Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

Pecson, Brian M.; Darby, Emily; Haas, Charles N.; Amha, Yamrot M.; Bartolo, Mitchel; Danielson, Richard E.; Dearborn, Yeggie; Giovanni, George di; Ferguson, Christobel; Fevig, Stephanie; Gaddis, Erica; Gray, Donald; Lukasik, George; Mull, Bonnie; Olivas, Liana; Olivieri, Adam W.; Qu, Yan; Consortium, SARS-CoV-2 Interlaboratory

doi:10.1039/d0ew00946f

Cited by 205 publications

(224 citation statements)

References 30 publications

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“…We conclude that pasteurization results in greater sensitivity and more precise estimates of SARS-CoV-2 prevalence. Similar outcomes have been reported elsewhere [17].…”

Section: Methodssupporting

confidence: 91%

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

Trujillo

Cheung

Gao

et al. 2021

Preprint

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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.

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“…We conclude that pasteurization results in greater sensitivity and more precise estimates of SARS-CoV-2 prevalence. Similar outcomes have been reported elsewhere [17].…”

Section: Methodssupporting

confidence: 91%

“…Our protocol performed strongly in a large-scale, nationwide comparative study of the reproducibility and sensitivity of 36 methods of quantifying SARS-CoV-2 in wastewater [17]. Our protocol is identified as 4S.1(H) in Table 3.…”

Section: Introductionmentioning

confidence: 99%

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

Trujillo

Cheung

Gao

et al. 2021

Preprint

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“…At this moment, investigation of SARS-CoV-2 genes with dPCR remains underexplored [30,31]. IWW samples from the tail of the second wave of the COVID-19 pandemic were analyzed with both qPCR and dPCR.…”

Section: Resultsmentioning

confidence: 99%

An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Jacobs

et al. 2021

Preprint

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Wastewater-based epidemiology could be applied to track down SARS-CoV-2 outbreaks at high spatio-temporal resolution and could potentially be used as an early-warning for emergence of SARS-CoV-2 circulation in the general population. Epidemiological surveillance of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW). Unlike predecessors, method development of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 loads (in combination with seeding IWW with a surrogate coronavirus). This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris,etc). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering pore sizes did not improve our limit of detection and quantification. Real-time polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E_Sarbeco-gene. This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. Interestingly, qPCR results were comparable with dPCR results suggesting that qPCR is a valid method. In this study, dPCR was also used as a proxy to assess the precision of qPCR. In this light, dPCR showed high variability at low concentration levels (100 copies/microliter), indicating that variability in bioanalytical assays for SARS-CoV-2 RNA might be substantial. On average, the N2-gene showed high in-sample stability in IWW for 10 days of storage at 4 degrees. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene proved to be less stable compared to the N2-gene and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene in IWW. Although WBE can already aid in filling some knowledge gaps in the epidemiological surveillance of SARS-CoV-2, future WBE studies should aim to further validate and standardize bioanalytical assays, especially with regards to methodological limitations.

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“…Colleges employed a range of viral concentration and RNA extraction methods depending on factors including expertise, availability, cost, and speed. A technical comparison of the different laboratory methods each college has been reported elsewhere (Pecson et al 2021).…”

Section: Description Of Wastewater Monitoring Approachesmentioning

confidence: 99%

Wastewater surveillance for SARS-CoV-2 on college campuses: Initial efforts, lessons learned and research needs

Harris-Lovett

Nelson

Beamer

et al. 2021

Preprint

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Background Wastewater surveillance for SARS-CoV-2 is an emerging approach to help identify the risk of a COVID-19 outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, nursing homes) scales. Objectives This research aims to understand the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. Methods This paper presents the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resource needs, and lessons learned from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Discussion Our analysis suggests that wastewater monitoring at colleges requires consideration of information needs, local sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

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Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

Abstract: The reproducibility and sensitivity of 36 methods for quantifying the genetic signal of SARS-CoV-2 in wastewater was evaluated in a nationwide interlaboratory assessment in the U.S.

Cited by 205 publications

References 30 publications

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

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

An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Wastewater surveillance for SARS-CoV-2 on college campuses: Initial efforts, lessons learned and research needs

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