Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples

  1. Kévin Cassinari
  2. Elodie Alessandri-Gradt
  3. Pascal Chambon
  4. Françoise Charbonnier
  5. Ségolène Gracias
  6. Ludivine Beaussire
  7. Kevin Alexandre
  8. Nasrin Sarafan-Vasseur
  9. Claude Houdayer
  10. Manuel Etienne
  11. François Caron
  12. Jean Christophe Plantier
  13. Thierry Frebourg

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Abstract

Background

Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity.

Methods

We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection.

Results

For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs.

Conclusion

Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.02.20166694: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All patients had a deep nasopharyngeal swabbing (Sigma Virocult® system -MWE, Corsham,UK), and then were asked to drool around 2 mL of saliva into a sterile 50 mL Falcon plastic tube (Thermo Fisher Scientific, Illkirch, France), after they gave informed consent.
    IACUC: The protocol was approved by the institutional ethics committee (2020T3-12_RIPH3 HPS_2020-A00920-39).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    RT-qPCR: After viral inactivation, two qualitative methods of RT-qPCR were used by the Virology laboratory of Rouen University Hospital for routine diagnosis, depending on the supply stock: (i) an automated method, using Abbott RealTime SARS-CoV-2 EUA test (Abbott Park, IL, USA), performed on 500 µl of nasopharyngeal samples and (ii) RNA extraction from 200 µl of sample (nasopharyngeal swab or saliva), performed using EZ1 DSP virus kit (Qiagen, Hilden, Germany) and EZ1 Advanced XL machine, then RT-qPCR on 10µl of extracted RNA, using RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) and performed on a CFX96™ Real-Time PCR Detection System (BioRad, Californie, USA).
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    RT-ddPCR: RT-ddPCR assays were performed using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad Laboratories, Hercules, CA, USA) and the QX200 ddPCR platform (Biorad).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    The specificity of each primer and probe was checked using the BLASTN program (https://blast.ncbi.nlm.nih.gov/) across a bank of 2045 viral genomic sequences.
    BLASTN
    suggested: (BLASTN, RRID:SCR_001598)

    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: 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

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  2. Version published to 10.1093/clinchem/hvaa323
  3. Version published to 10.1101/2020.08.02.20166694v1 on medRxiv