Redesigning SARS-CoV-2 clinical RT-qPCR assays for wastewater RT-ddPCR

COVID-19 wastewater surveillance has gained widespread acceptance to monitor community infection trends. Wastewater samples primarily differ from clinical samples by having low viral concentrations due to dilution, and high levels of PCR inhibitors. Therefore, wastewater samples should be processed by appropriately designed and optimized molecular workflows to accurately quantify targets. Digital PCR has shown to be more sensitive and resilient to environmental matrix inhibition. However, most SARS-CoV-2 assays have been designed for clinical use on RT-qPCR instruments, then adopted to digital PCR platforms. But it is unknown whether clinical RT-qPCR assays are adequate to use on digital PCR platforms. Here we designed an N and E gene multiplex (ddCoV_N and ddCoV_E) specifically for RT-ddPCR and benchmarked them against the nCoV_N2 and E_Sarbeco assays. ddCoV_N and ddCoV_E have equivalent limits of detections and wastewater sample concentrations to NCoV_N2 and E_Sarbeco but showed improved signal-to-noise ratios that eased interpretation and ability to multiplex. From GISAID downloaded unique sequences analyzed, 2.12% and 0.83% present a mismatch or would not be detected by the used primer/probe combination for the ddCoV_N and ddCoV_E, respectively.