基于数字PCR的非法转基因成分检测研究

With the diversified development of genetically modified organisms (GMOs) variety and traits, it brings new challenges for the detection of GMOs and their products. Detection and monitoring of un-authorized GMOs become the key and difficult point of GMO safety management. It is difficult to identify un-authorized GM ingredients mixed in legitimate GM products through existing detection methods. This project is scheduled to make an in-depth research on theory and technology of digital PCR nucleic acid measurement method, and explore absolute quantification method that does not rely on certified reference materials in GMO detection. By investigating linkage relationship of transgenic elements and flanking sequence which were adjacent in crops genomic DNA through digital PCR assays, the methods to identify un-authorized GM ingredients through analyzing copy number difference and linkage relationship of transgenic elements and flanking sequence will be established, which could solve the problems of discriminate un-authorized GMOs component mixed in legitimate agricultural products encountered in current GMO monitoring process.

转基因作物的多样化与复杂性给转基因成分检测带来诸多困难，非法商业化转基因作物的检测和监控已成为转基因生物安全管理的重点和难点。通过现有检测方法很难对混杂于合法转基因农产品中的非法转基因成分进行鉴别。本项目拟深入研究数字PCR在核酸测量中的理论与技术，探索在转基因检测中不依赖标准物质而对转基因含量进行绝对定量分析的方法；依据转基因作物基因组中处于相邻位置的外源元件和特异性旁侧序列，探索外源元件和特异性旁侧序列在PCR检测时连锁关系；建立通过分析外源元件和旁侧序列在数字PCR板检测中的拷贝数差异和连锁关系，来判断是否含有非法转基因成分的方法；解决当前转基因监测过程中遇到的非法或未知转基因成分释放到合法转基因农产品中的检测难题。

非法转基因成分的检测和监控目前仍然是转基因生物安全管理的重点和难点。本项目通过深入研究数字PCR在核酸测量中的理论与技术，建立了基于微流控芯片数字PCR和微滴式数字PCR直接对样品中的转基因含量进行绝对定量分析的方法，微流控芯片数字PCR分析的DNA模板量以300-1800拷贝为宜，微滴式数字PCR分析的DNA模板量以200-20000 拷贝为宜。以3个已批准商业化种植的油菜（GT73、OXY235、TOPAS）和1个本研究团队研发的转基因油菜Z7B10，分别模拟合法转基因成分和非法转基因成分，并混合配制形成盲样。利用P-FMV 35S、P-CaMV 35S、bar三个外源元件进行筛查检测，确定样品中含有转基因成分。利用转化体特异性检测方法，确定样品中具体含有的合法的转化体。利用数字PCR判断出样品中含有非法转基因成分。本项目的实施从基因的连锁性以及拷贝数精确定量角度出发，解决了当前转基因监测过程中遇到的非法或未知转基因成分释放到合法转基因农产品中的检测难题。本项目进一步比较了微流控芯片数字PCR和微滴式数字PCR在核酸精确定量方面的优缺点，为今后数字PCR在核酸精确定量方面提供理论基础和科学依据。