Development and performance evaluation of a low-cost in-house rRT-PCR assay in Ecuador for the detection of SARS-CoV-2

  1. Marco A. Salinas
  2. Diana F. Aguirre
  3. David I. De la Torre
  4. Jorge Pérez-Galarza
  5. Ronny J. Pibaque
  6. Paul A. Beltran
  7. Tatiana J. Veloz
  8. Lucy Y. Baldeón

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Abstract

Antecedents

Ecuador has had the greatest fatality rate from Coronavirus (COVID-19) in South America during the SARS-CoV-2 pandemic. To control the pandemic, it is necessary to test as much population as possible to prevent the spread of the SARS-CoV-2 infection. For the Ecuadorian population, accessing a PCR test is challenging, since commercial screening kits tend to be expensive. Objective: the objective of this study was to develop an in-house duplex rRT-PCR protocol for the detection of SARS-CoV-2 that contributes to the screening while keeping quality and low testing costs. Results: An in-house duplex rRT-PCR protocol based on the viral envelope (E) gene target of SARS-CoV-2 and a human ribonuclease P gene (RP) as an internal control is reported. The protocol was optimized to obtain primers E with an efficiency of up to 94.45% and detection of 100% of SARS-CoV-2 up to 15 copies per uL. The clinical performance was determined by a sensibility of 93.8% and specificity of 98.3%. Conclusion: we developed, standardized, and validated a low-cost, sensitive in-house duplex rRT-PCR assay that may be utilized in low-income countries.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.03.21260966: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    SPSS software version 23 was used for the analysis (IBM).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The limitation of the in-house duplex test is that it can only identify one viral gene (E) and one internal control gene (Rp). However, the kappa index (0.92) shows that our protocol is comparable to commercial kits (LightMix E / RdRp) that screen two or more viral genes. Furthermore, because the E gene is unique to all Sarbecoviruses and because SARS-CoV-2 is the only member of the family now circulating in humans, the WHO has recommended that the E gene be prioritized as a target. In this approach, a single viral genetic target suffices for case confirmation in the lab.

    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 10.1101/2021.08.03.21260966v1 on medRxiv