Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

  1. Jean Y. H. Lee
  2. Nickala Best
  3. Julie McAuley
  4. Jessica L. Porter
  5. Torsten Seemann
  6. Mark B. Schultz
  7. Michelle Sait
  8. Nicole Orlando
  9. Karolina Mercoulia
  10. Susan A. Ballard
  11. Julian Druce
  12. Thomas Tran
  13. Mike G. Catton
  14. Melinda J. Pryor
  15. Huanhuan L. Cui
  16. Angela Luttick
  17. Sean McDonald
  18. Arran Greenhalgh
  19. Jason C. Kwong
  20. Norelle L. Sherry
  21. Maryza Graham
  22. Tuyet Hoang
  23. Marion Herisse
  24. Sacha J. Pidot
  25. Deborah A. Williamson
  26. Benjamin P. Howden
  27. Ian R. Monk
  28. Timothy P. Stinear

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Abstract

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.

Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.

Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.

Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min ( sd ±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID 50 ml −1 ), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.

Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.28.067363: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingInterlaboratory comparison of N1-STOP-LAMP: A panel of 20 blinded clinical samples (13 positive and 7 negative) with cycle threshold (Ct) values previously established by E-gene RT-qPCR were aliquoted and distributed to three different laboratories for independent testing by N1-STOP-LAMP assay (Table S2)
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and SARS-CoV-2 RNA extraction: Vero cells (within 30 passages from the original American Type Culture Collection [ATCC] stock) were maintained in Minimal Essential Media (MEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 10 μM HEPES, 2 mM glutamine and antibiotics.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    After this quality control step, homology searches were then conducted using NCBI BLAST+ blastn using Genepuller.pl (https://github.com/tseemann/bioinfo-scripts/blob/master/bin/gene-puller.pl) to find the region in each of the remaining 2738 genomes matching the 5’ region of the N1 sequence.
    NCBI BLAST+
    suggested: (Japan Bioinformatics, RRID:SCR_012250)
    The alignment was visualised with Mesquite (v3.61).
    Mesquite
    suggested: (Mesquite, RRID:SCR_017994)
    Statistical analysis: Data analysis was managed using GraphPad Prism (v8.4.1).
    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.

    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 10.1099/jmm.0.001238 on Access Microbiology
  3. ScreenIT

    SciScore for 10.1101/2020.04.28.067363: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.BlindingInterlaboratory comparison of N1-STOP-LAMP A panel of 20 blinded clinical samples (13 positive and 7 negative) with cycle threshold (Ct) values previously established by E-gene RT-qPCR were aliquoted and distributed to three different laboratories for independent testing by N1-STOP-LAMP assay (Table S2).Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Briefly, serial dilutions of the stock virus were added to washed monolayers of Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, CVCL_0059
    Software and Algorithms
    SentencesResources
    After this quality control step , homology searches were then conducted using NCBI BLAST+ blastn using Genepuller . pl ( https://github.com/tseemann/bioinfo-scripts/blob/master/bin/gene-puller.pl ) to find the region in each of the remaining 2738 genomes matching the 5’ region of the N1 sequence.
    NCBI BLAST+
    suggested: (Japan Bioinformatics, SCR_012250)
    The alignment was visualised with Mesquite ( v3.61) .
    Mesquite
    suggested: (Mesquite, SCR_017994)
    Statistical analysis Data analysis was managed using GraphPad Prism ( v8.4.1) .
    GraphPad Prism
    suggested: (GraphPad Prism, SCR_002798)
    To assess the exclusivity criterion , we used a nucleotide BLAST search of the N1 region against the NCBI Genbank nt database and observed no non-SARS-CoV-2 sequence matches above 80 % nucleotide identity , in-line with FDA cross-reactivity requirement .
    BLAST
    suggested: (BLASTX, SCR_001653)
    OptiGene Genie II and III , BioRad CFX and ThermoFisher Quantstudio 7 .
    ThermoFisher Quantstudio
    suggested: (Primer Express Software, SCR_017376)
    BioRxiv 2020 . doi: https://doi.org/10.1101/2020.04.13.039941. 35 .
    BioRxiv
    suggested: (bioRxiv, SCR_003933)
    , showing inhibitory impact of 5 μL of a neat sample matrix on LAMP and the effect of diluting the UTM in water .
    LAMP
    suggested: (LAMP, SCR_001740)

    Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.04.28.067363v1 on bioRxiv
  5. Version published to 10.1101/2020.04.28.067363v2 on bioRxiv