title: Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.
abstract: 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.
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: David
authors.initials: D
authors.lastname: Dobnik
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Dejan
authors.initials: D
authors.lastname: Štebih
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Blejec
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Dany
authors.initials: D
authors.lastname: Morisset
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
mesh_terms: D018744:DNA, Plant / Q000032:analysis* / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics*; D003313:Zea mays / Q000737:chemistry / Q000235:genetics
chemical_list: D018744:DNA, Plant