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

PLoS One. 2021 Sep 23;16(9):e0257454. doi: 10.1371/journal.pone.0257454. eCollection 2021.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / virology
  • Costs and Cost Analysis / economics
  • Humans
  • New York City
  • Prevalence
  • RNA, Viral / genetics*
  • Real-Time Polymerase Chain Reaction / economics
  • Real-Time Polymerase Chain Reaction / methods
  • SARS-CoV-2 / genetics*
  • Wastewater / virology*

Substances

  • RNA, Viral
  • Waste Water

Grants and funding

JJD & DSS Project #17-2273 New York City Department of Environmental Protection https://www1.nyc.gov/site/dep/index.page The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. JJD Qiagen NGS Program grant Qiagen LLC https://www.qiagen.com/us/applications/next-generation-sequencing/rna-seq-grant-2021 The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.