Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater

• Published in: PloS one Vol. 16; no. 9; p. e0257454
• Main Authors: Trujillo, Monica, Cheung, Kristen, Gao, Anna, Hoxie, Irene, Kannoly, Sherin, Kubota, Nanami, San, Kaung Myat, Smyth, Davida S, Dennehy, John J
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 23-09-2021; Public Library of Science (PLoS)
• Subjects: Analysis; Asymptomatic; Biology; Biology and life sciences; Community colleges; Coronaviruses; Environmental protection; Enzymes; Funding; Genomes; Graduate studies; Health aspects; Lab Protocol; Laboratories; Medicine and health sciences; Pasteurization; Polyethylene glycol; Quantitation; Reproducibility; Research and analysis methods; Ribonucleic acid; RNA; RNA viruses; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Signal processing; Supervision; Troubleshooting; Viral diseases; Viruses; Wastewater; Workflow; United States; New York; United States > US; Texas; New York City New York
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0257454

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.