基于FMV35S的数字聚合酶链式反应方法定量检测转基因作物

A Digital Polymerase Chain Reaction Method Based on Figwort Mosaic Virus 35S Promoter(FMV 35S)for Quantitative Detection of Genetically Modified Crops

以含有FMV35S标记基因的大豆转化体MON87705为材料,利用实时聚合酶链式反应(real-timepolymerase chain reaction,real-time PCR)和数字PCR(digital PCR,dPCR)技术,建立一种转基因作物的定量检测方法。通过real-time PCR法对扩增体系测试,建立的检测方法对FMV 35S标记基因具有高度特异性;利用双重微滴式dPCR进行定量检测参数的测定,结果表明:在基因组DNA 0.005~20 ng/μL质量浓度范围,靶标基因的测量拷贝数与理论拷贝数具有良好的线性关系;dPCR方法对FMV 35S标记基因的检出限可达到5 copies,定量限为0.1%。利用两个dPCR平台,对不同含量的转基因作物的盲样进行测定,内标准基因和外源特异性片段的双重、三重测定,均获得准确的定量结果,数据可靠,再现性良好。因此本研究建立的基于筛选标记基因FMV 35S的转基因dPCR定量检测方法在满足转基因成分定量检测标准的参数要求的同时,能够进一步提高检测效率,可为转基因作物成分定量提供一种准确、简便的检测技术。

Using genetically modified(GM)soybean MON87705 containing the figwort mosaic virus 35S promoter(FMV 35S)as a marker gene,a quantitative method for detecting transgenic crops was established based on real-time fluorescent quantitative polymerase chain reaction(real-time PCR)and digital polymerase chain reaction(dPCR).The amplification system was tested by real-time PCR,revealing that the developed method was highly specific to FMV 35S.By determining quantitative parameters using duplex droplet digital PCR(ddPCR),it was found that within the genomic DNA concentration range of 0.005–20 ng/mL,the measured copy number of the target gene had a good linear correlation with the theoretical copy number;the limit of detection(LOD)and the limit of quantification(LOQ)of dPCR for FMV 35S were as low as 5 copies and 0.1%,respectively.Blind samples with different contents of MON87705 were tested on two dPCR platforms,indicating that accurate,reliable and reproducible results were obtained in the duplex and triple detection using a combination of internal reference genes and foreign specific fragments.Therefore,the quantitative dPCR method,which can meet the parameter requirements of the quantitative detection standard for genetically modified components with improved detection efficiency,represents an accurate and simple technology for the quantitative detection of genetically modified crops.