High-Level Multiplexing in Digital PCR with Intercalating Dyes by Coupling Real-Time Kinetics and Melting Curve Analysis

Digital polymerase chain reaction (dPCR) is a mature technique that has enabled scientific breakthroughs in several fields. However, this technology is primarily used in research environments with high-level multiplexing, representing a major challenge. Here, we propose a novel method for multiplexing, referred to as amplification and melting curve analysis (AMCA), which leverages the kinetic information in real-time amplification data and the thermodynamic melting profile using an affordable intercalating dye (EvaGreen). The method trains a system composed of supervised machine learning models for accurate classification, by virtue of the large volume of data from dPCR platforms. As a case study, we develop a new 9-plex assay to detect mobilized colistin resistant genes as clinically relevant targets for antimicrobial resistance. Over 100,000 amplification events have been analyzed, and for the positive reactions, the AMCA approach reports a classification accuracy of 99.33 ± 0.13%, an increase of 10.0% over using melting curve analysis. This work provides an affordable method of high-level multiplexing without fluorescent probes, extending the benefits of dPCR in research and clinical settings.