Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

  1. Maria Jose Lista
  2. Pedro M. Matos
  3. Thomas J. A. Maguire
  4. Kate Poulton
  5. Elena Ortiz-Zapater
  6. Robert Page
  7. Helin Sertkaya
  8. Ana M. Ortega-Prieto
  9. Edward Scourfield
  10. Aoife M. O’Byrne
  11. Clement Bouton
  12. Ruth E. Dickenson
  13. Mattia Ficarelli
  14. Jose M. Jimenez-Guardeño
  15. Mark Howard
  16. Gilberto Betancor
  17. Rui Pedro Galao
  18. Suzanne Pickering
  19. Adrian W. Signell
  20. Harry Wilson
  21. Penelope Cliff
  22. Mark Tan Kia Ik
  23. Amita Patel
  24. Eithne MacMahon
  25. Emma Cunningham
  26. Katie Doores
  27. Monica Agromayor
  28. Juan Martin-Serrano
  29. Esperanza Perucha
  30. Hannah E. Mischo
  31. Manu Shankar-Hari
  32. Rahul Batra
  33. Jonathan Edgeworth
  34. Mark Zuckerman
  35. Michael H. Malim
  36. Stuart Neil
  37. Rocio Teresa Martinez-Nunez

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Abstract

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .

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  1. Version published to 10.1371/journal.pone.0256813
  2. Version published to 10.1101/2020.04.22.20074351v4 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.04.22.20074351: (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

    No key resources detected.

    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

    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 10.1101/2020.04.22.20074351v3 on medRxiv
  5. ScreenIT

    SciScore for 10.1101/2020.04.22.20074351: (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.Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Recently , heat treatment of viral particles has been shown effective in inactivating SARS-CoV-2 [ 3 ] with 70°C 5min treatment rendering viral infectivity undetectable employing Vero-6 cells ( limit of detection of TCID50 assay is 100 TCID50/mL) .
    Vero-6
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, please follow this link.

  6. Version published to 10.1101/2020.04.22.20074351v2 on medRxiv
  7. Version published to 10.1101/2020.04.22.20074351v1 on medRxiv