An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

  1. Jayeshkumar Narsibhai Davda
  2. Keith Frank
  3. Sivakumar Prakash
  4. Gunjan Purohit
  5. Devi Prasad Vijayashankar
  6. Dhiviya Vedagiri
  7. Karthik Bharadwaj Tallapaka
  8. Krishnan Harinivas Harshan
  9. Archana Bharadwaj Siva
  10. Rakesh Kumar Mishra
  11. Jyotsna Dhawan
  12. Imran Siddiqi

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Abstract

With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

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    SciScore for 10.1101/2020.06.08.139477: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Sample Collection: NP swab samples were collected from patients suspected of being infected with SARS-CoV-2 and their contacts at different hospitals in the Hyderabad vicinity based on Indian Council of Medical Research (ICMR) guidelines (http://www.nie.gov.in/images/leftcontent_attach/COVID-SARI_Sample_collection_SOP_255.pdf) and in accordance with Institutional Ethics Committee guidelines.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    DNA was quantified by a Qubit dsDNA HS kit (Thermofisher Cat # Q32851).
    Thermofisher Cat
    suggested: None
    Images were edited using Adobe Photoshop and included removal of intervening lanes in the gel between samples and DNA marker indicated by a vertical line.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)

    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.
    • 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

    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 10.1101/2020.06.08.139477v1 on bioRxiv