Xu Xiaoli, Peng Cheng, Wang Xiaofu, Chen Xiaoyun, Wang Qiang, Xu Junfeng
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.
Transgenic Res. 2016 Dec;25(6):855-864. doi: 10.1007/s11248-016-9982-0. Epub 2016 Sep 8.
This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.
本研究评估了液滴数字PCR（ddPCR）作为一种用于确定玉米纯合性的工具的适用性，使用定量实时PCR（qPCR）作为参考技术。定量实时PCR通常用于确定转基因拷贝数或转基因生物的纯合性特征。然而，其有效性基于转基因和内参基因相同的反应效率。此外，为了保证准确性，应使用校准样本。液滴数字PCR是一种DNA分子计数技术，可直接对样品中靶标和参考DNA分子的绝对数量进行计数，与检测效率或外部校准物无关。使用靶标基因与单拷贝内参基因数量的比值可以轻松确定转基因的纯合性。在本研究中，qPCR和ddPCR方法均用于确定抗虫转基因玉米IE034的纯合性。两种方法都表现良好，但由于ddPCR方法具有绝对定量特性，因此更方便。
