Abstract
Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.
Highlights
The COVID-19 pandemic has motivated extensive scientific efforts to develop vaccines, therapeutics and diagnostics
To test the capacity of OM-505 collection tubes to completely inhibit viral infectivity, tissue culture supernatant containing SARS-CoV-2 was combined with OMNIgene sample stabilization fluid at a 1:1 ratio, incubated either at room temperature (22 ̊C) for 60 minutes or at 65 ̊C for 30 minutes, and tested whether infectious virus remained following treatment using a cytopathic effect (CPE) assay
Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples either DNA/RNA Shield or phosphate buffered saline (PBS), we found that a 3:1 ratio of DNA/ RNA Shield:saliva sample resulted in optimal detection of spiked-in MS2 following extraction (S2 Fig)
Summary
The COVID-19 pandemic has motivated extensive scientific efforts to develop vaccines, therapeutics and diagnostics. Saliva self-collection enabled increased population sampling capacity but, in turn, increased the demand for a rapid yet sensitive PCRbased testing protocol To address these challenges, we developed an automated procedure for extraction and detection of SARS-CoV-2 RNA in saliva as part of the Innovative Genomics Institute/UC Berkeley Free Asymptomatic Saliva Testing (IGI FAST) research study [6, 7]. We describe a robust, high-throughput saliva testing strategy that includes viral inactivation requirements for handling potentially infectious samples, a fully automated RNA-extraction-based detection method, and attempts at saliva sample pooling This procedure, which is suitable for at-home collection, enabled surveillance testing of 11,971 samples from asymptomatic individuals and identification of SARS-Cov-2-positive specimens even when the prevalence of viral infection was
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