柑橘转基因成分多重PCR检测体系的建立Establishment of a Multiplex PCR System for Detecting Transgenic Ingredients from Citrus
李政利;彭爱红;邹修平;何永睿;姚利晓;陈善春;
摘要(Abstract):
[目的]建立柑橘转基因成分的多重PCR检测体系。[方法]根据GenBank中pBI121质粒序列和柑橘(Citrus.)Actin基因序列,分别设计CaMV35S启动子、NOS启动子、NOS终止子特异引物和Actin基因的特异引物,建立能同时检测出4种序列的多重PCR检测体系,同时通过正交试验确定该体系的最佳引物浓度和比例及PCR反应体系中各因素的浓度及反应程序,并对该方法的灵敏度进行验证。[结果]试验得到的最佳MPCR反应体系为:10×buffer 2.5μl,25 mmol/L MgCl22.0μl;dNTP Mixture(2.5 mmol/L each)2.0μl,10μmol/L的Actin基因、35S启动子、NOS启动子、NOS终止子引物分别加入1.0、1.0、1.5、0.5μl,模板DNA 0.1μg,Taq DNA聚合酶1.25U,加ddH2O至25μl。PCR反应程序为:94℃预变性5 min;94℃30 s,64.1℃45 s,72℃50 s,31个循环;72℃10 min。试验中,经正交优化后的4重PCR反应灵敏度达0.1%。[结论]该研究建立的MPCR检测体系,理论上已能满足柑橘或其深加工产品的转基因成分检测。
关键词(KeyWords): 多重PCR;正交试验;检测;转基因成分
基金项目(Foundation): 重庆市重点实验室专项(CSTC);; “863”科技部农村领域国家科技计划课题(2011AA100205);; 农业部/公益性行业(农业)科研专项(201003067);; 教育部科学技术研究重点项目(109131)
作者(Author): 李政利;彭爱红;邹修平;何永睿;姚利晓;陈善春;
Email:
DOI: 10.13989/j.cnki.0517-6611.2012.16.120
参考文献(References):
- [1]CLIVE J.2011年全球生物技术/转基因作物商业化发展态势[J].中国生物工程杂志,2012,32(2):1-14.
- [2]HIDAKA T,OMURA M,UGAKI M,et al.Agrobacterium-mediated trans-formation and regeneration of Citrus spp.from suspension cells[J].Jpn JBreed,1990,40(2):199-207.
- [3]彭爱红,何永睿,许兰珍,等.柑桔转基因研究进展[J].热带作物学报,2011,32(7):1381-1387.
- [4]KLEPPIN L,SCHMIDT G,SCHRDER W.Cultivation of GMO in Germa-ny:support of monitoring and coexistence issues by WebGIS technology[J].Environmental Sciences Europe,2011,23:4.
- [5]TRAPMANN S,EMONS H.Reliable GMO analysis[J].Anal BioanalChem,2005,381:72-74.
- [6]EMONS H.GMO analysis-a complex and challenging undertaking[J].AnalBioanal Chem,2010,396:1949-1950.
- [7]邓汉超,尹长城,刘国振,等.转基因植物核酸成分检测技术研究进展[J].中国生物工程杂志,,2011,31(1):86-95.
- [8]CHAMBERCIAN J S,GIBBS R A,RANIER J E,et al.Detection screeningof the duchenne muscular dystrophy locus via mutiplex DNA amplication[J].Nucl Acids Res,1988,16:1141-1156.
- [9]CHAMBERLAIN J S,GIBBS R A,RANIER J E,et al.Deletion screeningof the Duchenne muscular dystrophy locus via multiplex DNA amplification[J].Nucleic Acids Res,1988,16(23):11141-11156.
- [10]GERMINI A,ZANETTI A,SALATI C,et al.Development of a seventargetmultiplex PCR for the simultaneous detection of transgenic soybean andmaize in feeds and foods[J].J Agric Food Chem,2004,52(11):3275-3280.
- [11]ONISHI M,MATSUOKA T,KODAMA T,et al.Development of a multi-plex polymerase chain reaction method method for simultaneous detectionof eight events of genetically modified maize[J].J Agric Food Chem,2005,53(25):9713-9721.
- [12]陈贞,芦春斌,杨梦婕,等.多重PCR检测转基因菜籽粕中的转基因成分[J].植物检疫,2011,25(3):35-38.
- [13]白月,才华,栾凤侠,等.多重PCR结合DHPLC方法检测番茄中转基因成分[J].作物杂志,2011(2):28-31.
- [14]范达,雷天刚,王军政,等.一种快速微量提取柑橘早期胚基因组DNA的方法[J].安徽农业科学,2011,39(9):5089-5090.
- [15]彭爱红,彭祝春,何永睿,等.根癌农杆菌介导甲型肝炎病毒(HAV)衣壳蛋白融合基因转化柑桔成年材料的研究[J].分子植物育种,2009,7(1):1-5.
- [16]郝玉芹,孙皆宜,李艾,等.正交优化多重PCR反应体系检测3种食源性致病菌的研究[J].安徽农业科学,2010,38(2):602-605.
- [17]JEYASEKARAN G,RAJ K,SHAKILA R,et al.Rapid detection of Salmo-nella enterica serovars by multiplex PCR[J].World J Microbiol Biotechn-ol,2011,27(4):953-959.
- [18]刘光明,苏文金,栾国彦,等.PCR方法检测食品中的转基因成分35S和NOS的研究[J].食品科学,2002,23(5):94-99.
- [19]BUSTAMANTE A V,SANSO A M,LUCCHESI P M,et al.Multiplex PCRassay for the detection of five putative virulence genes encoded in verotox-igenic escherichia coli Plasmids[J].Curr Microbiol,2011,62(5):1411-1415.
- [20]张明辉,高学军,于艳波,等.三重巢式PCR技术检测抗草甘膦转基因大豆深加工产品[J].农业生物技术学报,2006,14(5):752-756.
- [21]BRONZONIA R V M,MORELI M L,CRUZ A C R,et al.Multiplex nestedPCR for Brazilian alphavirus diagnosis[J].Transactions of the Royal So-ciety of Tropical Medicine and Hygiene,2004,98(8):456-461.