Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

中文翻译：

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用于单反应基因修饰（GM）植物检测和鉴定与花椰菜花叶病毒（CaMV）感染有关的假阳性GM植物的多重定量PCR

大多数转基因（GM）植物都包含一种来自植物病毒花椰菜花叶病毒（CaMV）的启动子P35S，并且许多都具有来源于根癌农杆菌的终止子TNOS。旨在检测转基因植物的检测方法通常针对P35S和/或TNOS DNA序列。但是，由于P35S启动子是从CaMV衍生的，因此这些检测方法可以从被这种天然存在的病毒感染的非转基因植物中产生假阳性。在这里，我们报告了一种检测方法的发展，该检测方法旨在在单个多重定量PCR（qPCR）反应中将受CaMV感染的植物与GM植物区分开。通过对质粒和植物DNA进行PCR和单重到多重qPCR的初步测试和优化后，设计了具有不同荧光波长的TaqMan qPCR探针以肌动蛋白（一种阳性对照植物基因）为靶标，P35S，P3（CaMV特异性基因）和TNOS。我们使用来自有机西洋菜，有机和转基因油菜的不同DNA提取物（有或没有CaMV感染）以及商业和工业样品测试了我们的四重qPCR分析的特异性。每个靶标的检出限（LOD）被确定为肌动蛋白1％，P35S 0.001％，P3和TNOS均为0.01％。对于本研究中测试的所有样品，该测定法能够在单个多重qPCR反应中将受CaMV感染的植物与GM植物区分开，这表明该方案具有广泛的适用性，并且可以轻松地通过qPCR平台转移给任何相关方。所有都带有或不带有CaMV感染，以及使用商业和工业样本。每个靶标的检出限（LOD）被确定为肌动蛋白1％，P35S 0.001％，P3和TNOS均为0.01％。对于本研究中测试的所有样品，该测定法能够在单个多重qPCR反应中将受CaMV感染的植物与GM植物区分开，这表明该方案具有广泛的适用性，并且可以轻松地通过qPCR平台转移给任何相关方。所有都带有或不带有CaMV感染，以及使用商业和工业样本。每个靶标的检出限（LOD）被确定为肌动蛋白1％，P35S 0.001％，P3和TNOS均为0.01％。对于本研究中测试的所有样品，该测定法能够在单个多重qPCR反应中将受CaMV感染的植物与GM植物区分开，这表明该方案具有广泛的适用性，并且可以轻松地通过qPCR平台转移给任何相关方。