A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study

  1. Michael P Coryell
  2. Mikhail Iakiviak
  3. Nicole Pereira
  4. Pallavi P Murugkar
  5. Jason Rippe
  6. David B Williams
  7. Taylor Heald-Sargent
  8. L Nelson Sanchez-Pinto
  9. Jairo Chavez
  10. Jessica L Hastie
  11. Rosa L Sava
  12. Christopher Z Lien
  13. Tony T Wang
  14. William J Muller
  15. Michael A Fischbach
  16. Paul E Carlson

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Abstract

No abstract available

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  1. Version published to 10.1016/s2666-5247(21)00059-8
  2. ScreenIT

    SciScore for 10.1101/2020.11.09.20228601: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All fecal samples were collected by the vendors with informed consent from healthy donors between 18 and 50 years of age with no use of antibiotics for at least 30-days prior to sample donation.
    Randomizationnot detected.
    BlindingThe recipient lab was blinded to the spike-in status of the specimens.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    A SARS-CoV-2 isolate obtained from BEI Resources (NIAID, NIH) was propagated in Vero E6 cells (ATCC, Cat# CRL-1586) cultured in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin and L-glutamine.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    All PCR runs were performed on the CFX-Connect 96-well real-time PCR detection system (Bio-Rad) and real-time fluorescence data were analyzed using CFX Manager software v. 3.1 (Bio-Rad).
    CFX Manager
    suggested: (CFX Manager, RRID:SCR_017251)
    Four different storage media were tested including PBS, Cary-Blair media, Stool Transport and Recovery (STAR) buffer (Roche Diagnostics), and DNA/RNA Shield (Zymo Research).
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Data analysis: All reported average Ct values with accompanying standard deviations were calculated in Excel, and were inclusive of any detected Ct values, regardless of whether they met the 40 Ct cutoff for positive detection.
    Excel
    suggested: None
    Results of viral stability experiments were graphed and analyzed with mixed-effects models for repeated measures with multiple comparison testing in GraphPad Prism V. 8.4.0 (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: We detected the following sentences addressing limitations in the study:
    With this in mind, we sought to assess the loss of sensitivity observed following storage in some conditions, which represents a potential limitation of this assay. To address these concerns, we evaluated the stability of virus spiked stool samples stored under a variety of conditions including different storage buffers and temperature. We assessed sample stability at a relatively high viral copy number in four storage buffers held at either 4°C or ambient temperature and found consistent detection among all media and storage temperatures. However, variation in detection signal (Ct value) suggests that both stool transport/dilution media and sample storage temperatures should be considered when developing protocols for collection, storage, and testing for SARS-CoV-2 RNA in stool. Among the media tested, DNA/RNA Shield performed best overall. It had similar baseline Ct values to PBS and provided better stability in samples stored at ambient temperature or frozen at −80°C. Notably, DNA/RNA Shield performed better at ambient temperature than at 4°C, which is consistent with the manufacturer’s recommendations for use and should be a consideration when using this product. Cary-Blair medium, a common substrate for stool transport, provided the least protection from freezing but otherwise performed similarly to PBS at 4°C. The results following freezing of spiked stool samples did show potential negative effects on sample stability. Notably, freezer storage of spiked specimens reduc...

    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.

  3. Version published to 10.1101/2020.11.09.20228601 on medRxiv