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

Trujillo, Mónica; Cheung, Kristen; Gao, Anna; Kannoly, Sherin; Kubota, Nanami; San, Kaung Myat; Smyth, Davida S.; Dennehy, John J.

doi:10.1371/journal.pone.0257454

Cited by 20 publications

(14 citation statements)

References 17 publications

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“…Wastewater (24 h composite samples) was collected from the inflow at 14 NYC wastewater treatment plants and RNA isolated according to our previously published protocol 2 . While samples have been obtained and processed on a weekly basis since June 2020, we report herein the outcome of sequencing runs performed approximately every 2 weeks between January and June 2021.…”

Section: Methodsmentioning

confidence: 99%

“…SARS-CoV-2 is shed in feces and can be detected by RT-qPCR in wastewater correlating to caseloads in sewersheds 1 – 3 . Consequently, municipalities and public health organizations have employed wastewater surveillance as a public health tool to make informed decisions about COVID-19 interventions 2 , 4 . However, the standard application of RT-qPCR does not provide genotype information and consequently cannot be used to monitor SARS-CoV-2 evolution and track variants of concern.…”

Section: Introductionmentioning

confidence: 99%

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Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

et al. 2022

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Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

et al. 2022

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“…During pasteurization, SARS-CoV-2 RNA in primary influent persisted, while infectious SARS-CoV-2 was rapidly inactivated at 50 and 70 °C . Others have reported no effect of pasteurization on SARS-CoV-2 RNA copy numbers or even increases in the copy number associated with pasteurization . Together these observations indicate pasteurization is a reasonable biosafety strategy to mitigate infection risks associated with infectious SARS-CoV-2 while preserving the genetic signal for SARS-CoV-2 RNA surveillance.…”

Section: Resultsmentioning

confidence: 96%

Within- and between-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater during Periods of Varying COVID-19 Prevalence and Positivity

Bivins

North

et al. 2021

ACS EST Water

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Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is being used to monitor Coronavirus Disease 2019 (COVID-19) trends in communities; however, within-and between-day variation of SARS-CoV-2 RNA concentration in primary influent remains largely uncharacterized. In the current study, grab sampling of primary influent was performed every 2 h over two 24-h periods at two wastewater treatment plants (WWTPs) in northern Indiana, USA. The recovery efficiency of endogenous SARS-CoV-2 RNA in wastewater was confirmed to be similar to the recovery efficiency of the process control, bovine respiratory syncytial virus (BRSV). Recovery-corrected SARS-CoV-2 RNA concentrations in primary influent indicate diurnal loading patterns and confirm monitoring dependent on grab samples should target daytime periods with high fecal loading. Importantly, manual compositing performed at the WWTP resulted in concentrations that were consistently lower than grab sample averages indicating potential bias. Uncorrected, recovery-corrected, and pepper mild mottle virus (PMMoV)-normalized SARS-CoV-2 RNA concentrations demonstrated an ordinal agreement with increasing clinical COVID-19 positivity but not COVID-19 cases. In areas where geolocated COVID-19 case data are not available, the COVID-19 positivity rate could provide a useful county-level metric for comparison with wastewater. Nonetheless, large variation both within-and between-days may preclude robust quantitative analyses beyond correlation.

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“… [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [19] , [20] , [21] , [22] , [25] , [27] , [28] , [29] , [30] , [31] .…”

Section: Uncited Referencesunclassified

Origin and evolutionary analysis of the SARS-CoV-2 Omicron variant

Sun

Lin

Dong

et al. 2022

Journal of Biosafety and Biosecurity

112

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly into new variants throughout the pandemic. The Omicron variant has more than 50 mutations when compared with the original wild-type strain and has been identified globally in numerous countries. In this report, we analyzed the mutational profiles of several variants, including the per-site mutation rate, to determine evolutionary relationships. The Omicron variant was found to have a unique mutation profile when compared with that of other SARS-CoV-2 variants, containing mutations that are rare in clinical samples. Moreover, the presence of five mouse-adapted mutation sites suggests that Omicron may have evolved in a mouse host. Mutations in the Omicron receptor-binding domain (RBD) region, in particular, have potential implications for the ongoing pandemic.

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

Cited by 20 publications

References 17 publications

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

Within- and between-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater during Periods of Varying COVID-19 Prevalence and Positivity

Origin and evolutionary analysis of the SARS-CoV-2 Omicron variant

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