Smart-Plexer: a breakthrough workflow for hybrid development of multiplex PCR assays

Developing multiplex PCR assays requires extensive experimental testing, the number of which exponentially increases by the number of multiplexed targets. Dedicated efforts must be devoted to the design of optimal multiplex assays ensuring specific and sensitive identification of multiple analytes in a single well reaction. Inspired by data-driven approaches, we reinvent the process of developing and designing multiplex assays using a hybrid, simple workflow, named Smart-Plexer, which couples empirical testing of singleplex assays and computer simulation to develop optimised multiplex combinations. The Smart-Plexer analyses kinetic inter-target distances between amplification curves to generate optimal multiplex PCR primer sets for accurate multi-pathogen identification. In this study, the Smart-Plexer method is applied and evaluated for seven respiratory infection target detection using an optimised multiplexed PCR assay. Single-channel multiplex assays, together with the recently published data-driven methodology, Amplification Curve Analysis (ACA), were demonstrated to be capable of classifying the presence of desired targets in a single test for seven common respiratory infection pathogens.