Development of a reproducible concentration method for monitoring SARS-CoV-2 in wastewater

Abstract

Monitoring the genetic signal of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through RNA titers in wastewater has emerged as a promising strategy for tracking community-scale prevalence of coronavirus disease 2019 (COVID-19), the resulting disease. Although many studies on SARS-CoV-2 in wastewater have been conducted around the world, a uniform procedure for concentrating the virus in wastewater is lacking. This study is the first to comprehensively evaluate how different methods for concentrating the suspended solids fraction in wastewater affects the SARS-CoV-2 signal. We additionally consider the effects of sample location in the wastewater treatment train (i.e., following preliminary or primary treatment), pasteurization, and RNA extraction method. Analysis of suspended solids obtained via centrifugation and vacuum filtration yield evidence that SARS-CoV-2 preferentially occurs in the solids as opposed to the liquid. Therefore, we assert that the recovery of SARS-CoV-2 from suspended solids in wastewater samples should be the focus of future experimental designs. Our data indicate that the SARS-CoV-2 signal is more reproducible among samples taken after primary clarification, as opposed to those taken after preliminary treatment. Additionally, we provide evidence that pasteurization of samples reduces the SARS-CoV-2 signal by approximately 50%. Finally, the results indicate that a magnetic particle approach to RNA extraction leads to an increased SARSCoV-2 signal as compared to a silica membrane approach.