玉米MON863质粒DNA标准物质的研制  

Development of Plasmid DNA Reference Material of Genetically Modified Maize ( Zea mays ) MON863

作　　者：马丽侠[1] 周李华[1] 叶德萍[1] 蒋子敬 姜展樾 蒲春如 杜娟兰 韩国全[2] MA Li-xia;ZHOU Li-hua;YE De-ping;JIANG Zi-jing;JIANG Zhan-yue;PU Chun-ru;DU Juan-lan;HAN Guo-quan(Institute of Biology,National Institute of Measurement and Testing Technology,Chengdu 610021,China;College of Food,Sichuan Agriculture University,Ya′an 625000,China)

机构地区：[1]中国测试技术研究院生物研究所,四川成都610021 [2]四川农业大学食品学院,四川雅安625000

出　　处：《化学试剂》2021年第12期1729-1734,共6页Chemical Reagents

基　　金：国家重点研发计划项目(2018YFC1603400)。

摘　　要：转基因成分检测是转基因生物安全应用和管理的重要技术支撑,转基因标准物质种类的不足可影响转基因生物检测方法的建立和标准化。选取玉米MON863中外源基因MON863与内标基因Adh1构建质粒标准物质,通过测序验证,以双重数字PCR技术对获得的质粒DNA进行均匀性及不同温度下(-20、4、25、37℃)短期(14 d)和-20℃长期(6个月)稳定性检验,联合8家实验室进行定值分析。结果显示,玉米MON863质粒DNA标准物质具备良好的均匀性,可以在-20℃稳定存放6个月;外源与内标基因的拷贝数比值为1.01,扩展不确定度为0.04;拷贝数为5.78×10^(10) copies/μL,扩展不确定度为0.29×10^(10) copies/μL。获得的标准物质适用于玉米MON863的方法学建立、定性定量检测和实验室质控等应用。The detection of genetically modified components is an important part of the safety application and management of genetically modified organisms.Due to the insufficient types of reference materials,the establishment and standardization of testing methods for genetically modified organisms are affected.The alcohol dehydrogenase 1(Adh1)gene of maize and MON863 event-specific gene were selected as target to construct plasmid standards.It was verified by sequencing.The homogeneity and short-term(fourteen days,-20,4,25,37℃)and long-term(six months,-20℃)stabilities of transgenic maize(Zea mays)plasmid DNA were evaluated using duplex digital PCR.Eight laboratories were combined for characterization analysis of reference materials,which were using MON863/Adh1 duplex digital PCR.The obtained experimental results show that the reference substance has good homogeneity and the effective period is at least 6 months(-20℃).The calculated average of all independent measurement results(8 laboratories)was assigned as the certified value.The uncertainty of the plasmid consisted of uncertainty components from characterization,homogeneity,and stability during short-term and long-term storages.The certified value of DNA copy number ratio was calculated to be 1.01 with an extended uncertainty of 0.04.DNA copy number was calculated to be 5.78×10^(10) copies/μL with an extended uncertainty of 0.29×10^(10) copies/μL.The plasmid reference material can be used for the qualitative detection,quantitative detection,and methodology establishment for GM MON863.

关 键 词：质粒DNA 标准物质 数字PCR 双重数字PCR 转基因玉米 

分 类 号：Q819[生物学—生物工程]