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

Elife. 2021 Mar 29;10:e65113. doi: 10.7554/eLife.65113.


Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. 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.

Keywords: SARS-CoV-2; infectious disease; microbiology; testing; university; virus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 / metabolism
  • COVID-19 / virology*
  • COVID-19 Testing
  • Carrier State / diagnosis*
  • Carrier State / virology*
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques / methods
  • RNA, Viral / genetics
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / isolation & purification*
  • Saliva / virology*
  • Sensitivity and Specificity
  • Specimen Handling / methods


  • RNA, Viral

Supplementary concepts

  • LAMP assay