转基因玉米MON863质粒DNA双重数字PCR定值方法的建立  

Method for determining the value of genetically modified maize MON863 plasmid DNA by duplex digital PCR

作　　者：马丽侠[1] 周李华[1] 蒋子敬 姜展樾 叶德萍[1] 蒲春如 杜娟兰 MA Lixia;ZHOU Lihua;JIANG Zijing;JIANG Zhanyue;YE Deping;PU Chunru;DU Juanlan(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,四川成都610021 [2]四川农业大学食品学院,四川雅安625000

出　　处：《中国测试》2020年第10期28-36,共9页China Measurement & Test

摘　　要：针对转基因玉米MON863质粒DNA,研究建立一套双重数字PCR(ddPCR)定值方法。优化引物探针浓度,退火温度等条件,考察方法特异性、线性范围、精密度。结果表明,外源基因MON863在1.6~6743.3 copies和内源基因Adh在1.1~6770.0 copies的浓度范围均呈线性关系,r^2为1;MON863基因和Adh基因的定量限分别为8.3 copies和7.9 copies;方法精密度小于5%。采用该方法对基体标准物质进行验证,误差小于10%。该方法每个反应体系都含有内源(VIC)和外源(FAM)基因,能实现内外源基因的同时检测,避免同一样品在不同反应体系造成的定值差异。结果显示该方法与单重数字PCR定值结果无显著差异,重复性优于单重数字PCR结果,方法准确可靠。In this study,a duplex PCR detection method for genetically modified maize of MON863 plasmid was established by droplet digital PCR(ddPCR)platform.The concentration of primer probe and annealing temperature were optimized,and the specificity,linear range and precision of the method were investigated.The results show that the exogenous gene MON863 was in a linear relationship between 1.6 to 6743.3 copies and the endogenous gene Adh was in the range of 1.1 to 6770.0 copies,and the linear coefficient was 1.The quantitative limits of MON863 was 8.3 copies and Adh was 7.9 copies.The precision was better than 5%and the specificity was good.We verified accurate quantification by determination of certified reference materials and the error is less than 10%,indicating that the method is accurate and reliable.The ddPCR duplex assay contains endogenous(VIC)and exogenous(FAM)genes simultaneously,which can compensated the error of Adh and MON863 systems in different wells.There is no significant variation between the duplex and singleplex digital PCR,but the repeatability is slightly better than singleplex digital PCR,so the duplex was accurate and reliable.

关 键 词：转基因玉米 质粒DNA 微滴数字PCR 双重数字PCR 

分 类 号：Q52[生物学—生物化学]