CN101962669A - Gene combination, primer and probe for detecting alcoholic liver disease susceptibility and application - Google Patents
Gene combination, primer and probe for detecting alcoholic liver disease susceptibility and application Download PDFInfo
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Abstract
The invention discloses a gene combination, a primer and a probe for detecting alcoholic liver disease susceptibility and application. The gene combination comprises a combination of three genes closely related with an alcoholic liver disease, namely an ADH2 gene of alcohol dehydrogenase, an ALDH2 gene of acetaldehyde dehydrogenase and a CYP2E1 gene of cytochrome P4502. The gene combination comprises the following SNP sites: rs1229984 site of the ADH2 gene, rs671 site of the ALDH2 gene and rs2031920 site of the CYP2E1 gene. Whether the detected crowds carry 'alcoholic liver disease susceptible genes' are comprehensively detected and analyzed by detecting a group of genes and sites related with the alcoholic liver disease susceptibility, using the specific primer and the probe and combining a mononucleotide extending technique and a micro array chip technique so as to screen the alcoholic liver disease susceptible crowd from the crowds, change unhealthy lifestyles and fulfill the purpose of preventing.
Description
Technical field
The present invention relates to a kind of assortment of genes, primer, probe and purposes, in particular for detecting the assortment of genes, primer, probe and the purposes of alcoholic liver disease susceptible.
Background technology
(alcoholic liver disease is because the hepatic injury of drinking and causing for a long time shows as 3 kinds of forms, i.e. alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis ALD) to alcoholic liver disease.These three kinds of forms can separately or be mixed and be existed.According to statistics, the whole world has 1,500 ten thousand-2,000 ten thousand people excessive drinking approximately, and wherein about 10%-20% (1,500,000～4,000,000) has alcoholic liver disease in various degree.In China, along with improving constantly of living standards of the people, the improvement of economic condition, the sickness rate of ALD also is ascendant trend year by year in recent years, and alcohol has become the second largest reason that causes liver injury after virus.
Drink back ethanol mainly in little intestinal absorption, and wherein 90%～95% at intrahepatic metabolism.Ethanol is that main MEOS is decomposed into acetaldehyde by intracytoplasmic ADH and the intravital CYP2EI of particulate in liver.Under normal conditions, with ADH be main working; The chronic habitual alcohol user then induces CYP2EI after taking in alcohol, and ethanol carries out metabolism by CYP2EI.The acetaldehyde that generates, mainly metabolism is an acetate through ALDH, and the latter enters tricarboxylic acid cycle, and metabolism is carbonic acid gas and water.ADH, ALDH and CYP2EI all have genetic polymorphism, the individual enzymic activity difference of expressing of different genes, thereby influence body to alcoholic acid susceptibility.
Up to now, ADH clearly has 7 kinds of isozymes (ADH1～7), and wherein ADH2 plays an important role in the alcohol metabolism process of liver.Human ADH2 gene is positioned at karyomit(e) No. 4, and 3 allelotrope are arranged: ADH2*1 type allelotrope is the most common in American-European blood lineage crowd, and its expression product lacks alcohol dehydrogenase activity, induces metabolism alcoholic acid ability lower; ADH2*2 type allelotrope is most commonly in Asia blood lineage crowd, and the biological activity of its expression product is 40 times of ADH2*1, and the individual metabolism alcoholic acid speed that therefore has ADH2*2 genotype enzyme is fast.ADH2*3 is detected in African blood lineage's crowd.The individual alcohol metabolism speed of ADH2*2 genotype is very fast, cause accumulating of its meta-bolites acetaldehyde easily, excessive acetaldehyde not only can directly damage liver cell in the body, causes fat to be piled up in liver cell, can also generate a large amount of cytokines by activation Kupffer cell and cause inflammation.
In the human ALDH gene family 12 members are arranged, wherein ALDH2 is and the main metabolic enzyme closely related and that have polymorphism of being addicted to drink, and at asian population the height genetic polymorphism is arranged.The ALDH2 gene is positioned at karyomit(e) 12q24.2, and 2 allelotrope ALDH2*1 and ALDH2*2 are arranged.ALDH2*1 is a wild-type, the expression product biologically active, and the ALDH2*2 genotype base occurs at the 12nd exon and replaces that (G → A) causes the 487th replacement that L-glutamic acid and Methionin take place of ALDH2, thereby make ALDH2 forfeiture enzymic activity, acetaldehyde is accumulated in vivo, and excessive acetaldehyde can increase the ill risk of ALD in the body.
MEOS is the oxydase system of one group of mixing functions, and its katalysis depends on the participation of Cytochrome P450, and wherein CYP2EI and alcohol metabolism are closely related, are considered to the tumor susceptibility gene of alcoholic liver disease.The CYP2EI gene is positioned on human No. 10 karyomit(e), and long 11413bp comprises 9 exons and 8 introns.There are 6 kinds of restriction fragment length polymorphisms in the CYP2EI gene: Tap I, Rsa I, Dra I, Msp I Rsa I and Pst I polymorphic and 5 '-end control region are polymorphic.Wherein be positioned at-5 '-end control region Rsa I in 1019bp site is positioned at the transcription factor calmodulin binding domain CaM, may influence genetic expression, when origination point sudden change C → T, cause the increase of transcribing of liver CYP2EI, thereby promotion alcohol metabolism, produce the reactive oxygen species of liver injury, relevant with alcoholic liver damage.
Prior art detects alcoholic liver disease 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 and site relevant with the alcoholic liver disease 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 " alcohol tumor susceptibility gene ", Susceptible population screens from the crowd with alcohol, 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 assortment of genes that is used to detect the alcoholic liver disease susceptible, it comprises the combination with alcoholic liver disease three genes in close relations: the CYP2E1 gene of the ADH2 gene of ethanol dehydrogenase, the ALDH2 gene of acetaldehyde dehydrogenase and Cytochrome P450 2E1.
Comprise following SNP site: the rs2031920 site of the rs1229984 site of ADH2 gene, the rs671 site of ALDH2 and CYP2E1.
The described assortment of genes that is used to detect the alcoholic liver disease 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 alcoholic liver disease 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 3 sites simultaneously:
At rs1229984 site: TTTCTGAATCTGAACAGCTTCTC
TAAARTCACAGGAAGGGGG
At rs671 site: AGCCCCAACAGGCCCTGA
AAGATGTCGGGRAGTGGC
At rs2031920 site: ATGACTTTTATTTTCTTCATTTCTCATC
TAACTGGCAATATATAGRAGTTCTTAATTC。
The described specific probe that is used to detect the assortment of genes design of alcoholic liver disease susceptible, described specific probe sequence is as follows, can detect somatotype to 3 sites simultaneously:
At rs1229984 site: YAGATGGTGGCTGTAGGAATCTGTC
At rs671 site: GGTCCCACACTCACAGTTTTCACTT
At rs2031920 site: CCAGTTTCTCCCTCGCTGTTTTTAT.
Described primer or probe are used for detecting the alcoholic liver disease tumor susceptibility gene by the mononucleotide elongation technology in conjunction with micro-array chip technology specificity.
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
The present invention is used to detect the assortment of genes of alcoholic liver disease susceptible, and it comprises the combination with alcoholic liver disease three genes in close relations: the CYP2E1 gene of the ADH2 gene of ethanol dehydrogenase, the ALDH2 gene of acetaldehyde dehydrogenase and Cytochrome P450 2E1.Comprise following SNP site: the rs2031920 site of the rs1229984 site of ADH2 gene, the rs671 site of ALDH2 and CYP2E1.
The described assortment of genes that is used to detect the alcoholic liver disease susceptible, be used at described site design specific primer to and probe.
Primer of the present invention is the rs2031920 site of the rs671 site of rs1229984 site, ALDH2 at the ADH2 gene and CYP2E1 and designing, and can be used for carrying out the multiplex PCR amplification, can amplify the gene fragment in above-mentioned 3 sites simultaneously.Designing this class primer is that those skilled in the art can be unlabored.Preferably, primer described in the present invention is for having following sequence:
At rs1229984 site: TTTCTGAATCTGAACAGCTTCTC
TAAARTCACAGGAAGGGGG
At rs671 site: AGCCCCAACAGGCCCTGA
AAGATGTCGGGRAGTGGC
At rs2031920 site: ATGACTTTTATTTTCTTCATTTCTCATC
TAACTGGCAATATATAGRAGTTCTTAATTC。
Auele Specific Primer can synthesize with the synthetic technology of routine, and it is right to those skilled in the art will appreciate that primer of the present invention is not limited to these 3 primers.
Probe of the present invention is the rs2031920 site of the rs671 site of rs1229984 site, ALDH2 at the ADH2 gene and CYP2E1 and designing, and can detect somatotype to 3 sites simultaneously.Designing this class probe is that those skilled in the art can be unlabored.Preferably, probe described in the present invention is for having following sequence:
At rs1229984 site: YAGATGGTGGCTGTAGGAATCTGTC
At rs671 site: GGTCCCACACTCACAGTTTTCACTT
At rs2031920 site: CCAGTTTCTCCCTCGCTGTTTTTAT.
Specific probe can synthesize with the synthetic technology of routine, those skilled in the art will appreciate that specific probe of the present invention is not limited to this 3 probes.
Probe described in the present invention refers in particular to the extension primer in the mononucleotide elongation technology, below will be stated as the extension primer among the embodiment.
Below in conjunction with specific embodiment the present invention is described in further detail:
1.DNA extraction:
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.
2.PCR amplification
The extracting genome DNA liquid of getting the person under inspection adds in 96 orifice plates, carries out multiplex PCR amplification: reaction cumulative volume 5ul, 10M mol/L dNTPs 0.0375ul wherein, 10 * PCR Buffer 0.5ul, 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; Hold4 ℃.
Amplimer:
1F TTTCTGAATCTGAACAGCTTCTC
1R TAAARTCACAGGAAGGGGG
2F AGCCCCAACAGGCCCTGA
2R AAGATGTCGGGRAGTGGC
3F ATGACTTTTATTTTCTTCATTTCTCATC
3R TAACTGGCAATATATAGRAGTTCTTAATTC
3. purifying
In pcr amplification product, add 2ul Exo I-SAP-IT purified reagent (available from USB).96 orifice plates are put into the PCR instrument carry out purifying, purifying procedure: 37 ℃ of 30min, 96 ℃ of 10min, Hold4 ℃.
4. primer extension reaction
Add the extension mixture in the PCR product behind the purifying: extension Dilution Buffer3.76ul, extend primer mixed solution (1010uM/L each) 0.03ul, 20 * Extension Mix 0.2ul, archaeal dna polymerase 0.02ul supplies water to 7ul.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.
Extend primer sequence:
1P YAGATGGTGGCTGTAGGAATCTGTC
2P GGTCCCACACTCACAGTTTTCACTT
3P CCAGTTTCTCCCTCGCTGTTTTTAT
Above extension probes 5 ' end has and micro-array chip address primer corresponding address sequence.
5. after extension finished, adding hybridization solution can carry out hybridization with micro-array chip.
42 ℃ of incubation temperature, incubation time 2 hours.
6. laser scanning detects fluorescent signal: in 3 detection site, all be variation when homozygous, ill risk is the highest, and 2 is the homozygous person that makes a variation, and ill risk is taken second place, and risk was minimum when 1 variation was isozygotied.
The present invention makes up with alcoholic liver disease susceptible, the closely-related gene of generation 3, shows described 3 gene: ADH2, ALDH2 and CYP2E1 and site by a large amount of garbled datas: the rs2031920 site of the rs1229984 site of ADH2 gene, the rs671 site of ALDH2 and CYP2E1.All have the site to undergo mutation as ADH2, ALDH2 and CYP2EI3 gene, the possibility of then suffering from alcoholic hepatitis is higher; Undergo mutation as ADH2 and ALDH2 gene, the possibility of then suffering from alcohol type fatty liver is higher; Undergo mutation as the ALDH2 gene, the possibility of then suffering from the disease of being addicted to drink is higher.
The method that the present invention adopts mononucleotide elongation technology and micro-array chip technology to combine, at above-mentioned 3 sites, design one cover multiple PCR primer, 3 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, 3 pleomorphism sites have been selected in the key gene searching that the present invention is directed to the different ill approach of alcoholic liver disease, 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 alcoholic liver disease 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. assortment of genes that is used to detect the alcoholic liver disease susceptible, it is characterized in that it comprises the combination with alcoholic liver disease three genes in close relations: the CYP2E1 gene of the ADH2 gene of ethanol dehydrogenase, the ALDH2 gene of acetaldehyde dehydrogenase and Cytochrome P450 2E1.
2. the assortment of genes that is used to detect the alcoholic liver disease susceptible according to claim 1 is characterized in that, comprises following SNP site: the rs2031920 site of the rs1229984 site of ADH2 gene, the rs671 site of ALDH2 and CYP2E1.
3. the assortment of genes that is used to detect the alcoholic liver disease susceptible as claimed in claim 2, be used at described site design specific primer to and probe.
4. the Auele Specific Primer that is used to detect the assortment of genes design of alcoholic liver disease susceptible as claimed in claim 2, it is characterized in that, 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 3 sites simultaneously:
At rs1229984 site: TTTCTGAATCTGAACAGCTTCTC
TAAARTCACAGGAAGGGGG
At rs671 site: AGCCCCAACAGGCCCTGA
AAGATGTCGGGRAGTGGC
At rs2031920 site: ATGACTTTTATTTTCTTCATTTCTCATC
TAACTGGCAATATATAGRAGTTCTTAATTC。
5. the specific probe that is used to detect the assortment of genes design of alcoholic liver disease susceptible as claimed in claim 2 is characterized in that described specific probe sequence is as follows, can detect somatotype to 3 sites simultaneously:
At rs1229984 site: YAGATGGTGGCTGTAGGAATCTGTC
At rs671 site: GGTCCCACACTCACAGTTTTCACTT
At rs2031920 site: CCAGTTTCTCCCTCGCTGTTTTTAT.
6. as claim 4 or 5 described primer or probes, be used for detecting the alcoholic liver disease tumor susceptibility gene in conjunction with micro-array chip technology specificity by the mononucleotide elongation technology.
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| RU2565036C2 (en) * | 2012-05-04 | 2015-10-10 | Федеральное государственное бюджетное учреждение науки Институт молекулярной биологии им. В.А. Энгельгардта Российской академии наук | Method for analysing genetic polymorphism for determining disposition to schizophrenia and alcoholism |
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| CN105506089A (en) * | 2015-12-30 | 2016-04-20 | 广州金域体检门诊部有限公司 | Primer and method for detecting ALDH2 gene*2 polymorphism |
| CN105524987A (en) * | 2015-12-30 | 2016-04-27 | 广州金域检测科技股份有限公司 | Primers and method for simultaneously detecting ALDH2 gen *2 polymorphism and ADH1B gene *2 polymorphism |
| CN105803104A (en) * | 2016-05-27 | 2016-07-27 | 刘鹏飞 | Reagent system and kit for detecting alcohol or nitroglycerin metabolism and application of reagent system and kit |
| CN106987623A (en) * | 2017-03-20 | 2017-07-28 | 杭州迪安医学检验中心有限公司 | A kind of primer of pyrosequencing joint PCR sequencing PCR detection alcohol metabolism gene and its application |
| CN106987623B (en) * | 2017-03-20 | 2021-01-26 | 杭州迪安医学检验中心有限公司 | Primer for detecting alcohol metabolism gene by pyrosequencing combined sequencing method and application thereof |
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Application publication date: 20110202 |