1. Microbiology and Infectious Disease
Download icon

Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

  1. Qing Yang
  2. Nicholas R Meyerson
  3. Stephen K Clark
  4. Camille L Paige
  5. Will T Fattor
  6. Alison R Gilchrist
  7. Arturo Barbachano-Guerrero
  8. Benjamin G Healy
  9. Emma R Worden-Sapper
  10. Sharon S Wu
  11. Denise Muhlrad
  12. Carolyn J Decker
  13. Tassa K Saldi
  14. Erika Lasda
  15. Patrick Gonzales
  16. Morgan R Fink
  17. Kimngan L Tat
  18. Cole R Hager
  19. Jack C Davis
  20. Christopher D Ozeroff
  21. Gloria R Brisson
  22. Matthew B McQueen
  23. Leslie A Leinwand
  24. Roy Parker
  25. Sara L Sawyer  Is a corresponding author
  1. University of Colorado Boulder, United States
  2. Howard Hughes Medical Institute, University of Colorado, United States
  3. University of Colorado, Boulder, United States
Research Article
  • Cited 0
  • Views 394
  • Annotations
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.

Metrics

  • 394
    Page views
  • 95
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organism

    1. Part of

    COVID-19: A Collection of Articles

    Edited by Diane M Harper et al.
  2. Further reading

Further reading

    1. Epidemiology and Global Health
    2. Medicine
    3. Microbiology and Infectious Disease

    COVID-19: A Collection of Articles

    Edited by Diane M Harper et al.
    Collection

    eLife has published the following articles on SARS-CoV-2 and COVID-19.

    1. Microbiology and Infectious Disease

    Functional insights from a surface antigen mRNA-bound proteome

    Larissa Melo do Nascimento et al.
    Research Article Updated

    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.

    1. Epidemiology and Global Health
    2. Microbiology and Infectious Disease

    Heterogeneity in transmissibility and shedding SARS-CoV-2 via droplets and aerosols

    Paul Z Chen et al.
    Research Article

    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.