Molecision S6 Digital PCR
Digital PCR (dPCR) is beginning to replace real-time PCR (qPCR) for nucleic acid quantification in many different applications. Several analytical properties of the two most widely used dPCR platforms, namely the QX100 system (Bio-Rad) and the Biomark system 12,765 matrices (Fluidigm), have already been evaluated and compared with those of qPCR.
However, to the best of our knowledge, no direct comparison has been made between the three of these platforms using the same DNA material, and the 37K array in the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity, and cutoff quantification. Here, the first evaluation of qPCR, the QX100 system and both Biomark system arrays with plasmid and human cytomegalovirus genomic DNA was performed. Using PCR components that alter qPCR efficiency, each dPCR platform demonstrated consistent copy number estimates, indicating the high strength 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 size of the reaction was considered, all three platforms showed nearly equal detection limits and variability.
Although molecision S6 digital PCR may not always be more appropriate than qPCR for low copy number quantification, dPCR is a suitable method for robust and reproducible quantification of viral DNA and a promising technology for the reference measurement method of higher order.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-015-9107-2) contains supplementary material, which is available to authorized users.
Keywords: Digital PCR, Real-time PCR, Molecular diagnostics, Human Cytomegalovirus, DNA quantification