Abstract
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.
Highlights
Disease screening is one of the most basic and powerful tools in the public health arsenal
We designed our saliva stabilization solution to include a chelating agent (1 mM ethylenediaminetetraacetic acid [EDTA] final concentration) and Proteinase K to inhibit RNases, both of which help preserve virion RNA and to increase sensitivity. (Note that Proteinase K will inhibit the Reverse transcription (RT)-LAMP reaction if it does not go through a heat inactivation step prior to that reaction.) the saliva stabilization solution contains TCEP, which aids in RNA stabilization by breaking disulfide bonds present in RNases and helps to reduce saliva viscosity
(2) We optimized a saliva stabilization solution that allows for analysis of a broad range of naturally acidic saliva samples while maintaining compatibility with a colorimetric RT-LAMP assay
Summary
Disease screening is one of the most basic and powerful tools in the public health arsenal. Screening tests identify unknown illness in apparently healthy or asymptomatic individuals. In the case of dangerous pathogens, screening tests serve to direct potential carriers of the pathogen into the healthcare system for confirmatory testing and to alert them that they could possibly infect others while they await confirmatory results. If dangerous pathogens are spreading at high rates, individuals will need to be screened frequently. As such, screening tests should operate with minimal requirements for laboratory equipment and labor, such that they are community deployable and do not burden the critical pipelines for diagnostics. In the current SARS-CoV-2 pandemic, body temperature is a ubiquitous screening test being used on apparently healthy people around the world.
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