Multiplex Quantification of 12 European Union Authorized Genetically Modified Maize Lines with Droplet Digital Polymerase Chain Reaction
Multiplex Quantification of 12 European Union Authorized Genetically Modified Maize Lines with Droplet Digital Polymerase Chain Reaction
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DOI：
10.1021/acs.analchem.5b01208
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发表时间：
2015-08-18
影响因子：
7.4
通讯作者：
Zel, Jana
中科院分区：
文献类型：
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作者：
Dobnik, David;Spilsberg, Bjorn;Zel, Jana
许多国家对食品和饲料产品中存在的转基因生物进行了监管。欧盟（EU）已对每种成分含有超过0.9%授权转基因生物的产品的标签实施了门槛。随着转基因生物的数量随着时间的推移而增加，基于标准曲线的单纯定量聚合酶链反应（qPCR）分析不再具有足够的成本效益，尽管广泛使用基于PCR的初始筛选。新开发的转基因生物检测方法，也是多重方法，主要集中在筛选和检测，而不是定量。在液滴数字PCR（ddPCR）技术的基础上，开发了用于量化所有12个欧盟授权转基因玉米系（2015年4月1日）的多重检测方法。由于12个GM靶标中的一些具有高序列相似性，因此需要两个单独的多重测定。在两种测定（4重和10重）中，转基因用一种荧光报告物标记，内源基因用另一种荧光报告物标记（GMO浓度=转基因/内源基因比率）。结果表明，这两种多重检测方法都能产生特定的结果，并且定量限、重复性和真实度等性能参数符合国际转基因生物定量方法的建议。此外，对于含有转基因生物的样品，与qPCR相比，通量和成本效益显著提高。因此，可以得出结论，多重ddPCR检测可以应用于12个欧盟授权的转基因玉米品系的常规定量。在新授权的情况下，事件可以容易地添加到现有的多重测定中。所提出的定量多路复用的原理可以应用于任何其他领域。
Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.