Clinical Assessment and Validation of a Rapid and Sensitive SARS-CoV-2 Test Using Reverse Transcription Loop-Mediated Isothermal Amplification Without the Need for RNA Extraction

  1. Melis N Anahtar
  2. Graham E G McGrath
  3. Brian A Rabe
  4. Nathan A Tanner
  5. Benjamin A White
  6. Jochen K M Lennerz
  7. John A Branda
  8. Constance L Cepko
  9. Eric S Rosenberg

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Abstract

Background

Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive coronavirus disease 2019 (COVID-19) testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect severe acute resrpiratory syndrome coronavirus 2 (SARS-CoV-2) in 40 minutes from sample collection to result.

Methods

We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica particle–based concentration method. Amplification was performed with 2 SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted.

Results

Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3 000 000 copies/mL), with sensitivities of 50% (95% CI, 28%–72%) and 59% (95% CI, 43%–73%) in samples collected in universal transport medium and saline, respectively, compared with quantitative polymerase chain reaction (qPCR). Adding an upfront RNase inactivation step markedly improved the limit of detection to at least 25 000 copies/mL, with 87.5% (95% CI, 72%–95%) sensitivity and 100% specificity (95% CI, 87%–100%). Using both inactivation and purification increased the assay sensitivity by 10-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics.

Conclusions

By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity, especially in resource-limited settings.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.12.20095638: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was approved by the Partners Human Research Committee at the Massachusetts General Hospital.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    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:
    There are several limitations of this assay. The assay is qualitative and does not provide a semi-quantitative cycle threshold number. Additionally, the visual interpretation affords substantial flexibility, but it can also be prone to user errors. Objective color measurements can be performed by measuring the absorbance at 432 and 560 nm, or potentially with a smartphone application. It is critical to use the positive and negative controls as interpretative aids to avoid misinterpreting an orange intermediate color change as positive. The N gene primer set is more likely to give subtle background color changes and additional primer sets will be tested in the future. Like any nucleic acid amplification test, systems must be in place to avoid environmental and sample contamination with post-amplification products. One such precaution is to refrain from opening the reaction vessel after amplification; reactions should be discarded or transferred to a sealed container for later reference, since the color change remains stable for days to weeks. While the assay generally requires very little infrastructure, the operator must abide by laboratory biosafety guidelines and the procedure is most safely performed within a biosafety class II cabinet, although we recognize in many setting this may not be possible [13]. Additionally, the RT-LAMP master mix currently requires storage at -20 °C, which is not ideal for low-resource or remote settings, but this may be ameliorated by lyophiliz...

    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 10.1093/ofid/ofaa631
  3. Version published to 10.1101/2020.05.12.20095638 on medRxiv