利用微滴式数字PCR技术分析转基因玉米抗除草剂标记基因EPSP拷贝数

Analysis of the Copy Number of Herbicide Resistant Marker Gene EPSP in Transgenic Maize by Droplet Digital PCR

基于微滴式数字PCR(Droplet digital PCR,ddPCR)平台,以转基因玉米为例,建立了转基因作物(Genetically modified crops,GM crops)外源基因拷贝数分析方法,对待测样品进行了快速鉴定,并从T_0转基因玉米株系中鉴定出多个单拷贝单株。对该方法与实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)方法在分析结果的准确性方面进行了比较,从试验数据可以看出,2种检测方法的结果比较一致,单拷贝检测结果高度一致;但是ddPCR试验操作更加简便,试验结果可重复性强,试验数据更加准确可靠。研究表明,ddPCR方法是一种更加便捷、快速和准确的外源基因拷贝数分析新方法,基于其在准确性和灵敏度方面的显著优势,将会在转基因作物的外源基因拷贝数分析中得到广泛的应用。

The droplet digital PCR （ddPCR） method to evaluate the exogenous gene copy number in genetically modified crops （GM crops） as the example from genetically modified maize was developed,and some T0 transgenic maize plants with single copy of EPSP gene were selected from all the samples to be detected.The results were also compared with those from quantitative real-time PCR （qRT-PCR）,as could be seen from the experimental datas,the results from qRT-PCR（TaqMan） and ddPCR for EPSP were consistent,especially the single copy detection results of both in a high degree of consistency.And the ddPCR method was easy to operate,the result was repeatable and accurate.Therefore,the ddPCR would be well developed as a novel method for estimating transgenic copy number with high accuracy,which might be widely used in the exogenous genes copy number analysis in GM crops in the near future.