THE COMPARISON OF THREE REAL-TIME PCR KITS FOR SARS-COV-2 DIAGNOSIS REVEALS DISCREPANCIES ON THE IDENTIFICATION OF POSITIVE COVID-19 CASES AND DISPERSION ON THE VALUES OBTAINED FOR THE DETECTION OF SARS-COV-2 VARIANTS

  1. Álvaro Santibáñez
  2. Roberto Luraschi
  3. Carlos Barrera-Avalos
  4. Eva Vallejos-Vidal
  5. Javiera Alarcón
  6. Javiera Cayunao
  7. Andrea Mella
  8. Maximiliano Figueroa
  9. Felipe Hernández
  10. Bárbara Plaza
  11. Ailen Inostroza-Molina
  12. Daniel Valdés
  13. Mónica Imarai
  14. Claudio Acuña-Castillo
  15. Felipe E. Reyes-López
  16. Ana María Sandino

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Abstract

The COVID-19 pandemic has generated a huge challenge and threat to public health throughout the world population. Reverse transcription associated with real-time Polymerase Chain Reaction (RT-qPCR) has been the gold-standard molecular tool for diagnosis and detection of the SARS-CoV-2. Currently, it is used as the main strategy for testing, traceability, and control of positive cases For this reason, the on-top high demand for reagents has produced stock-out on several occasions and the only alternative to keep population diagnosis has been the use of different RT-qPCR kits. Therefore, we evaluate the performance of three of the commercial RT-qPCR kits currently in use for SARS-CoV-2 diagnosis in Chile, consisting in: TaqMan 2019-nCoV Assay Kit v1 (Thermo). Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2 (BGI), and LightCycler® Multiplex RNA Virus Master (Roche). Results of quantification cycle (Cq) and relative fluorescence units (RFU) obtained from their RT-qPCR reactions revealed important discrepancies on the total RNA required for the identification of SARS-CoV-2 genes and diagnosis. Marked differences between kits in samples with 30>Cq value< 34 was observed. Samples with positive diagnoses for Covid-19 using the Thermo Fisher kit had different results when the same samples were evaluated with Roche and BGI kits. The displacement on the Cq value for SARS-CoV-2 identification between the three different RT-qPCR kits was also evident when the presence of single nucleotide variants was evaluated in the context of genomic surveillance. Taken together, this study emphasizes the special care adjusting RT-qPCR reaction conditions of the different kits must be taken by all the laboratories before carrying out the detection of SARS-CoV-2 genes from total RNA nasopharyngeal swab (NPS) samples.

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    SciScore for 10.1101/2021.07.13.21260484: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: 2.6 Ethics statement: All the experimental procedures included in this study was authorized by the Ethical Committee of the University of Santiago of Chile (No. 226/2021) and the Scientific Ethical Committee of the Central Metropolitan Health Service, Ministry of Health, Government of Chile (No. 370/2021), and following the Chilean law in force.
    Consent: Verbal consent was detailed by the health professional assigned by CESFAM for this purpose.
    Sex as a biological variablenot detected.
    RandomizationIn order to get the maximum representation of values in the curve, we used for the 10-fold serial dilutions a reference pool made from randomized ten total RNA NPS-extracted samples with a Cq value around 20.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All the RT-qPCR reactions were performed on the Agilent AriaMx Real-Time PCR System (Agilent Technologies, Part. No. G8830A).
    Agilent AriaMx
    suggested: (Agilent AriaMx Real-time PCR System, RRID:SCR_019469)
    We determined the slope by linear regression in GraphPad Prism and defined the required levels for PCR efficiency (E) and R-squared (R2) as>95 % and>0.95, respectively.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Prism 8 statistical software was used to analyze and plot the data obtained.
    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: 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.

  2. Version published to 10.1101/2021.07.13.21260484v1 on medRxiv