Comparison of RT-qPCR and Digital PCR Methods for Wastewater-Based Testing of SARS-CoV-2

Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson’s r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provides support for the application of all three methods in wastewater-based epidemiology, with additional guidelines for the use of RT-qPCR. Impact Statement PCR-based assays are the current standard for sensitive, specific, rapid pathogen quantification in environmental samples, including wastewater. The increased availability of multiple digital PCR technologies necessitates side-by-side comparison between platforms, including traditional qPCR, to guide the application of these methods. Specifically, this work can inform interpretation of wastewater SARS-CoV-2 PCR data, as reported to public health agencies for pandemic response. ### Competing Interest Statement Raymond John Abayan, Kristin Loomis, and Monica Herrera are employees of Bio-Rad Laboratories, which commercializes equipment and assays for ddPCR. ### Funding Statement Catena Foundation and NSF-GRFP to A. Hinkle, H.D. Greenwald, and L.C. Kennedy. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the study are available in the supplementary tables