油菜转化体鉴定阳性质粒分子pYCID-1905的研制及应用

转基因油菜是我国转基因生物安全监管的重要对象,为了解决检测机构常常面临的标准物质(样品)缺乏困境,统计我国批准进口和正在申请安全证书的11个转基因油菜品种,分析分子特征和相应的检测标准方法,确定每个转基因油菜品种的检测靶标序列。将11个转基因油菜品种的检测靶标序列通过基因合成的方法融合构建到pUC18载体上,研制出阳性质粒分子pYCID-1905,为转基因油菜的转化体鉴定检测提供了通用的阳性对照分子。结果发现,该质粒分子可同时用作国家标准(GB/T)、农业农村部公告、进出口检验检疫标准(SN/Y)和欧盟标准中普通PCR方法和实时荧光PCR方法的阳性对照,可以解决转基因油菜检测中缺乏标准样品的难题。

Development and application of reference plasmid pYCID-1905 for identification of rapeseed events

To monitor and detect the presence of genetically modified rapeseed in China,reference materials are strongly needed.This study collected 11 transgenic rapeseed varieties that China had approved for import and would approve for import.Target sequences for event-specific identification of 11 transgenic rapeseed varieties were determined by analyzing their molecular characteristics and corresponding standard methods.Their sequences were fused and inserted into pUC18 vector by gene synthesis as reference plasmid pYCID-1905,which provided a general positive control molecule for the identification of transgenic rapeseed events.Results showed that the plasmid molecule can be used as positive control for both conventional PCR methods and real-time PCR methods in national standards(GB/T),declaration of the Ministry of Agriculture and Rural Affairs,entry-exit inspection and quarantine industry standards(SN/T),and European Union standards.