Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification

  1. Laura E. Lamb
  2. Sarah N. Bartolone
  3. Elijah Ward
  4. Michael B. Chancellor

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Abstract

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  1. Version published to 10.1371/journal.pone.0234682
  2. ScreenIT

    SciScore for 10.1101/2020.02.19.20025155: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Patient samples: All studies were approved by Beaumont Health’s Institutional Review Board (IRB approval # 2020-040 and 2016-044).
    Consent: All study participants gave their informed consent to participate.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    To improve specificity, areas of divergence of COVID-19 (GenBank MN908947) with the sequence-related coronavirus, Bat Severe Acute Respiratory Syndrome (SARS)-like coronavirus (GenBank KY417152.1), were identified using BLAST 2 (Basic Local Alignment Search Tool; NCBI).
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    RT-LAMP primers were designed using LAMP Designer 1.15 (Premier Biosoft), and blasted using Primer-BLAST (NCBI) against genomes of interest.
    LAMP
    suggested: (LAMP, RRID:SCR_001740)
    Primer-BLAST
    suggested: (Primer-BLAST, RRID:SCR_003095)
    No template control or COVID-19 was added to SYBRSelect Master Mix (ThermoFisher), and 10 µM forward and reverse primers with a total reaction volume of 20 µL.
    ThermoFisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)

    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: We detected the following sentences addressing limitations in the study:
    This study has several limitations. First, COVID-19 is Biosafety level 3 so our laboratory was unable to work directly with the virus or with infected samples. As such, all the experiments presented here used a nucleotide oligo of COVID-19 corresponding to the GenBank MN908947 sequence. Similarly, we were unable to directly test related coronaviruses and instead used nucleotide oligos from the same region of those viruses. However, these do represent a proof-of-feasibility for this assay, and the primers were further evaluated for specificity by BLASTing them to related coronavirus sequences. Although RT-LAMP reactions are highly specific, it is not a quantitative test. However other groups are working at improving the read outs of RT-LAMP assays including the use of smartphone-integrated sensors to make interpretation of the assay even more user-friendly.8 RT-LAMP reactions can have a higher rate of false positives compared to qRT-PCR; we did not experience this in any of our no template negative control reactions, negative control samples, or other samples containing other coronaviruses. We also took the precautions of having a lateral work flow for experiments and we included a Thermolabile Uracil-DNA Glycosylase (UDG) in all reactions to prevent possible carry-over contamination from previous reactions. This study was not powered to determine sensitivity in a clinical population. Lastly, this is a rapidly developing area of study and all the information presented at the t...

    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 10.1101/2020.02.19.20025155 on medRxiv