Protocol for Safe, Affordable, and Reproducible Isolation and Quantitation of SARS-CoV-2 RNA from Wastewater

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. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step increases the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Tracking Cryptic SARS-CoV-2 Lineages Detected in NYC Wastewater

“…Wastewater was collected from the inflow at 14 NYC wastewater treatment plants and RNA isolated according to our previously published protocol 2 . Briefly, 250 mL from 24-hr composite raw sewage samples obtained from NYC WWTPs were centrifuged at 5,000 x g for 10 min at 4°C to pellet solids.…”

“…SARS-CoV-2 is shed in feces and can be detected in wastewater correlating to caseloads in sewersheds 1,2 . Since January of 2021, we sequenced SARS-CoV-2 RNA isolated from all 14 NYC WWTPs approximately twice per month 3 .…”

Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing

“…SARS-CoV-2 is shed in feces of infected individuals [1,2], and SARS-CoV-2 RNA can be extracted and quantified from community wastewater to provide estimates of SARS-CoV-2 community prevalence [3,4]. This approach is especially powerful since it randomly samples all community members and can detect viruses shed by individuals whose infections are not recorded, such as asymptomatic individuals, those who abstain from testing, or those who test at home [5,6]. Additionally, SARS-CoV-2 RNA isolated from wastewater can be sequenced using high-throughput sequencing technologies to define the composition of variants in the community [7][8][9].…”

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing

“…SARS-CoV-2 is shed in feces of infected individuals [1,2], and SARS-CoV-2 RNA can be extracted and quantified from community wastewater to provide estimates of SARS-CoV-2 community prevalence [3,4]. This approach is especially powerful since it randomly samples all community members and can detect viruses shed by individuals whose infections are not recorded, such as asymptomatic individuals, those who abstain from testing, or those who test at home [5,6]. Additionally, SARS-CoV-2 RNA isolated from wastewater can be sequenced using high-throughput sequencing technologies to define the composition of variants in the community [7][8][9].…”

“…Interestingly, polymorphisms in these lineages show considerable overlap with the Omicron VOC, suggesting convergent evolution due to similar selective pressures. 6 Here we describe an expanded set of cryptic lineages from multiple locations around the United States. While each sewershed contains its own signature lineages and at least some of the lineages appear to have diverged independently from one another, we present evidence that some likely shared a common ancestor.…”