Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

  1. Aravind Natarajan
  2. Alvin Han
  3. Soumaya Zlitni
  4. Erin F. Brooks
  5. Summer E. Vance
  6. Marlene Wolfe
  7. Upinder Singh
  8. Prasanna Jagannathan
  9. Benjamin A. Pinsky
  10. Alexandria Boehm
  11. Ami S. Bhatt

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Abstract

COVID-19 patients shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. In our benchmarking study, we recommend a standardized protocol for the preservation, extraction and detection of viral RNA from stool. This protocol includes a preservative, viral RNA extraction steps, and PCR-based quantification methods to maximize yield and detection of SARS-CoV-2 RNA. Our protocol takes advantage of commercially available reagents and equipment to maximize ease of access and consistency across studies. Additionally, we apply an attenuated bovine coronavirus vaccine as a spike-in control, and synthetic RNA standards to improve standardization and reliability of the assay. While we recommend both ddPCR and RT-qPCR-based assays, we acknowledge that ddPCR may be prohibitively expensive due to the necessity of specialized equipment and reagents. This protocol was developed with a focus on SARS-CoV-2 RNA, but may apply to other coronaviruses as well. We estimate that this protocol takes between 6 to 8 hours total to quantify the viral RNA load in a fecal sample.

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  1. Version published to 10.21203/rs.3.pex-1601/v1
  2. Version published to 10.1038/s41467-021-25576-6
  3. ScreenIT

    SciScore for 10.1101/2021.04.10.21255250: (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
    Next, we took two biopsy punches using the Integra Miltex Biopsy Punches with Plunger System (Thermo Fisher Scientific; Catalog # 12-460-410) from each of the relevant stool samples and transferred these to microcentrifuge tubes corresponding to the respective samples.
    Thermo Fisher Scientific; Catalog
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2021.04.10.21255250v1 on medRxiv