微滴式数字PCR和实时荧光PCR在豆奶饮料转基因成分检测中的应用

Application of Digital PCR and Real-time PCR on Screening Detection of GM Components of Soybean Milk

为探索数字PCR在转基因成分筛选检测中的应用,解决现行转基因食品标准实施中的问题,完善我国转基因食品的检测监管体系。本文选取转基因启动子Ca MV35s和终止子NOS基因为靶标,大豆内源基因Lectin为内标,以转基因大豆标准品分别测定数字PCR和实时荧光PCR的浓度和含量检测低限,并应用于30批次豆奶饮料转基因成分实测。结果表明,数字PCR在转基因筛查的浓度检测低限达到0.04ng/反应,含量检测低限达到0.05%,均优于实时荧光PCR(0.2ng/反应,1%),且能够满足最严欧盟转基因标识阈值0.9%的检测要求。在豆奶饮料实际应用中发现,26批次样品两个平台检测结果一致为阴性,1批次一致为阳性,3批次Ct值在34.59～38.28之间的样品经数字PCR检测得到确切结果,说明数字PCR可辅助确认RT-PCR可疑结果,解决现行标准判定难题。

In order to solve the implementation problems of current GM food standards and improve the inspection and supervision of GM foods in China, the application of digital PCR(dPCR) in the screening detection of genetically modified organisms(GMOs) was developed. In this study, the transgenic promoter Ca MV35 s and the terminator NOS genes were selected as targets and soybean endogenous gene Lectin as internal standard, and the detection limit were determined using transgenic soybean standard on real-time PCR(RT-PCR) and ddPCR systems. Two methods were both applied to GM screening detection for 30 batched of soybean milk. The results showed that the concentration detection limit of ddPCR reached 0.04 ng/reaction, and the limit of content detection reached 0.05%, which were both superior to RT-PCR(0.2 ng/reaction, 1%). In addition, 0.05% was lower than the most stringent labeling threshold level 0.9% of the European Union(EU). In the practical application of soybean milk, 26 batches of samples were consistently negative and 1 was positive on both of the two platforms, while 3 batches of samples with Ct values between 34.59~38.28 were confirmed by ddPCR, indicating that dPCR could help confirm the uncertain results of RT-PCR and slove the current standard judgment problems.