ABSTRACT
Droplet digital polymerase chain reaction (ddPCR) is a third generation of PCR that was recently developed to overcome the limitation of direct quantification observed in real-time quantification PCR (qPCR). Recent studies have shown that ddPCR is more sensitive than the gold standard reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. In combination with multiplexing, multiple RT-ddPCR assays can be developed to directly quantify different SARS-CoV-2 nucleic acid targets within a single sample, significantly saving on cost and time. Since ddPCR is tolerant to a number of inhibitors unlike qPCR, it can be used to detect and quantify samples from complex environments like wastewater. Here we present three one-step RT-ddPCR protocols on how to develop simplex (one target), duplex (two targets), and triplex probe mix (three targets) assays for SARS-CoV-2 detection and quantification. The assays can be used for diagnosis or other research-related SARS-CoV-2 applications.