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

  1. Monica Trujillo
  2. Kristen Cheung
  3. Anna Gao
  4. Irene Hoxie
  5. Sherin Kannoly
  6. Nanami Kubota
  7. Kaung Myat San
  8. Davida S. Smyth
  9. John J. Dennehy

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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.

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  1. Version published to 10.1371/journal.pone.0257454
  2. ScreenIT

    SciScore for 10.1101/2021.02.16.21251787: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    We used both the Swift Normalase® Amplicon Panel (SNAP) SARS-CoV-2 Panel kit as well as the Qiagen QIAseq® SARS-CoV-2 Primer Panel and QIAseq FX DNA Library kit and have obtained SARS-CoV-2 sequences from several of our wastewater treatment plants.
    SNAP
    suggested: (SNAP, RRID:SCR_007936)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  3. Version published to 10.1101/2021.02.16.21251787 on medRxiv