转基因玉米ND207转化事件特异性定性PCR检测方法及其标准化  

Event-specific PCR detection method of transgenic maize ND207 and its standardization

作　　者：常丽娟[1] 梁晋刚 宋君[1] 刘文娟[1] 付成平[3] 代晓航[1] 王东[1] 魏超[1] 熊梅 CHANG Li-Juan;LIANG Jing-Gang;SONG Jun;LIU Wen-Juan;FU Cheng-Ping;DAI Xiao-Hang;WANG Dong;WEI Chao;XIONG Mei(Institute of Quality Standard and Testing Technology Research,Sichuan Academy of Agricultural Sciences,Chengdu 610066,Sichuan,China;Development Center of Science and Technology,MARA,Beijing 100176;Institute of Remote Sensing and Digital Agriculture,Sichuan Academy of Agricultural Sciences,Chengdu 610066,Sichuan,China)

机构地区：[1]四川省农业科学院农业质量标准与检测技术研究所,四川成都610066 [2]农业农村部科技发展中心,北京100176 [3]四川省农业科学院遥感与数字农业研究所,四川成都610066

出　　处：《作物学报》2023年第7期1818-1828,共11页Acta Agronomica Sinica

基　　金：2020年农业国家标准和行业标准制修订项目计划(农质标函(2020)128号);四川省自然科学基金(面上)项目“2022NSFSC0140”;四川省农业科学院“1+9”揭榜挂帅科技攻关项目(1+9KJGG006)——生物安全前沿技术资助。

摘　　要：转基因玉米ND207是中国农业大学研发的转mCry1Ab和mCry2Ab基因的抗虫玉米,本研究的目的是建立ND207转化事件特异性定性PCR检测方法。根据研发者提供的引物进行PCR扩增,PCR产物测序分析,获得了5’端旁侧序列262 bp,包括109 bp的载体序列和153 bp的玉米基因组序列, 3’端旁侧序列316 bp,包括76 bp的载体序列和240bp的玉米基因组序列。针对两端旁侧序列设计14条引物,组成25对引物组合进行引物筛选,选中3’端一对最佳引物优化PCR反应体系及反应条件,建立ND207的转化事件特异性定性PCR检测方法, PCR产物片段大小为166 bp。经过测试,该方法的检出限为0.1%,相当于20个拷贝的ND207特异分子片段。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试,循环验证报告显示:该方法符合国家标准方法的各项要求,可在检测行业推广应用。ND207转化事件特异性定性PCR检测方法的建立,为我国ND207及其衍生品系的安全监管提供技术支撑。Transgenic maize ND207 is an insect-resistant maize with mCry1Ab and mCry2Ab genes developed by China Agricultural University.The objective of this study is to develop the event-specific PCR detection method for ND207.The PCR amplification was performed according to the primers provided by the ND207 developer.The sequence of insertion site of ND207 was sequenced and obtained 262 bp of the 5'-flanking sequence,including 109 bp vector sequence and 153 bp maize genome sequence,316 bp of the 3'-flanking sequence,including 76 bp vector sequence and 240 bp maize genome sequence.Fourteen primers were designed at both ends to form 25 primer pairs.The best primer pair at the 3'end was selected to optimize the PCR reaction system and reaction condition.The event-specific qualitative PCR detection method of ND207 was established and the PCR product size was 166 bp.After testing,the results showed that the detection limit of this method was 0.1%,equivalent to 20 copies of ND207 specific molecule fragment.Eight GMO safety testing institutions in China tested specificity,detection limit,reproducibility of the method,and the circular verification report revealed that the method met the requirement of the national standard method,which could be promoted and applied in the testing industry.The established event-specific qualitative PCR detection method of ND207 provides the technical support for the safety supervision of ND207 and its derivatives in China.

关 键 词：转基因玉米 ND207 旁侧序列 转化事件特异性 定性PCR 

分 类 号：S513[农业科学—作物学]