适于转基因油菜RT73品系特异性检测的标准分子构建及其适用性鉴定  

Applicability of a Reference Molecule Suitable for Event-specific Analysis of Genetically Modified Rapeseed RT73

作　　者：李俊毅[1,2] 李想[2] 褚庆华[2] 吕蓉[2] 张舒亚[2] 潘良文[2] 黄新[3] 朱水芳[3] 

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]上海出入境检验检疫局,上海200135 [3]中国检验检疫科学研究院动植物检疫研究所,北京100029

出　　处：《生物技术通报》2011年第4期76-83,共8页Biotechnology Bulletin

基　　金：国家科技支撑项目(2008BAK41B01);国家转基因重大专项(2009ZX08012-002B);上海市科委创新平台服务项目(10DZ2294102);国家"863"项目(2006AA10Z442);上海检验检疫局科研项目(HK003-2010)

摘　　要：针对目前转基因产品检测标准物质缺乏的难题,构建适于转基因油菜RT73品系特异性检测的标准分子pEASY-RT73,其包含转基因油菜RT73品系3'端侧翼序列,油菜内标准基因HMG和PEP片段。对其在定性和定量检测中的适用性进行了实验室内部验证,结果表明,标准分子pEASY-RT73高度特异于转基因油菜RT73品系。以标准分子为标准品的普通PCR扩增中,3个目标片段的检测下限(LOD)均为10拷贝。实时荧光PCR检测的LOD均为25拷贝,定量下限(LOQ)均为50拷贝。以标准分子为标准品构建的标准曲线反应效率介于0.96-1.02间,相关系数均大于0.999。分别以PEP和HMG为内标准基因对5个盲样的测试结果显示,测量值与设定值间偏差(Bias)介于-14.13%-14.29%间,SD小于0.20,RSD小于18.0%。因此,标准分子pEASY-RT73可很好地替代植物来源阳性标准品用于转基因油菜RT73及其来源产品品系特异性检测。Due to deficiency of reference materials for GMO analyses,a reference molecule pEASY-RT73 suitable for event-specific analysis of GM rapeseed RT73 was developed.The applicability of pEASY-RT73,which contains three DNA fragments,i.e.3′flanking sequence of RT73,and two endogenous gene fragments of HMG and PEP,in identification and quantification of RT73 was also validated in our lab.Results showed that pEASY-RT73 was highly specific to rapeseed RT73 detection.In the qualitative PCR analysis using pEASY-RT73 as the calibrator,limit of detection（LOD）for three targets are all 10 copies.In the real-time PCR,LOD for three targets were all 25 copies,and LOQs were all 50 copies.The reaction efficiency of three standard curves using pEASY-RT73 as the calibrator was between 0.96 to 1.02,and the squared regression coefficients（R2）were all higher than 0.999.Five blind samples analyses using PEP and HMG as endogenous gene,respectively,showed that bias between measured value and true value was between-14.13% and 14.29%,SD and RSD values were lower than 0.20 and 18.0%,respectively.Therefore,the developed reference molecule pEASY-RT73 could be used to qualitatively and quantitatively analyze GM rapeseed RT73 and its derived products as a good alternative of reference material derived from plant materials.

关 键 词：PCR RT73 标准分子 品系特异性检测 实时荧光PCR 

分 类 号：S565.4[农业科学—作物学]