Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.
Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.
复制标题
DOI：
10.1038/srep35451
复制
发表时间：
2016-10-14
影响因子：
4.6
通讯作者：
Žel J
中科院分区：
文献类型：
--
作者：
Dobnik D;Štebih D;Blejec A;Morisset D;Žel J
数字PCR技术的优点到目前为止已经有很好的记录。实现该技术更好的成本效率的一种方法是在多路复用策略中使用它。包括两个荧光滤光片的液滴数字PCR平台至少支持双重反应，并且随着一些发展和优化，更高的多重化是可能的。本研究不仅显示了液滴数字PCR中多重检测的发展，而且还首次对这种多重数字PCR中的几个参数进行了全面评估。开发了两种4重测定用于定量8种不同的DNA靶标（7种遗传修饰的玉米事件和玉米内源基因）。每次测定，两个靶标用一种荧光团标记，两个用另一种标记。由于目前的分析软件不支持超过双链体的分析，因此开发了一种新的基于R和Shiny的Web应用程序分析工具（http：//bit.ly/ddPCRmulti），可自动分析4-plex结果。总之，这两种开发的多重检测适用于定量转基因玉米事件，并且相同的方法可用于需要准确和可靠定量多个DNA靶标的任何其他领域。
The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.
登录
查看更多内容
影响因子：
7.4
作者：
Dobnik, David;Spilsberg, Bjorn;Zel, Jana
通讯作者：
Zel, Jana
影响因子：
3.4
作者：
Peccoud, J;Jacob, C
通讯作者：
Jacob, C
影响因子：
7.4
作者：
McDermott, Geoffrey P.;Do, Duc;Lowe, Adam J.
通讯作者：
Lowe, Adam J.
影响因子：
9.3
作者：
Huggett, Jim F.;Foy, Carole A.;Bustin, Stephen A.
通讯作者：
Bustin, Stephen A.
影响因子：
3.7
作者：
Lievens, A.;Jacchia, S.;Querci, M.
通讯作者：
Querci, M.