摘要摘 要 数字PCR是一种不依赖于外部标准曲线而对目标DNA/RNA进行绝对定量的测量技术，在转基因生物的定量检测、外源基因拷贝数分析以及标准物质定值方面具有广阔的应用前景。本研究基于3种数字PCR平台：微流控芯片数字PCR(chamber digital PCR, cdPCR)、QX100?微滴数字PCR(droplet digital PCR, ddPCR)和QuantStudio? 3D 数字PCR(3D digital PCR, 3D-dPCR)，分别测定转基因油菜(Brassica campestris)多靶标质粒DNA标准分子pCAN的外源品系特异性序列拷贝数和內源标准基因磷酸烯醇丙酮酸羧化酶基因(phosphoenolpyruvate carboxylase, PEP)拷贝数，二者之比即为pCAN标准分子的特性量值。实验结果表明，三种数字PCR平台对多靶标质粒分子的各靶标的量值测定结果均接近真实值1(copies/copies)，且反应体系稳定，重复性良好，相对标准差(relative standard deviation, RSD)均小于25%，说明这三种数字PCR技术均能准确地对质粒标准物质进行定值。同时，本研究建立的基于cdPCR、ddPCR和3D-dPCR平台的质粒标准物质的定值方法也为其他形式标准物质的定值提供新的方法借鉴。
Abstract：Abstract Digital PCR is a new absolute quantitative method that does not rely on the external standard curves, and it shows broad application prospects in quantified detection of genetically modified organisms (GMOs), evaluation the exogenous gene copy number of GMOs and validation the certified values of GM reference materials. In this study, three digital PCR platforms including chamber digital PCR (cdPCR), droplet digital PCR (ddPCR) and 3D digital PCR (3D-dPCR) were used to quantify the copy number of exogenous fragments and endogenous gene phosphoenolpyruvate carboxylase (PEP) of GM canola (Brassica campestris) multi-targets plasmid reference molecule, and the certified value were determined by the mean copy number ratios of exo-gene/endo-gene. The results indicated that the certified values from three dPCR technology were close to the true value of 1 (copies/copies) and the relative standard deviations were (RSD) below 25%, The three dPCR all had stable reaction systems and good repeatability and showed high accuracy in value determination of plasmid DNA reference materials. Meanwhile, the plasmid value determination methods based on cdPCR, ddPCR and 3D-dPCR platforms provide a new model for validation of other reference materials' certified values.
