An Open One-Step RT-qPCR for SARS-CoV-2 detection

  1. Ariel Cerda
  2. Maira Rivera
  3. Grace Armijo
  4. Catalina Ibarra-Henriquez
  5. Javiera Reyes
  6. Paula Blázquez-Sánchez
  7. Javiera Avilés
  8. Aníbal Arce
  9. Aldo Seguel
  10. Alexander J. Brown
  11. Yesseny Vásquez
  12. Marcelo Cortez-San Martín
  13. Francisco A. Cubillos
  14. Patricia García
  15. Marcela Ferres
  16. César A. Ramírez-Sarmiento
  17. Fernán Federici
  18. Rodrigo A. Gutiérrez

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Abstract

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

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  1. Version published to 10.1371/journal.pone.0297081
  2. Version published to 10.1101/2021.11.29.21267000v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.11.29.21267000: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: All methods and procedures were performed following the relevant guidelines and regulations approved by the Ethics Committee of the Universidad de Santiago of Chile.
    Consent: Informed consent was obtained from all participants and/or their legal guardians.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    The Taq DNA Polymerase (pET-28a_6H-TAQ_E602D) was obtained from Dr. Robert Tjian
    pET-28a_6H-TAQ_E602D
    suggested: RRID:Addgene_166944)
    In addition, NdeI and BamHI were used to clone it in a pET-19 vector with N-terminal 10x His-tag.
    pET-19
    suggested: None
    Software and Algorithms
    SentencesResources
    The analysis was performed using GraphPad Prism 8 software.
    GraphPad Prism
    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: 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.11.29.21267000v1 on medRxiv