微滴式数字PCR定量检测转基因玉米品系VCO-01981-5

摘要

建立基于QX100微滴式数字聚合酶链式反应(polymerase chain reaction,PCR)平台的我国未批准转基因玉米品系VCO-01981-5的二重微滴式数字PCR定量检测方法.该方法选择基因组中单拷贝的玉米内源基因hmg和VCO-01981-5品系边界序列为定量靶序列,分别设计不同的PCR扩增引物和TaqMan探针,并对两种探针用不同的荧光进行标记,然后将上述探针和引物置于同一个PCR反应体系中以同时定量两个靶标序列.特异性实验结果显示该法只有VCO-01981-5品系的两个靶序列才都有扩增信号.灵敏度、线性和准确性实验结果显示在定量结果相对标准偏差不大于25％时,最低可稳定定量5个拷贝的VCO-01981-5品系特异性序列分子和4个拷贝的内源基因hmg分子;而在高达50 ng模板DNA以下范围内,PCR反应模板量与测定样品拷贝数之间呈高度正相关,相关系数达0.99以上;平均误差小于10％.结果表明本研究建立的该玉米品系定量方法特异性强,稳定性好,精确性、准确性以及灵敏度高,定量范围广,可用于进、出口农产品和食品中该转基因玉米品系成分的定量检测.此外,该法还可为其他转基因玉米品系及其他转基因作物品系建立类似定量检测方法提供参考.%In this paper,a duplex droplet digital polymerase chain reaction (ddPCR) method was developed to detect the content of unauthorized genetically modified (GM) maize event VCO-01981-5 based on a QX100 ddPCR platform.Towards this goal,PCR primers and TaqMan probes were designed based on the maize endogenous gene hmg and VCO-01981-5 event specific boundary sequences,and the probes were labeled with different chromophores so that the two targets could be detected in one single droplet.Analysis of the specificity of the method showed that both endogenous gene hmg and foreign sequence could be detected only in event VCO-01981-5.Moreover,the sensitivity,linearity and accuracy were evaluated and it was found that when relative standard deviation (RSD) was 25％ or less,5 copies of boundary sequences and 4 copies of gene hmg could be quantitatively detected,and the quantitative results (target copy) were highly correlated with the amount of DNA template up to 50 ng (R2 ≥ 0.99),with an average error of less than 10％.All the above results indicated that the established method in this study was very specific,stable,accurate and sensitive,and had a wide quantitative range in quantitative analysis of GM maize VCO-01981-5.Therefore,this method can be applied practically in quantitatively determining this GM maize event in imported and exported agricultural product and foods.Besides,this reported method can be used as a reference to establish a quantitative method for the detection of other GM maize events and GM events of other plants.