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首页> 《中国测试》期刊 >本期导读>转基因玉米MON863质粒DNA双重数字PCR定值方法的建立

转基因玉米MON863质粒DNA双重数字PCR定值方法的建立

204    2020-10-27

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作者:马丽侠1, 周李华1, 蒋子敬1, 姜展樾1, 叶德萍1, 蒲春如2, 杜娟兰2

作者单位: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


关键词:转基因玉米;质粒DNA;微滴数字PCR;双重数字PCR


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1.6~6 743.3 copies和内源基因Adh在1.1~6 770.0 copies的浓度范围均呈线性关系,r2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。


Method for determining the value of genetically modified maize MON863 plasmid DNA by duplex digital PCR
MA Lixia1, ZHOU Lihua1, JIANG Zijing1, JIANG Zhanyue1, YE Deping1, PU Chunru2, DU Juanlan2
1. National Institute of Measurement and Testing Technology, Chengdu 610021, China;
2. College of Food, Sichuan Agriculture University, Ya′an 625000, China
Abstract: 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.
Keywords: genetically modified maize;plasmid DNA;droplet digital PCR;duplex digital PCR
2020, 46(10):28-36  收稿日期: 2020-06-15;收到修改稿日期: 2020-07-21
基金项目:
作者简介: 马丽侠(1990-),女,河南周口市人,助理研究员,主要从事生物测试技术研究工作
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