转基因玉米MON87427/zSSIIb 二重微滴数字PCR方法建立及应用

实施转基因产品定量标识制度需要建立准确可靠的定量检测技术和方法。数字PCR（dPCR）不依赖标准物质,实现对DNA分子的绝对定量,已成功用于转基因含量检测和标准物质定值。为建立可靠的dPCR方法,获得准确测量结果,本研究以耐除草剂玉米MON87427为材料,探索建立二重微滴数字PCR（ddPCR）的策略。以构建的聚合MON87427转化体和5个玉米内标基因的重组质粒pUC57-M为质控样品,将5个不同的玉米内标基因分别与MON87427组合,通过优化退火温度,根据阳性微滴与阴性微滴分辨率、中等信号强度雨滴数量及测量值与理论值的一致性等,确定了二重ddPCR组合为MON87427/zSSIIb,最适退火温度为58.4℃。MON87427/zSSIIb 二重ddPCR的动力学范围为10～60 000拷贝。对盲样进行定量检测,二重ddPCR的定量结果与荧光定量PCR有良好的可比性,表明MON87427/zSSIIb 二重ddPCR可取代qPCR方法进行转基因玉米MON87427的定量检测及标准物质定值。

Development and application of MON87427/zSSIIb duplex droplet digital PCR method

Digital PCR(dPCR),an absolute quantitative method of DNA molecules,has been successfully applied for genetically modified organisms(GMO)detection and reference material characterization.In this paper,a duplex ddPCR method was established for quantification of MON87427 content with reference plasmid pUC57-M harboring MON87427-specfic sequence and 5 maize reference gene sequences as quality control.By comparing the 5 reference genes,zSSIIb was determined to combine with MON87427 for duplex ddPCR assay according to the resolution of positive droplets and negative droplets,the number of raindrops with medium signal intensity,and the consistency between measured value and theoretical value.The optimal annealing temperature for MON87427/zSSIIb duplex ddPCR was determined at 58.4℃,and the duplex ddPCR displayed good linearity over the range from 10 to 60000 copies.The quantitative results of blinded samples by duplex ddPCR were well comparable with those by real-time quantitative PCR(qPCR),indicating that the MON87427/zSSIIb duplex ddPCR might replace the qPCR method for quantification of transgenic maize MON87427 and characterization of MON87427 reference materials.