转基因甜菜GTSB77双重微滴数字PCR定量检测方法

Event-specific quantitative determination method for genetically modified sugarbeet GTSB77 using duplex droplet digital polymerase chain reaction

为实现转基因甜菜品系GTSB77的标识管理和精准定量,根据GTSB77的3'端边界序列和甜菜谷氨酰胺合成酶(GS)基因设计引物探针建立双重微滴数字PCR检测方法。结果显示:建立的转基因甜菜GTSB77数字PCR检测方法特异性强,在20μL反应体系中定量下限(LOQ)均为1.84拷贝/μL,检测下限(LOD)均为0.61拷贝/μL,可应用于转基因甜菜GTSB77成分的精确定量检测。

For implementation of genetically modified sugarbeet GTSB77 labeling regulations and precise quantification,primer pairs and probes based on the 3′flanking sequence of transgene GTSB77 and endogenous glutamine synthetase(GS)gene were designed and then the duplex droplet digital PCR(ddPCR)detection method for GTSB77 was established in this study.Results showed that the developed ddPCR method was specific for GTSB77.The limit of quantitation(LOQ)and the limit of detection(LOD)of transgenes and endogenous gene were 1.84 copies/μL and 0.61 copies/μL of GTSB77 haploid genomic DNA in 20μL reaction system,respectively.The precision and trueness were all in the acceptable range.It was concluded that the duplex droplet digital PCR assays could be applied for precise quantification of sugarbeet GTSB77.