基于RNAi技术的转基因玉米逆转录数字PCR检测方法

Reverse Transcription Digital PCR Detection Method for RNAi-Based Transgenic Maize

针对基于RNAi技术的转基因玉米品系,研究并建立了一套逆转录芯片式数字PCR(dPCR)定量检测该品系玉米双链RNA的方法。研究内容主要包括RNA提取方法、引物探针设计、逆转录方法、dPCR反应条件及体系等方面的探索和优化。该方法的绝对定量限为2.5 copies/μL,RSD为12.4%;检测低浓度实际样品达21.7 copies/μL时,相对偏差为2.7%,RSD为14.6%,满足国际上转基因定量结果RSD≤25%的要求。将该方法用于基于RNAi技术转基因作物的定量检测,将为我国相关转基因作物的安全评价提供了精确可靠的技术手段。

Aiming at the transgenic maize based on RNAi technology,this paper studied and established a set of reverse transcription chip digital PCR(dPCR)method of quantitatively detecting dsRNA in this transgenic maize.The research contents mainly included exploration and optimization of RNA extraction methods,primer probe design,reverse transcription method,dPCR reaction conditions and compositions,etc.The limit of absolute quantification of this method is 2.5 copies/μL,and the RSD is 12.4%.The relative deviation is 2.7%,and the RSD is 14.6%.When detecting the actual sample of low concentration is 21.7 copies/μL,which meets the international requirement of transgenic quantitative result RSD≤25%.Applying the method in the quantitative detection of genetically modified crops based on RNAi technology will provide accurate and reliable technical means for the safety evaluation of related genetically modified crops in China.