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

Citation

Dennehy, John (2021), Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater, Dryad, Dataset, https://doi.org/10.5061/dryad.zkh189396

Abstract

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 BSL1+ 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.

Methods

Cts of SARS-CoV-2 RNA extracted from NYC wastewater processed using different techniques were converted to N1 copies/L using RT-qPCR standard curves. 

Usage Notes

Rows represent independent samples obtained from different wastewater treatment plants on the same date. Columns under the same treatment heading represent repeated RT-qPCR assay measurements of the same sample. 

Funding

New York City Department of Environmental Protection, Award: 58359-02-02

New York City Department of Environmental Protection, Award: 58359-02-02