Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

  1. Tuna Toptan
  2. Sebastian Hoehl
  3. Sandra Westhaus
  4. Denisa Bojkova
  5. Annemarie Berger
  6. Björn Rotter
  7. Klaus Hoffmeier
  8. Jindrich Cinatl
  9. Sandra Ciesek
  10. Marek Widera

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Abstract

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.20.052258: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: In accordance to the decision of the Committee on Biological Agents (ABAS) and Central Committee for Biological Safety (ZKBS) all work involving infectious SARS-CoV-2 and SARS-CoV was performed under biosafety level 3 (BSL-3) conditions in a BSL-3 facility.
    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
    SARS-CoV-2 was propagated in Caco-2 cells using MEM, while SARS-CoV was grown in Vero cells both using with 1% FCS Virus stocks were stored at −80°C.
    Caco-2
    suggested: None
    For intracellular RNA testing, 1x 105 Vero cells were seeded per well in a 12-well plate.
    Vero
    suggested: None
    Illumina NGS Sequencing of SARS-CoV-2 isolates: Caco2 cells were infected with different viral strains (FFM1-FFM7) at an MOI 0.01.
    Caco2
    suggested: None
    Software and Algorithms
    SentencesResources
    The PCR runs were analyzed with Bio-Rad CFX Manager software version 3.1
    Bio-Rad CFX Manager
    suggested: None
    CFX
    suggested: None
    Primer3 and Geneious Prime® software version 2020.0.5 (Biomatters Ltd.) were used to design primer and probes matching the consensus sequence.
    Geneious Prime®
    suggested: (Geneious, RRID:SCR_010519)
    Oligo characteristics were calculated using a modified version of Primer3 2.3.7.
    Primer3
    suggested: (Primer3, RRID:SCR_003139)
    Illustration were made with Geneious software.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)

    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.
    • No funding statement was detected.
    • 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.

  2. Version published to 10.3390/ijms21124396
  3. ScreenIT

    SciScore for 10.1101/2020.04.20.052258: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIn accordance to the decision of the Committee on Biological Agents ( ABAS ) and Central Committee for Biological Safety ( ZKBS ) all work involving infectious SARS-CoV-2 and SARS-CoV was performed under biosafety level 3 ( BSL-3 ) conditions in a BSL-3 facility .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
    SARS-CoV-2 was propagated in Caco-2 cells using MEM , while SARS-CoV was grown in Vero cells both using with 1 % FCS Virus stocks were stored at −80°C .
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, CVCL_0025
          <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For time point analysis , Caco2 cells were infected with SARS-CoV-2 ( 0.01 MOI ) in MEM media supplemented with 1 % FCS , at 37°C , 5 % CO2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Caco2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR runs were analyzed with Bio-Rad CFX Manager software version 3.1</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Bio-Rad CFX Manager</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CFX</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primer3 and Geneious Prime® software version 2020.0.5 ( Biomatters Ltd. ) were used to design primer and probes matching the consensus sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Geneious Prime®</b></div>
        <div>suggested: (Geneious, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010519">SCR_010519</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Oligo characteristics were calculated using a modified version of Primer3 2.3.7 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Primer3</b></div>
        <div>suggested: (Primer3, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003139">SCR_003139</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Illustration were made with Geneious software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Geneious</b></div>
        <div>suggested: (Geneious, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010519">SCR_010519</a>)</div>
      </div>
    </td></tr></table>

    Results from Barzooka: We also found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  4. Version published to 10.1101/2020.04.20.052258v1 on bioRxiv