基于微滴式数字PCR平台，建立了一种在同一个微滴反应体系中同时检测油菜样品中两种靶标序列的双重微滴式数字PCR（duplex-ddPCR）定量分析方法。试验结果表明，内参基因和品系特异性基因均能特异性扩增出来，所建立的RF1品系duplex-ddPCR方法特异性好。在单位体系内参和外源基因拷贝数位于18~23077的区间内可以呈现出良好的线性，r2=0.999。经验证，本方法的定量检测限（LOQ）和最低检测限（LOD）分别为18拷贝/反应和3.7拷贝/反应。精密度试验结果表明两组浓度的DNA样品所得到的平行试验结果的标准偏差（relative standard deviations，RSD）介于8.40%~24.50%，准确度试验结果显示标准偏差RSD值介于5.97%~12.64%，均达到了对方法精密度RSD和准确度RSD小于25%的要求。综上所述，本研究所建立的duplex-ddPCR定量分析方法可用于转基因油菜RF1的定量检测。

A duplex droplet digital PCR (duplex-ddPCR) quantitative analysis method for the the simultaneous detection of genetically modified rapeseed Rf1 was established based on the droplet digital PCR platform. The results showed that both endogenous reference gene and Rf1 event-specific gene could be specifically amplified, and the established duplex-ddPCR method for RF1 event-specific rapeseed had good specificity. In unit system, the reference gene number and the exogenous gene copy number showed a good linearity (r2=0.999) in the range of 18~23077 copies, andthe LOQ and LOD were 18 copies and 3.7 copies, respectively. The precision test results showed that the relative standard deviations (RSD) of endogenous PEP and RF1 content were between 8.40% and 24.50%, respectively, and the accuracy test results showed that the relative standard deviations (RSD) were between 5.97% and 12.64%, respectively. The RSD of precision and accuracy were all less than 25%, which met the detecting requirements. In conclusion, the duplex-ddPCR quantitative analysis method established in this study could be used for the quantitative detection of genetically modified rapeseed RF1.

出入境检验检疫行业标准计划项目（2015B218k）；珠海出入境检验检疫局科技计划项目（ZH2016-15）