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首页> 外文期刊>BMC Biotechnology >Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection
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Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection

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

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Abstract BackgroundMost 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.ResultsHere 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.ConclusionsThis 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.
机译：摘要背景大多数转基因（GM）植物都含有一种来自植物病毒花椰菜花叶病毒（CaMV）的启动子P35S，并且有许多终止子TNOS是根癌土壤杆菌（Agrobacterium tumefaciens）衍生的。旨在检测转基因植物的分析通常针对P35S和/或TNOS DNA序列。但是，由于P35S启动子是从CaMV衍生而来的，所以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结果。在此，我们报告了一种旨在区分被CaMV感染的植物与转基因植物的分析方法的开发。在单个多重定量PCR（qPCR）反应中。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，以靶向肌动蛋白（一种阳性对照植物基因），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（有无CaMV感染）和商业和工业样品测试了我们的四重qPCR分析的特异性。确定每个靶标的检出限（LOD）分别为肌动蛋白1％，P35S 0.001％，P3和TNOS均为0.01％。结论该测定法能够通过一次多重鉴定将受CaMV感染的植物与GM植物区分开这项研究中测试的所有样品的qPCR反应，表明该方案可广泛应用，并且可以轻松地通过qPCR平台转移到任何有关方面。

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