Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

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 the gold standard, nasopharyngeal swab specimens. 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.

Analysis of Four Different Transport and Preservation Medium Kits for Sars-Cov-2 Diagnosis From Nasopharyngeal Swab by Real-Time Pcr: Adapting to the Constantly Increasing Demand of Sampling Processing and Stock-Outs During the Pandemic

“…This agent has been widely used for sample collection and transport of various infectious agents [21][22][23]. Indeed, the group of Hamilton et al 2021 [24] reported this reagent improves the detection of SARS-CoV-2 RNA in saliva samples, by detecting the Spike protein at low Cq cycles compared to samples preserved without DNA/RNA Shield™ . Similarly, the group of Coryell et al 2021 [25] reported that DNA/RNA Shield™ gave the best results in terms of viral detection.…”

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus

“…We designed and implemented a saliva specimen collection pipeline that minimized exposure risk and maximized ease of use for participants. In parallel, we developed a high-throughput automated qPCR-based laboratory procedure for testing saliva for the presence of SARS-CoV-2 genetic material [2].…”

“…tube with the station worker. From here, the samples were brought back to the IGI Diagnostics Lab, where they underwent the procedures outlined in [2].…”

“…This outcome could be attributable to several factors, including test sensitivity; however, we speculate that it likely reflects a difference in demographics and associated exposure risk between our campus' study cohort and the broader population of the City of Berkeley. Overall, IGI FAST featured a high number (n = 761, 6.4%) of "specimen insufficient" results, making it a difficult test to further scale through pooled testing [2].…”

“…We found saliva to be a challenging matrix for nucleic acid extraction in general and observed that viscous samples were often more likely to fail, leading to a high specimen insufficient rate. These technical issues are further discussed in our companion manuscript [2], and made the possibility of pooling to increase surveillance capacity impractical, a strategy we were able to test by virtue of operating under an IRB rather than as a clinical requisition. Third, 20% of survey respondents indicated that producing a sufficient amount of saliva was either "somewhat difficult" or "extremely difficult."…”