A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point-of-care

  1. Diem Hong Tran
  2. Hoang Quoc Cuong
  3. Hau Thi Tran
  4. Uyen Phuong Le
  5. Hoang Dang Khoa Do
  6. Le Minh Bui
  7. Nguyen Duc Hai
  8. Hoang Thuy Linh
  9. Nguyen Thi Thanh Thao
  10. Nguyen Hoang Anh
  11. Nguyen Trung Hieu
  12. Cao Minh Thang
  13. Van Van Vu
  14. Huong Thi Thu Phung

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Abstract

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.

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  1. Version published to 10.1101/2020.05.24.113423v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.05.24.113423: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All participants were confirmed negative with SARS-CoV-2 by RT-PCR provided their informed consent to participate in the trial.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Primer specificity analysis: The reference genomes of SARS-CoV-2 and related species were downloaded from NCBI (https://www.ncbi.nlm.nih.gov).
    https://www.ncbi.nlm.nih.gov
    suggested: (GENSAT at NCBI - Gene Expression Nervous System Atlas, RRID:SCR_003923)
    The primer sequences were aligned to genomes of different coronaviruses to calculate the number of mismatches using Geneious Prime 2020.0.3 (https://www.geneious.com).
    https://www.geneious.com
    suggested: (Geneious, RRID:SCR_010519)
    The software FastPCR available at http://primerdigital.com/fastpcr.html was used for in silico PCR analysis.
    http://primerdigital.com/fastpcr.html
    suggested: (FastPCR, RRID:SCR_003155)

    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

    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.

  3. Version published to 10.1101/2020.05.24.113423v1 on bioRxiv