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)

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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    Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites’ variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2 (cyclin F-box protein 2), an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle, and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.

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    Background: Which virological factors mediate overdispersion in the transmissibility of emerging viruses remains a longstanding question in infectious disease epidemiology.

    Methods: Here, we use systematic review to develop a comprehensive dataset of respiratory viral loads (rVLs) of SARS-CoV-2, SARS-CoV-1 and influenza A(H1N1)pdm09. We then comparatively meta-analyze the data and model individual infectiousness by shedding viable virus via respiratory droplets and aerosols.

    Results: The analyses indicate heterogeneity in rVL as an intrinsic virological factor facilitating greater overdispersion for SARS-CoV-2 in the COVID-19 pandemic than A(H1N1)pdm09 in the 2009 influenza pandemic. For COVID-19, case heterogeneity remains broad throughout the infectious period, including for pediatric and asymptomatic infections. Hence, many COVID-19 cases inherently present minimal transmission risk, whereas highly infectious individuals shed tens to thousands of SARS-CoV-2 virions/min via droplets and aerosols while breathing, talking and singing. Coughing increases the contagiousness, especially in close contact, of symptomatic cases relative to asymptomatic ones. Infectiousness tends to be elevated between 1-5 days post-symptom onset.

    Conclusions: Intrinsic case variation in rVL facilitates overdispersion in the transmissibility of emerging respiratory viruses. Our findings present considerations for disease control in the COVID-19 pandemic as well as future outbreaks of novel viruses.

    Funding: Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program, NSERC Senior Industrial Research Chair program and the Toronto COVID-19 Action Fund.