MDA技术在转基因检测中的研究与应用
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摘要
PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。
PCR technology is the major genetically modified(GM) crops detection method, but the purity and quality of the template is the major limitation, furthermore, it is not suitable for the detection of the sample that has low purity and quality. In this paper, we develop a novel detection method based on multiple displacement amplification.The limit of detection reaches to 10 copies. More over, the method have also detected certified reference material of GM maize Bt11 by single particle derived from maize powder. The result showed the detection rate of endogenous reference gene is up to 70%. The ratio of the detected event specific sequence to endogenous reference gene was in agreement with the certified values. The results provide a novel method for detection of traceable GMsamples,meanwhile, it also provides a potential solution for the detection of stacked GM crops.
PCR technology is the major genetically modified(GM) crops detection method, but the purity and quality of the template is the major limitation, furthermore, it is not suitable for the detection of the sample that has low purity and quality. In this paper, we develop a novel detection method based on multiple displacement amplification.The limit of detection reaches to 10 copies. More over, the method have also detected certified reference material of GM maize Bt11 by single particle derived from maize powder. The result showed the detection rate of endogenous reference gene is up to 70%. The ratio of the detected event specific sequence to endogenous reference gene was in agreement with the certified values. The results provide a novel method for detection of traceable GMsamples,meanwhile, it also provides a potential solution for the detection of stacked GM crops.
引文
Ba llantyne K.N.,van Oorschot R.A.,Mitchell R.J.,and Koukoulas I.,2006,Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping,Analytical Biochemistry,355(2):298-303
Bhoge R.K.,Chhabra R.,Randhawa G.,Sathiyabama M.,and Sngh M.,2015,Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification,Food Control,55:18-30
Dean F.B.,Hosono S.,Fang L.,Wu X.,Faruqi A.F.,Bray-Ward P.,Sun Z.,Zong Q.,Du Y.,Du J.,Driscoll M.,Song W.,Kingsmore S.F.,Egholm M.,and Lasken R.S.,2002,Comprehensive human genome amplification using multiple displacement amplification,Proceedings of the National Academy of Sciences,99(8):5261-5266
Forte V.T.,Di Pinto A.,Martino C.,Tantillo G.M.,Grasso G.,and Schena F.P.,2005,A general multiplex-PCR assay for the general detection of genetically modified soya and maize,Food Control,16(6):535-539
Fukuta S.,Mizukami Y.,Ishida A.,Ueda J.,Hasegawa M.,Hayashi I.,Hashimoto M.,and Kanbe M.,2004,Real-time loop-mediated isothermal amplification for the CaMV-35S promoter as a screening method for genetically modified organisms,European Food Research and Technology,218(5):496-500
Hawkins T.L.,Detter J.C.,and Richardson P.M.,2002,Whole genome amplification-applications and advances,Current Opinion in Biotechnology,13(1):65-67
Hellani A.,Coskun S.,Benkhalifa M.,Tbakhi A.,Sakati N.,AlOdaib A.,and Ozand P.,2004,Multiple displacement amplification on single cell and possible PGD applications,Molecular Human Reproduction,10(11):847-852
Hellani A.,Coskun S.,Tbakhi A.,and Al-Hassan S.,2005,Clinical application of multiple displacement amplification in preimplantation genetic diagnosis,Reproductive Biomedicine Online,10(3):376-380
Huang J.W.,and Shang X.,2012,Application of multiplex ligation-dependent probe amplifcation in the molecular diagnosis of genetic disease,Fenzi Zhengduan Yu Zhiliao Zazhi(Journal of Molecular Diagnostics and Therapy),4(4):274-278(黄际卫,商璇,2012,多重连接依赖式探针扩增技术在遗传病分子诊断中的应用,分子诊断与治疗杂志,4(4):274-278)
Hughes S.,Arneson N.,Done S.,and Squire J.,2005,The use of whole genome amplification in the study of human disease,Progress in Biophysics and Molecular Biology,88(1):173-189
Hughes S.,Yoshimoto M.,Beheshti B.,Houlston R.S.,Squire J.A.,and Evans A.,2006,The use of whole genome amplification to study chromosomal changes in prostate cancer:insights into genome-wide signature of preneoplasia associated with cancer progression,BMC Genomics,7(1):65
Jin W.J.,Jia S.R.,and Peng Y.F.,2004,Comparison of labling policy of enetically modified products in different countries and territories,Nongye Shengwu JishuXuebao(Journal of Agricultural Biotechnology),12(1):1-7(金芜军,贾士荣,彭于发,2004,不同国家和地区转基因产品标识管理政策的比较,农业生物技术学报,12(1):1-7)
Lasken R.S.,and Egholm M.,2003,Whole genome amplification:abundant supplies of DNA from precious samples or clinical specimens,Trends in Biotechnology,21(12):531-535
Liang X.Y.,Trentham-Dietz A.,Titus-Ernstoff L.,Newcomb P.A.,Welch R.A.,Hutchinson A.A.,Hampton J.M.,Sutcliffe C.B.,Haines J.L.,and Egan K.M.,2007,Whole-genome amplification of oral rinse self-collected DNA in a population-based case-control study of breast cancer,Cancer Epidemiology and Prevention Biomarkers,16(8):1610-1614
Man X.H.,Xu Y.,Wand Z.N.,Lu Z.,Xu M.D.,Jiang L.,Luo Y.,Xu H.M.,and Zhang X.,2006,Loss of heterozygosity at chromosome 8p21-p23 in adenocarcinoma of gastric cardia,Yichuan(Hereditas),28(6):641-645(满晓辉,徐岩,王振宁,吕志,徐米多,姜莉,罗阳,徐惠绵,张学,2006,贲门癌中染色体8p21-p23杂合性丢失的研究,遗传,28(6):641-645)
Maragh S.,Jakupciak J.P.,Wagner P.D.,Rom W.N.,Sidransky D.,Srivastava S.,and O'Connell C.,2008,Multiple strand displacement amplification of mitochondrial DNA from clinical samples,BMC Medical Genetics,9(1):7-9
Michikawa Y.,Sugahara K.,Suga T.,Ohtsuka Y.,Ishikawa K.,Ishikawa A.,Shiomi N.,Shiomi T.,Iwakawa M.,Imai T.,2008,In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level,Analytical Biochemistry,383(2):151-158
Moreano F.,Ehlert A.,Busch U.,and Engel K.H.,2006,Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNAfrom two genetically modified organisms,European Food Research and Technology,222(5-6):479-485
Morisset D.,Dobnik D.,Hamels S.,譕el J.,and Gruden K.,2008a,NAIMA:target amplification strategy allowing quantitative on-chip detection of GMOs,Nucleic Acids Research,36(18):e118-e118
Morisset D.,Stebih D.,Cankar K.,譕el J.,and Gruden K.,2008b,Alternative DNA amplification methods to PCR and their application in GMO detection:a review,European Food Research and Technology,227(5):1287-1297
Randhawa G.J.,Chhabra R.,Bhoge R.K.,and Singh M.,2015,Visual and real-time event-specific loop-mediated isothermal amplification based detection assays for Bt cotton events MON531 and MON15985,Journal of AOAC International,98(5):1207-1214
Renwick P.J.,Trussler J.,Ostad-Saffari E.,Fassihi H.,Black C.,Braude P.,and Abbs S.,2006,Proof of principle and first cases using preimplantation genetic haplotyping-a paradigm shift for embryo diagnosis,Reproductive Biomedicine Online,13(1):110-119
Renwick P.,and Ogilvie C.M.,2007,Preimplantation genetic diagnosis for monogenic diseases:overview and emerging issues,Expert Review of Molecular Diagnostics,7(1):33-43
Roth L.,Zagon J.,Laube I.,Holst-Jensen A.,and Broll H.,2008,Generation of reference material by the use of multiple displ-acement amplification(MDA)for the detection of genetically modified organisms(GMOs),Food Analytical Methods,1(3):181-189
Shen X.,Xu Y.,Zhong Y.,Zhou C.,Zeng Y.,Zhuang G.,and Li T.,2011,Preimplantation genetic diagnosis forα-andβ-double thalassemia,Journal of Assisted Reproduction and Genetics,28(10):957-964
Shen X.T.,Xu Y.W.,Zhong Y.P.,Zeng Y.H,Wang J.,Ding C.H.,Xing W.J.,and Zhou C.Q.,2013,Combination of multiple displacement amplification with short tandem repeat polymorphismin preimplantation genetic diagnosis,Beijing Daxue Xuebao(Yixueban)(Journal of Peking University(Health Sciences)),45(6):852-858(沈晓婷,徐艳文,钟依平,曾艳红,王静,丁晨晖,邢卫杰,周灿权,2013,多重置换扩增结合短串联重复序列在植入前遗传学诊断中的应用,北京大学学报(医学版)),45(6):852-858)
Spits C.,Le Caignec C.,De Rycke M.,Van Haute L.,Van Steirteghem A.,Liebaers I.,and Sermon K.,2006,Optimization and evaluation of single-cell whole-genome multiple displacement amplification,Human Mutation,27(5):496-503
Wang S.K.,Huang L.,and He B.,2015,Multiple displacement amplification and its application in preimplantation genetic diagnosis of thalassemia.,Zhongguo Linchuang Xinyixue(Chinese Journal of New Clinical Medicine),8(10):1004-1007(王世凯,黄莉,何冰,2015,多重置换扩增在地中海贫血植入前遗传学诊断中的应用,中国临床新医学,8(10):1004-1007)
Zhen Z.,Zhang M.H.,Liu Y.,Ao J.X.,Yu Y.B.,Li L.,Qu B.,Chou Y.W.,Gao X.J.,and Zhang K.J.,2015,Development of loop-mediated isothermal amplification(LAMP)method for rapid detection of transgenic maize NK603,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),20(3):24-29(甄贞,张明辉,刘营,敖金霞,于艳波,李璐,曲波,2015,转基因玉米NK603品系成分LAMP快速PCR检测方法的建立,中国农业大学学报,20(3):24-29)
Zhou D.,Wang C.,Li Z.,Chen Y.,Gao S.,Guo J.,and Que Y.,2016,Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay,Frontiers in Plant Science,7:279
Zhou D.G.,Guo J.L.,Xu L.P.,Gao S.W.,Lin Q.L.,Wu Q.B.,Wu L.G.,and Que Y.W.,2014,Establishment and application of a loop-mediated isothermal amplification(LAMP)system for detection of cry1Ac transgenic sugarcane,Scientific Reports,4:4912-4915
Bhoge R.K.,Chhabra R.,Randhawa G.,Sathiyabama M.,and Sngh M.,2015,Event-specific analytical methods for six genetically modified maize events using visual and real-time loop-mediated isothermal amplification,Food Control,55:18-30
Dean F.B.,Hosono S.,Fang L.,Wu X.,Faruqi A.F.,Bray-Ward P.,Sun Z.,Zong Q.,Du Y.,Du J.,Driscoll M.,Song W.,Kingsmore S.F.,Egholm M.,and Lasken R.S.,2002,Comprehensive human genome amplification using multiple displacement amplification,Proceedings of the National Academy of Sciences,99(8):5261-5266
Forte V.T.,Di Pinto A.,Martino C.,Tantillo G.M.,Grasso G.,and Schena F.P.,2005,A general multiplex-PCR assay for the general detection of genetically modified soya and maize,Food Control,16(6):535-539
Fukuta S.,Mizukami Y.,Ishida A.,Ueda J.,Hasegawa M.,Hayashi I.,Hashimoto M.,and Kanbe M.,2004,Real-time loop-mediated isothermal amplification for the CaMV-35S promoter as a screening method for genetically modified organisms,European Food Research and Technology,218(5):496-500
Hawkins T.L.,Detter J.C.,and Richardson P.M.,2002,Whole genome amplification-applications and advances,Current Opinion in Biotechnology,13(1):65-67
Hellani A.,Coskun S.,Benkhalifa M.,Tbakhi A.,Sakati N.,AlOdaib A.,and Ozand P.,2004,Multiple displacement amplification on single cell and possible PGD applications,Molecular Human Reproduction,10(11):847-852
Hellani A.,Coskun S.,Tbakhi A.,and Al-Hassan S.,2005,Clinical application of multiple displacement amplification in preimplantation genetic diagnosis,Reproductive Biomedicine Online,10(3):376-380
Huang J.W.,and Shang X.,2012,Application of multiplex ligation-dependent probe amplifcation in the molecular diagnosis of genetic disease,Fenzi Zhengduan Yu Zhiliao Zazhi(Journal of Molecular Diagnostics and Therapy),4(4):274-278(黄际卫,商璇,2012,多重连接依赖式探针扩增技术在遗传病分子诊断中的应用,分子诊断与治疗杂志,4(4):274-278)
Hughes S.,Arneson N.,Done S.,and Squire J.,2005,The use of whole genome amplification in the study of human disease,Progress in Biophysics and Molecular Biology,88(1):173-189
Hughes S.,Yoshimoto M.,Beheshti B.,Houlston R.S.,Squire J.A.,and Evans A.,2006,The use of whole genome amplification to study chromosomal changes in prostate cancer:insights into genome-wide signature of preneoplasia associated with cancer progression,BMC Genomics,7(1):65
Jin W.J.,Jia S.R.,and Peng Y.F.,2004,Comparison of labling policy of enetically modified products in different countries and territories,Nongye Shengwu JishuXuebao(Journal of Agricultural Biotechnology),12(1):1-7(金芜军,贾士荣,彭于发,2004,不同国家和地区转基因产品标识管理政策的比较,农业生物技术学报,12(1):1-7)
Lasken R.S.,and Egholm M.,2003,Whole genome amplification:abundant supplies of DNA from precious samples or clinical specimens,Trends in Biotechnology,21(12):531-535
Liang X.Y.,Trentham-Dietz A.,Titus-Ernstoff L.,Newcomb P.A.,Welch R.A.,Hutchinson A.A.,Hampton J.M.,Sutcliffe C.B.,Haines J.L.,and Egan K.M.,2007,Whole-genome amplification of oral rinse self-collected DNA in a population-based case-control study of breast cancer,Cancer Epidemiology and Prevention Biomarkers,16(8):1610-1614
Man X.H.,Xu Y.,Wand Z.N.,Lu Z.,Xu M.D.,Jiang L.,Luo Y.,Xu H.M.,and Zhang X.,2006,Loss of heterozygosity at chromosome 8p21-p23 in adenocarcinoma of gastric cardia,Yichuan(Hereditas),28(6):641-645(满晓辉,徐岩,王振宁,吕志,徐米多,姜莉,罗阳,徐惠绵,张学,2006,贲门癌中染色体8p21-p23杂合性丢失的研究,遗传,28(6):641-645)
Maragh S.,Jakupciak J.P.,Wagner P.D.,Rom W.N.,Sidransky D.,Srivastava S.,and O'Connell C.,2008,Multiple strand displacement amplification of mitochondrial DNA from clinical samples,BMC Medical Genetics,9(1):7-9
Michikawa Y.,Sugahara K.,Suga T.,Ohtsuka Y.,Ishikawa K.,Ishikawa A.,Shiomi N.,Shiomi T.,Iwakawa M.,Imai T.,2008,In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level,Analytical Biochemistry,383(2):151-158
Moreano F.,Ehlert A.,Busch U.,and Engel K.H.,2006,Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNAfrom two genetically modified organisms,European Food Research and Technology,222(5-6):479-485
Morisset D.,Dobnik D.,Hamels S.,譕el J.,and Gruden K.,2008a,NAIMA:target amplification strategy allowing quantitative on-chip detection of GMOs,Nucleic Acids Research,36(18):e118-e118
Morisset D.,Stebih D.,Cankar K.,譕el J.,and Gruden K.,2008b,Alternative DNA amplification methods to PCR and their application in GMO detection:a review,European Food Research and Technology,227(5):1287-1297
Randhawa G.J.,Chhabra R.,Bhoge R.K.,and Singh M.,2015,Visual and real-time event-specific loop-mediated isothermal amplification based detection assays for Bt cotton events MON531 and MON15985,Journal of AOAC International,98(5):1207-1214
Renwick P.J.,Trussler J.,Ostad-Saffari E.,Fassihi H.,Black C.,Braude P.,and Abbs S.,2006,Proof of principle and first cases using preimplantation genetic haplotyping-a paradigm shift for embryo diagnosis,Reproductive Biomedicine Online,13(1):110-119
Renwick P.,and Ogilvie C.M.,2007,Preimplantation genetic diagnosis for monogenic diseases:overview and emerging issues,Expert Review of Molecular Diagnostics,7(1):33-43
Roth L.,Zagon J.,Laube I.,Holst-Jensen A.,and Broll H.,2008,Generation of reference material by the use of multiple displ-acement amplification(MDA)for the detection of genetically modified organisms(GMOs),Food Analytical Methods,1(3):181-189
Shen X.,Xu Y.,Zhong Y.,Zhou C.,Zeng Y.,Zhuang G.,and Li T.,2011,Preimplantation genetic diagnosis forα-andβ-double thalassemia,Journal of Assisted Reproduction and Genetics,28(10):957-964
Shen X.T.,Xu Y.W.,Zhong Y.P.,Zeng Y.H,Wang J.,Ding C.H.,Xing W.J.,and Zhou C.Q.,2013,Combination of multiple displacement amplification with short tandem repeat polymorphismin preimplantation genetic diagnosis,Beijing Daxue Xuebao(Yixueban)(Journal of Peking University(Health Sciences)),45(6):852-858(沈晓婷,徐艳文,钟依平,曾艳红,王静,丁晨晖,邢卫杰,周灿权,2013,多重置换扩增结合短串联重复序列在植入前遗传学诊断中的应用,北京大学学报(医学版)),45(6):852-858)
Spits C.,Le Caignec C.,De Rycke M.,Van Haute L.,Van Steirteghem A.,Liebaers I.,and Sermon K.,2006,Optimization and evaluation of single-cell whole-genome multiple displacement amplification,Human Mutation,27(5):496-503
Wang S.K.,Huang L.,and He B.,2015,Multiple displacement amplification and its application in preimplantation genetic diagnosis of thalassemia.,Zhongguo Linchuang Xinyixue(Chinese Journal of New Clinical Medicine),8(10):1004-1007(王世凯,黄莉,何冰,2015,多重置换扩增在地中海贫血植入前遗传学诊断中的应用,中国临床新医学,8(10):1004-1007)
Zhen Z.,Zhang M.H.,Liu Y.,Ao J.X.,Yu Y.B.,Li L.,Qu B.,Chou Y.W.,Gao X.J.,and Zhang K.J.,2015,Development of loop-mediated isothermal amplification(LAMP)method for rapid detection of transgenic maize NK603,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),20(3):24-29(甄贞,张明辉,刘营,敖金霞,于艳波,李璐,曲波,2015,转基因玉米NK603品系成分LAMP快速PCR检测方法的建立,中国农业大学学报,20(3):24-29)
Zhou D.,Wang C.,Li Z.,Chen Y.,Gao S.,Guo J.,and Que Y.,2016,Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay,Frontiers in Plant Science,7:279
Zhou D.G.,Guo J.L.,Xu L.P.,Gao S.W.,Lin Q.L.,Wu Q.B.,Wu L.G.,and Que Y.W.,2014,Establishment and application of a loop-mediated isothermal amplification(LAMP)system for detection of cry1Ac transgenic sugarcane,Scientific Reports,4:4912-4915