A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2

  1. Federico Fuchs Wightman
  2. Micaela A. Godoy Herz
  3. Juan C. Muñoz
  4. José N. Stigliano
  5. Laureano Bragado
  6. Nicolas Nieto Moreno
  7. Marcos Palavecino
  8. Lucas Servi
  9. Gonzalo Cabrerizo
  10. José Clemente
  11. Martín Avaro
  12. Andrea Pontoriero
  13. Estefanía Benedetti
  14. Elsa Baumeister
  15. Fabian Rudolf
  16. Federico Remes Lenicov
  17. Cybele Garcia
  18. Valeria Buggiano
  19. Alberto R. Kornblihtt
  20. Anabella Srebrow
  21. Manuel de la Mata
  22. Manuel J. Muñoz
  23. Ignacio E. Schor
  24. Ezequiel Petrillo

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Abstract

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  1. Version published to 10.1080/15476286.2021.1926648
  2. ScreenIT

    SciScore for 10.1101/2020.12.16.20246678: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationAnonymized and randomized samples were either inactivated and transported to be extracted at Instituto de Fisiología,
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All candidate pairs were checked for dimer formation in both Thermo Fisher Primer Analyzer and Beacon Designer.
    Thermo Fisher Primer
    suggested: (Resource Identification Portal, RRID:SCR_004098)
    Alternatively, the GeneFinder RT-qPCR Kit was used for some experiments (REF: IFMR-45, https://www.fda.gov/media/137116/download).
    GeneFinder
    suggested: (GENEFINDER, RRID:SCR_009190)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The main limitation we encountered when switching to intercalating dyes for detection was unspecific amplification. Unlike labeled probes, which have an extra layer of specificity, negative samples analyzed with SYBR Green-based qPCRs may sometimes exhibit unspecific sigmoidal fluorescence curves with high Ct values due to, for example, primer dimer amplification. While the wide screening of primer pairs that we performed resulted in an amplicon where this issue was greatly diminished, it came at the cost of analyzing and selecting a specific threshold to consider a sample positive for each qPCR mix. The thresholds were selected to exclude Cts arising from occasional amplification of negative controls (lacking any template). As described above, this did not have a major effect on the sensitivity of detection. It is worth mentioning that a negative result on this kind of test does not rule out completely the possibility of actual SARS-CoV-2 infection. Other variables, like the progression of the disease at the moment of the swab or the quality of the sample, also influence the final result of the test. A proper diagnosis must take into account all these variables and not only the result of the qPCR. The work presented here clearly broadens the possibilities regarding the use of reagents and equipment. Furthermore, this method based on intercalating dyes displays similar diagnostic power to widely used qPCRs based on labeled probes. Hence, combining these alternatives for SARS-...

    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 10.1101/2020.12.16.20246678 on medRxiv