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 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.

Saliva versus Upper Respiratory Swabs

“…Traditionally, upper respiratory swabs have been considered a better specimen for SARS-CoV-2 detection. 8 , 10 , 11 , 15 , 19 , 25 , 28 Meta-analyses have shown that the sensitivity of NP swab is approximately 98%, higher than the sensitivity of saliva. 38 , 40 In our study, however, the sensitivity of upper respiratory swab was 83.7% for AN in the University cohort and 75.8% for NP/OP in hospitalized patients, lower than the sensitivity of saliva in both populations.…”

“…The reported clinical sensitivity of saliva compared to NP swab ranges widely from 60.7% to 100%. 6 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 Some studies have reported that saliva has a significantly lower detection rate than swab specimens while others show similar detection rates between the two specimens. 8 , 10 , 11 , 15 , 19 , 25 , 28 , 31 Saliva specimens could generate a false negative result in asymptomatic individuals, when collected later in the disease course in symptomatic patients, or when using testing methods that lack an RNA extraction step.…”

“… 6 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 Some studies have reported that saliva has a significantly lower detection rate than swab specimens while others show similar detection rates between the two specimens. 8 , 10 , 11 , 15 , 19 , 25 , 28 , 31 Saliva specimens could generate a false negative result in asymptomatic individuals, when collected later in the disease course in symptomatic patients, or when using testing methods that lack an RNA extraction step. 20 , 31 From the technical side, saliva can be difficult to manipulate due to variable viscosity, which complicates automation of sample manipulation and PCR amplification.…”

“… 20 , 31 From the technical side, saliva can be difficult to manipulate due to variable viscosity, which complicates automation of sample manipulation and PCR amplification. 1 , 15 , 17 , 32 …”

Saliva as an alternative specimen to nasopharyngeal swabs for COVID-19 diagnosis: Review

“…Although it is not noted in the studies that used POPS examined for this review, this may be a particular issue with this specimen type over oral saliva as the potential mixing with sputum during throat clearing to obtain the specimen could increase its viscosity. This is especially an issue when trying to automate the diagnostic protocol to increase testing speed and capacity, where one study that developed a robotic RNA extraction step noted significant issues in using saliva specimens for this due to the inherent variable nature and viscosity of saliva specimens [76]. In many studies, and indeed in most saliva diagnostic tests that have been granted EUAs by the US FDA, stabilisers and preservatives are added to saliva specimens in order to minimise RNA degradation and thus maximise SARS-CoV-2 detection [16].…”