Platinum chloride-based viability RT-qPCR for SARS-CoV-2 detection in complex samples

  1. Enric Cuevas-Ferrando
  2. Walter Randazzo
  3. Alba Pérez-Cataluña
  4. Irene Falcó
  5. David Navarro
  6. Sandra Martin-Latil
  7. Azahara Díaz-Reolid
  8. Inés Girón-Guzmán
  9. Ana Allende
  10. Gloria Sánchez

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Abstract

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum.

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  1. Version published to 10.1038/s41598-021-97700-x
  2. ScreenIT

    SciScore for 10.1101/2021.03.22.21253818: (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
    Significant differences in median cycle threshold (Ct) were determined by using either one- or two way(s) ANOVA followed by Dunnett’s multiple comparisons test on GraphPad Prism version 8·02 (GraphPad Software, US).
    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:
    Above all, as some individuals reportedly remain PCR positive weeks after SARS-CoV-2 infection recovery, knowing whether viral RNA in these persistent carriers is contagious provides key insights for quarantine policy, to safely discontinue self-isolation and contact tracing as essential public health measures to definitively prevent transmission.(6,26,32) Besides some limitations, the proposed viability RT-qPCR effectively reduced the amplification signals of non-infectious and free RNA of SARS-CoV-2 in complex matrices finally providing a better estimation of the infectiousness of samples. Thus, mathematical models derived from laboratory scale experiments comparing viability RT-qPCR and viral replication could correlate viral load and infectivity, finally providing relevant tools of interest based on rapid molecular assay for prevention strategies and risk assessment.

    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 10.1101/2021.03.22.21253818 on medRxiv