1. Microbiology and Infectious Disease
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Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

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Cite this article as: eLife 2021;10:e65113 doi: 10.7554/eLife.65113

Abstract

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Qing Yang

    Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    Qing Yang, Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9053-3158
  2. Nicholas R Meyerson

    Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    Nicholas R Meyerson, Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein..
  3. Stephen K Clark

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  4. Camille L Paige

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    Camille L Paige, Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein..
  5. Will T Fattor

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  6. Alison R Gilchrist

    Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  7. Arturo Barbachano-Guerrero

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  8. Benjamin G Healy

    Department of Mechanical Engineering, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  9. Emma R Worden-Sapper

    Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  10. Sharon S Wu

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  11. Denise Muhlrad

    Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, United States
    Competing interests
    No competing interests declared.
  12. Carolyn J Decker

    Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, United States
    Competing interests
    No competing interests declared.
  13. Tassa K Saldi

    Integrated Physiology and Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  14. Erika Lasda

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  15. Patrick Gonzales

    Integrative Physiology, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  16. Morgan R Fink

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0567-3234
  17. Kimngan L Tat

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  18. Cole R Hager

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  19. Jack C Davis

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  20. Christopher D Ozeroff

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  21. Gloria R Brisson

    Wardenburg Health Center, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  22. Matthew B McQueen

    Integrated Physiology, University of Colorado, Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
  23. Leslie A Leinwand

    BioFrontiers Institute, University of Colorado Boulder, Boulder, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1470-4810
  24. Roy Parker

    Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8412-4152
  25. Sara L Sawyer

    Department of Molecular Cellular and Developmental Biology, University of Colorado Boulder, Boulder, United States
    For correspondence
    ssawyer@colorado.edu
    Competing interests
    Sara L Sawyer, Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein.Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6965-1085

Funding

Burroughs Wellcome Fund (PDEP)

  • Nicholas R Meyerson

Burroughs Wellcome Fund (PATH)

  • Sara L Sawyer

National Institutes of Health (DP1-DA-046108)

  • Sara L Sawyer

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: This study was approved by the University of Colorado Boulder Institutional Review Board. Saliva samples for assay development were collected under protocol 20-0068. Adult participants were consented verbally and donated up to 2mL of whole saliva for use as a reagent in optimization and limit of detection experiments. Data on human subjects is aggregated from University of Colorado Boulder operational COVID-19 surveillance testing activities. For this reason, the research herein did not fall under IRB purview.

Reviewing Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Publication history

  1. Received: November 23, 2020
  2. Accepted: March 26, 2021
  3. Accepted Manuscript published: March 29, 2021 (version 1)
  4. Version of Record published: April 20, 2021 (version 2)

Copyright

© 2021, Yang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Further reading

Further reading

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    Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

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