title: Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.
abstract: Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.|Jožef Stefan International Postgraduate School, Jamova 39, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.
authors.forename: Jernej
authors.initials: J
authors.lastname: Pavšič
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
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Milavec
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000821:virology; D004279:DNA, Viral / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
chemical_list: D004279:DNA, Viral
keywords: DNA quantification; Digital PCR; Human cytomegalovirus; Molecular diagnostics; Real-time PCR