from the subject population, changing unhealthy habits and customs and achieving the purpose of prevention.

Description

Be used to detect the test kit of gastric cancer susceptibility

Technical field

The present invention relates to the test kit that a kind of disease susceptibility detects usefulness, especially a kind of test kit that detects gastric cancer susceptibility, by detecting the mononucleotide polymorphism site (SNP) of mthfr gene, CYP2E1 metabolic enzyme gene, XRCC1 and ADPRT repair enzyme genetic, predict individual susceptibility to cancer of the stomach.

Background technology

Cancer of the stomach is one of the most common malignant tumor of digestive tract tumour, is common disease, the frequently-occurring disease of serious threat human health life.According to statistics, the whole world has 900,000 people newly to be diagnosed as cancer of the stomach every year approximately, and in China, cancer of the stomach accounts for the 1st of malignant tumor of digestive tract, the 3rd of whole body malignant tumour.According to the data of Ministry of Health's health information statistics center calendar year 2001 announcement, malignant tumour is China city resident's the 1st cause of the death, and mortality ratio is 135.59/10 ten thousand (24.9%).Wherein, cancer of the stomach occupies the 3rd in the malignant tumour cause of the death.The generation of cancer of the stomach, development are the same with other malignant tumours, belong to that multifactor, multistep is rapid, multistage and polygene change process.In recent years, the single nucleotide polymorphism of some gene (SNP) receives much attention in the research of malignant tumour susceptibility, Methylene tetrahydrofolate reductase gene (Methylenetetrahydrofolate reductase is thought in research, MTHFR) C677T site (rs1801133), the rs2031920 site of CYP2E1, the X line is repaired cross complementary group 1 (X-ray repaircross-complementing group 1, XRCC1) (Adenosine diphosphate ribotransferase, Ala762Val site (rs1136410) ADPRT) increases the individual risk that gets a cancer of the stomach for Arg399Gln site (rs25487) and adenosine diphosphate ribose based transferase.

MTHFR is positioned karyomit(e) 1p36.3, and whole coding head of district 1980bp comprises 11 exons, its cDNA sequence total length 2.2Kb.The coded product of this gene is to contain the homodimer that 656 amino-acid residues, relative molecular weights are approximately 150kDa, its aminoacid sequence high conservative.Mthfr gene has the various mutations type, and different mutation types produces different influences to the enzymic activity of MTHFR with thermostability.So far, found to have on the mthfr gene nearly 30 SNP sites, wherein existing 18 cSNP land at the GenBank database.Nineteen ninety-five, the someone has found the C677T site of MTHFR.This site is positioned on the folate binding site of mthfr gene the 4th exon.Mthfr gene the 677th bit base cytosine(Cyt) (C) is replaced by thymus pyrimidine (T), thereby makes the L-Ala (Ala) of a high conservative become Xie Ansuan (Val).The C677T site is positioned at the MTHFR catalysis region, and this sudden change directly influences the activity and the thermotolerance of Methylene tetrahydrofolate reductase, and the enzymic activity that shows as in various degree reduces and reduces with thermotolerance.

Folic acid is the synthetic and methylated important precursor of nucleotide DNA, and folic acid deficiency is synthesized by interference DNA and methylated, and then influences the generation of cancer.Discover among the crowd that the variation of mthfr gene C677T site causes enzymic activity to descend, total folate level is reduced, particularly 5-methyl tetrahydrofolate level reduces, 5,50-subunit tetrahydrofolic acid (THFA) gather and cell in folic acid derivatives constitute and change.Studies show that mthfr gene 677TT genotype polymorphism is a risk factors of gastric cancer.

Most of chemical substances needs just producing carcinogenic effect after the activation under the effect of metabolic enzyme, and main metabolic enzyme has oxydase (I phase), as Cytochrome P450 oxydase (CYPs).It is interior through CYP450 catalysis that procarcinogen enters human body, is transformed into biomacromolecule in the electrophilic compound attack cells, forms the DNA affixture, starts carcinogenic or mutagenesis process; Because the gene pleiomorphism of these enzymes causes enzyme molecular structure, function and level and changes, can influence the ability of cell deactivation carcinogens and mutagenic compound, and then be considered to relevant with tumor susceptibility.CYP2E1 is wherein a kind of, and (Debrisoquine Hydroxylase DBH), mainly participates in nitrosamine and precursor and the metabolism in vivo of lower molecular weight halogenated hydrocarbon compound in the food to coding N-nitrosodimethylamine D-demethylase.This assignment of genes gene mapping is in human chromosomal 10q24.3-ter, and long 11.4kb contains 9 exons.The rs2031920 site of CYP2E1, be positioned at the 5-control region by due to the displacement of C → T, it is relevant that research thinks that this loci polymorphism and CYP2E1 metabolic enzyme transcriptional activity strengthen, and the expression of its mutator gene type is than wild gene type height, and the TT genotype increases the risk of suffering from the esophageal carcinoma.

In recent years, DNA-repair gene receives much attention in the research of malignant tumour susceptibility.The dna damage reparation is the complex process that a plurality of enzymes and protein participate in, in case genes involved is undergone mutation, will cause whole genome DNA repair ability to descend, and may cause the generation and the development of the malignant tumour that comprises cancer of the stomach.The X line is repaired cross complementary gene 1 (X-ray repair cross-complementinggroup 1, XRCC1) be first be separated to influence the mammalian genes of cell to ionizing rays susceptibility, the reparation of its wide participation dna damage is to study more a kind of DNA-repair gene at present.XRCC1 is positioned human chromosomal 19q13.2-13.3, and size is 33kb, comprises 17 exons, the protein of 1 70kD that is made up of 633 amino acid that encodes.XRCC1 is as scafffold proteins, by direct and archaeal dna polymerase β, dna ligase III and poly adenosine diphosphate (ADP) (adenosinediphosphate, ADP) ribose polymerase (poly ADP-ribose polymerase, PARP) form mixture, fellowship is repaired and the single-strand break reparation because of the base excision that ionizing rays and oxidative damage cause, the stability of keeping gene is brought into play very crucial effect.The rs25487 site of XRCC1 gene, cause corresponding amino-acid residue Arg399Gln (change of G → A), this polymorphism changes the XRCC1 activity of proteins, mutator gene type AA is considered to relevant with the morbidity of cancer of the stomach.

ADPRT is the core protein member that (BER) system is repaired in the base excision.ADPRT is conjugated protein as a kind of DNA, by identification DNA splitting of chain and make multiple nuclear receptor protein (comprising self) that poly ADP riboseization take place, thereby participates in the BER process.When ADPRT is incorporated into DNA splitting of chain place, can causes self poly ADP riboseization and activate.Activated ADPRT and albumen support XRCC1 interact, and raise archaeal dna polymerase-β and dna ligase III formation complex body and finish the dna damage repair process.ADPRT has a plurality of pleomorphism sites, and is maximum with the research in rs1136410 site.The rs25487 polymorphic site of XRCC1 is positioned at the interactional BRCT-structural domain with ADPRT just, therefore the interaction of this gene-gene has the biology foundation, the rs1136410 polymorphic site of ADPRT and the variation of the rs25487 polymorphic site of XRCC1 have interaction, both make a variation simultaneously and reduce base excision repairing effect, the individual susceptibility to cancer of influence.

Prior art detects cancer of the stomach Susceptible population, adopts hybridization hybrid chip method or sheet glass chip method, and utilizes the taqman probe, and this method accuracy rate is low, false negative and false positive rate are higher, and can not batch detection; Another kind of direct sequencing, though the accuracy rate height, can not be realized batch detection equally at its cost height, therefore existing method all can't satisfy the requirement of extensive genescreen.

Summary of the invention

Technical problem to be solved by this invention is, by detecting one group of gene relevant and site with gastric cancer susceptibility, utilize Auele Specific Primer and probe, by the mononucleotide elongation technology in conjunction with the micro-array chip technology, comprehensive detection and analysis are subjected to the inspection crowd whether to carry " cancer of the stomach tumor susceptibility gene ", Susceptible population screens from the crowd with cancer of the stomach, changes bad living habit, reaches the purpose of prevention.

In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of detection kit that is used to detect the cancer of the stomach susceptible, it comprises the combination with cancer of the stomach 4 genes in close relations: mthfr gene, CYP2E1 metabolic enzyme gene, XRCC1 and ADPRT repair enzyme genetic.

Comprise following SNP site: the rs1801133 site of mthfr gene, the rs2031920 site of CYP2E1, the rs25478 site of XRCC1 gene, the rs1136410 site of ADPRT gene.

The described assortment of genes that is used to detect the cancer of the stomach susceptible, be used at described site design specific primer to and probe.

The described Auele Specific Primer that is used to detect the assortment of genes design of cancer of the stomach susceptible, the right sequence of described Auele Specific Primer is as follows, is used to carry out the multiplex PCR amplification, can amplify the gene fragment in above-mentioned 4 sites simultaneously:

At rs1801133 site: CACAAAGCGGAAGAATGTGTC (SEQ ID NO:1)

GTCATCCCTATTGGCAGGTTAC(SEQ?ID?NO：2)

At rs2031920 site: ACAAGTGATTTGGCTGGATTGT (SEQ ID NO:3)

AGAGAATGTTTTTCATTCTGTCTTCT(SEQ?ID?NO：4)

At rs25478 site: CAGTCTGACTCCCCTCCAGATT (SEQ ID NO:5)

TGCTGGACTGTCACCGCAT(SEQ?ID?NO：6)

At rs1136410 site: TCTCTGCATGTAGGTTTTCTCTG (SEQ ID NO:7)

AGCAGACTGTAGGCCACCTC(SEQ?ID?NO：8)

The described specific probe that is used to detect the assortment of genes design of cancer of the stomach susceptible, described specific probe sequence is as follows, can detect somatotype to 4 sites simultaneously:

At the rs1801133 site:

ACGCACGTCCACGGTGATTTGAAAAGCTGCGTGATGATGAAATCG(SEQ?ID?NO：9)

At the rs2031920 site:

GGATGGCGTTCCGTCCTATTAAGATTCATTGTTAATATAAAAGTA(SEQ?ID?NO：10)

At the rs25478 site:

CGTGCCGCTCGTGATAGAATCGCATGCGTCGGCGGCTGCCCTCCC(SEQ?ID?NO：11)

At the rs1136410 site:

AGCGATCTGCGAGACCGTATTTTCTTTTGCTCCTCCAGGCCAAGG(SEQ?ID?NO：12)

Described primer or probe are used for detecting the sick tumor susceptibility gene of cancer of the stomach by the mononucleotide elongation technology in conjunction with micro-array chip technology specificity.

The component and the content of this test kit comprise:

250ul 10 * PCR reaction buffer,

20ul 10mM dNTP mixed solution,

450ul 25mM MgCl2 solution,

45ul (5U/ul) Taq archaeal dna polymerase,

50ul Auele Specific Primer (8) mixed solution (10uM each),

15ul specificity extension probes (4) mixed solution (10uM),

90ul?ExoI(10U/ul)，

450ul?SAP(1U/ul)，

140ul 10 * SAP reaction buffer,

1700ul?Extension?Dilution?Buffer，

90ul?10×Extension?Mix，

10ul?DNA?polymerase，

3500ul?Hybridization?Solution，

200ul?Hybridization?Additive，

2500ul?20×Wash?Buffer?1，

800ul?64×Wash?Buffer?2，

One 384 hole 12 heavy micro-array chips

Deionized water 20ml,

This test kit detects for 380 person-portions and uses, and the test kit storage temperature is-20 ℃.

The invention has the beneficial effects as follows: accuracy is up to more than 99% as a result for (1) somatotype, and good reproducibility does not have the false positive influence; (2) detect a plurality of SNP site simultaneously, it is low to detect cost; (3) flux height can once detect 384 samples; (4) easy and simple to handle; (5) highly sensitive, each DNA consumption that detects is 2ng only.The present invention in conjunction with the micro-array chip technology, utilizes Auele Specific Primer and probe that the gene type accuracy rate of single nucleotide polymorphism (SNP) is surpassed 99% by the mononucleotide elongation technology.

Embodiment

Below in conjunction with specific embodiment the present invention is described in further detail.The experiment of unreceipted actual conditions is amplified in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The extraction of 1DNA:

Get person under inspection's peripheric venous blood, the cell pyrolysis liquid that adds equal volume, twice of cracking, abundant cracking white corpuscle, centrifugal abandoning adds Proteinase K damping fluid and Proteinase K (50ug/ml) behind the supernatant, hatched 10 minutes for 65 ℃, isopropanol precipitating, 75% washing with alcohol twice are dissolved in after drying in an amount of TB solution.

The 2PCR amplification

The extracting genome DNA liquid of getting the person under inspection adds in 96 orifice plates, carries out the multiplex PCR amplification:

Reaction cumulative volume 5ul, 10M mol/L dNTPs 0.0375ul wherein, 10 * PCR Buffer0.5ul, 25mmol/L MgCl ₂1ul, template DNA 30ng, 6 heavy primer mixed solution 10uM/L each0.025ul, Amplitaq Gold (5U/ul) 0.1ul supplies water to 5ul.Place on the PCR instrument and react: pre-94 ℃ of 1min of sex change; 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 1min, 40 circulations; 4 ℃ of Hold.

The amplimer mixed solution comprises following primer:

1F CACAAAGCGGAAGAATGTGTC(SEQ?ID?NO：1)

1R GTCATCCCTATTGGCAGGTTAC(SEQ?ID?NO：2)

2F ACAAGTGATTTGGCTGGATTGT(SEQ?ID?NO：3)

2R AGAGAATGTTTTTCATTCTGTCTTCT(SEQ?ID?NO：4)

3F CAGTCTGACTCCCCTCCAGATT(SEQ?ID?NO：5)

3R TGCTGGACTGTCACCGCAT(SEQ?ID?NO：6)

4F TCTCTGCATGTAGGTTTTCTCTG(SEQ?ID?NO：7)

4R AGCAGACTGTAGGCCACCTC(SEQ?ID?NO：8)

3 purifying

In pcr amplification product, add 0.2ul ExoI, 1ul SAP, 0.3ul 10 * SAP reaction buffer and 1.5ul deionized water.96 orifice plates are put into the PCR instrument carry out purifying, purifying procedure: 37 ℃ of 30min, 96 ℃ of 10min, 4 ℃ of Hold.

4 primer extension reactions

Add the extension mixture in the PCR product behind the purifying: extension Dilution Buffer3.76ul, extension probes mixed solution (1010uM/L each) 0.03ul, 20 * Extension Mix 0.2ul, archaeal dna polymerase 0.02ul, deionized water 3ul.96 orifice plates that the extension mixture is housed are put into the PCR instrument once more carry out extension, response procedures: 96 ℃ of 3min of Hold; 94 ℃ of 20sec, 40 ℃ of 11sec, 46 circulations; 4 ℃ of Hold.

The extension probes mixed solution comprises following probe:

1P ACGCACGTCCACGGTGATTTGAAAAGCTGCGTGATGATGAAATCG(SEQ?ID?NO：9)

2P GGATGGCGTTCCGTCCTATTAAGATTCATTGTTAATATAAAAGTA(SEQ?ID?NO：10)

3P CGTGCCGCTCGTGATAGAATCGCATGCGTCGGCGGCTGCCCTCCC(SEQ?ID?NO：11)

4P AGCGATCTGCGAGACCGTATTTTCTTTTGCTCCTCCAGGCCAAGG(SEQ?ID?NO：12)

Above extension probes 5 ' end has and micro-array chip address primer corresponding address sequence.

After 5 extensions finished, adding hybridization solution can carry out hybridization with micro-array chip.

42 ℃ of incubation temperature, incubation time 2 hours.

6 laser scannings detect fluorescent signal

It is significant to detect the leucocythemia susceptibility that above-mentioned 3 gene pairss are predicted, comparison is individual simultaneously.The present invention will make up with leukemia susceptible, the closely-related gene of generation, show described 3 genes by a large amount of garbled datas: mthfr gene, CYP2E1 metabolic enzyme gene, XRCC1 and ADPRT repair enzyme genetic, all have the site to undergo mutation as four genes, the probability of then suffering from acute cancer of the stomach is the highest; The probability that does not have gene to have the site to undergo mutation to get a cancer of the stomach is less.

The method that the present invention adopts mononucleotide elongation technology and micro-array chip technology to combine, at above-mentioned 4 sites, design one cover multiple PCR primer, 4 gene fragments in SNP site are carried in amplification simultaneously in an amplified reaction, according to the sequences Design oligonucleotide probe of upstream, SNP site 5 ' end 23-25bp, 3 ' end is positioned at snp5 ' end 1bp place, upstream behind this probe and the sequence hybridization.Base ddNTP who carries fluorescence in the probe end combination that polysaccharase carries according to SNP during detection, according to bonded ddNTP difference, its entrained fluorescence color is also inequality, hold the basis and the different designs of micro-array chip binding site that the Tag address sequence of different 20bp is arranged at 5 ' of probe, with sequence complementation corresponding on the micro-array chip, sequence hybridization on probe after the extension and the micro-array chip on the corresponding zone, different probes is combined in the different zones of chip, by detecting the fluorescence of correspondence position, reach the purpose of carrying out a plurality of SNP somatotypes simultaneously.

Disease be a very complicated process, be subjected to the common influence of a plurality of albumen and gene, wherein the change of any one enzyme all can influence the susceptibility of disease.The method of traditional gene test disease is subjected to the restriction of method, often can only analyze the polymorphism of one to two gene, can only cover a part of ill risk population, for the ill risk of the disease early warning timely that causes owing to the polymorphism on other genes, therefore the accuracy that detects can significantly reduce, and the person under inspection can not comprehensively understand the susceptible situation of self-disease.

The present invention is directed to the different ill approach of a kind of disease and detect a plurality of relevant genes and its pleomorphism site, can more accurately more fully detect the ill risk of the different ill approach of person under inspection, can propose pointed and Health ﹠ Fitness Tip effectively to the person under inspection, avoid the generation of its disease timely and effectively.

Because the detection method that the present invention uses can high-throughput, the detection SNP of automatization, significantly reduced the detection cost, can a plurality of genes of the ill approach of difference be detected simultaneously, 4 pleomorphism sites have been selected in the key gene searching that the present invention is directed to the different ill approach of leukemia, can more accurately more fully detect the ill risk of the different ill approach of person under inspection, can propose pointed Health ﹠ Fitness Tip, avoid the generation of its disease timely and effectively to the person under inspection.The present invention passes through the mononucleotide elongation technology in conjunction with the micro-array chip technology, utilize Auele Specific Primer and probe that the gene type accuracy rate of single nucleotide polymorphism (SNP) is surpassed 99%, it is significant to detect the gastric cancer susceptibility that above-mentioned 3 gene pairss detect, comparison is individual simultaneously.

In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.

Claims (6)

1. test kit that is used to detect gastric cancer susceptibility, it is characterized in that, comprise: the Auele Specific Primer in the rs25478 site of the rs1801133 site of mthfr gene, the rs2031920 site of CYP2E1, XRCC1 gene, the rs1136410 site of ADPRT gene and corresponding extension probes, the Taq enzyme, the dNTP mixed solution, MgCl ₂Solution, 10 * PCR reaction buffer, ExoI, SAP, 10 * SAP reaction buffer, Extension Dilution Buffer, 10 * Extension Mix, DNA polymerase, Hybridization Solution, Hybridization Additive, 20 * Wash Buffer 1,64 * Wash Buffer 2, deionized water, micro-array chip.

2. the test kit that is used to detect gastric cancer susceptibility according to claim 1, it is characterized in that: described Auele Specific Primer is to being rs 1801133 sites at mthfr gene, the rs2031920 site of CYP2E1, the rs25478 site of XRCC1 gene, the rs1136410 site of ADPRT gene can specific amplification goes out to comprise that the primer of dna fragmentation in above-mentioned SNP site is right.

3. the test kit that is used to detect gastric cancer susceptibility according to claim 1, it is characterized in that: described specificity extension probes is the rs1801133 site at mthfr gene, the rs2031920 site of CYP2E1, the rs25478 site of XRCC1 gene, the rs1136410 site of ADPRT gene and designing can be extended and microarray technology detect the probe of these four SNPs loci gene types by mononucleotide.

4. the test kit that is used to detect gastric cancer susceptibility according to claim 1, it is characterized in that: contained Auele Specific Primer has SEQ ID NO:1 to being selected from, the primer of nucleotide sequence shown in 2 to, have a SEQ ID NO:3, the primer of nucleotide sequence shown in 4 to, have a SEQ ID NO:5, the primer of nucleotide sequence shown in 6 to and have SEQ ID NO:7, the primer of nucleotide sequence shown in 8 is right.

5. the test kit that is used to detect gastric cancer susceptibility according to claim 1 is characterized in that: contained specificity extension probes is selected from has the extension probes shown in the SEQ ID NO:9,10,11 and 12.

6. the test kit that is used to detect gastric cancer susceptibility according to claim 1, it is characterized in that: the component of test kit and content comprise 250ul 10 * PCR reaction buffer, 20ul 10mM dNTP mixed solution, 450ul 25mM MgCl2 solution, 45ul 5U/ul Taq archaeal dna polymerase, 8 Auele Specific Primer mixed solutions of every 10uM of 50ul, 4 specificity extension probes mixed solutions of every 10uM of 15ul, 90ul10U/ul ExoI, 450ul 1U/ul SAP, 140ul 10 * SAP reaction buffer, 1700ulExtension Dilution Buffer, 90ul 10 * Extension Mix, 10ul DNApolymerase, 3500ul Hybridization Solution, 200ul HybridizationAdditive, 2500ul 20 * Wash Buffer 1,800ul 64 * Wash Buffer 2, deionized water 20ml, confession 380 person-portions detect 384 holes, the 12 heavy micro-array chips of using, and the test kit storage temperature is-20 ℃.