一种基因编辑位点特异性PCR方法的开发和应用  

Development and application of a gene editing site-specific PCR method

作　　者：张洪文 赵圣博 闫晓红[1] 李俊[1] 翟杉杉 肖芳 高鸿飞 李允静[1] 吴刚[1] 武玉花[1] ZHANG Hong-wen;ZHAO Sheng-bo;YAN Xiao-hong;LI Jun;ZHAI Shan-shan;XIAO Fang;GAO Hong-fei;LI Yun-jing;WU Gang;WU Yu-hua(Oil Crops Research Institute,Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Im-provement of Oil Crops,Ministry of Agriculture and Rural Affairs,Wuhan 430062,China)

机构地区：[1]中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,湖北武汉430062

出　　处：《中国油料作物学报》2021年第1期77-89,共13页Chinese Journal of Oil Crop Sciences

基　　金：转基因生物新品种培育专项(2018ZX0801105B)。

摘　　要：为了对基因组编辑产品进行精准定性和定量检测,以水稻SP1基因的编辑植株为材料,在编辑位点上下游设计通用引物,在编辑位点处设计基因编辑位点特异性TaqMan探针,建立了编辑位点特异性PCR方法。利用该方法可准确鉴定特异基因组编辑产品,检测灵敏度达到5~10拷贝,可在实时荧光PCR(qPCR)和微滴数字PCR(ddPCR)平台上对基因组编辑产品进行定量检测。由于数字PCR的微反应单元可消除野生型DNA对通用引物的竞争性消耗,与qPCR的定量结果相比,ddPCR定量结果具有更高的定量准确性。Genome editing technology has been successfully used in agricultural breeding,it is urgent to establish accurate identification and quantification technology based on molecular characteristics of genome-edited products.In this study,SP1 gene-edited rice was used as materials to develop an editing site-specific PCR method by designing universal primer pair upstream and downstream of the editing sites,and gene editing site-specific TaqMan probes targeting at the editing sites.The results demonstrated that this method accurately identified specific genome-edited products with a detection sensitivity of 5-10 copies.The quantitative detection of genome-edited products was performed by both real-time PCR(qPCR)and droplet digital PCR(ddPCR).Since thousands of independent droplets of ddPCR might eliminate the competitive consumption of universal primers by wild-type DNA,the quantitative results of ddPCR showed higher quantitative accuracy than those of qPCR.

关 键 词：基因组编辑作物 基因编辑位点特异性PCR 身份鉴定 定量检测 

分 类 号：Q789[生物学—分子生物学]