On-Site Viral Inactivation and RNA Preservation of Gargle and Saliva Samples Combined with Direct Analysis of SARS-CoV-2 RNA on Magnetic Beads

Abstract

Samples of nasopharyngeal swabs (NPS) are commonly used for the detection of SARS-CoV-2 and diagnosis of COVID-19. As an alternative, self-collection of saliva and gargle samples minimizes transmission to healthcare workers and relieves the pressure of resources and healthcare personnel during the pandemic. This study aimed to develop an enhanced method enabling simultaneous viral inactivation and RNA preservation during on-site self-collection of saliva and gargle samples. Our method involves the addition of saliva or gargle samples to a newly formulated viral inactivation and RNA preservation (VIP) buffer, concentration of the viral RNA on magnetic beads, and detection of SARS-CoV-2 using reverse transcription quantitative polymerase chain reaction directly from the magnetic beads. This method has a limit of detection of 25 RNA copies per 200 μL of gargle or saliva sample and 9–111 times higher sensitivity than the viral RNA preparation kit recommended by the United States Centers for Disease Control and Prevention. The integrated method was successfully used to analyze more than 200 gargle and saliva samples, including the detection of SARS-CoV-2 in 123 gargle and saliva samples collected daily from two NPS-confirmed positive SARS-CoV-2 patients throughout the course of their infection and recovery. The VIP buffer is stable at room temperature for at least 6 months. SARS-CoV-2 RNA (65 copies/200 μL sample) is stable in the VIP buffer at room temperature for at least 3 weeks. The on-site inactivation of SARS-CoV-2 and preservation of the viral RNA enables self-collection of samples, reduces risks associated with SARS-CoV-2 transmission, and maintains the stability of the target analyte.

An efficient method to enhance recovery and detection of SARS-CoV-2 RNA in wastewater

“…Secondly, we used our in-house developed VIP-Mag method, which concentrated pure RNA with high integrity. Our VIP buffer, which includes reagents such as 2-Mercaptoethanol, guanidinium isothiocyanate, Triton X-100, proteinase K, and glycogen, effectively lysed the SARS-CoV-2 viral particles and denatured RNases ( Liu et al, 2022 , Ramón-Nuńez et al, 2017 ). The extracted RNA was then captured on magnetic beads, and any remaining RT-qPCR inhibitors were removed through repeated washing of the beads (Appendix A Fig.…”

“…The EMs containing the captured SARS-CoV-2 particles were directly used to extract viral RNA. The viral inactivation and preservation (VIP) buffer, developed in-house ( Liu et al, 2022 ), was used for RNA extraction. The EMs were vortexed thoroughly in 600 µL of the VIP buffer, placed on a heating block for 10 min at 55°C, centrifuged for 2 min at 13,000 ×g , and the final supernatant was transferred to a new tube.…”

“…The supernatant containing the extracted SARS-CoV-2 RNA is then incubated with the in-house developed magnetic beads mixture. The details on the preparation and optimization of the magnetic beads buffer can be found in our previous publication ( Liu et al, 2022 ). 400 μL of the magnetic beads suspension and 200 μL of 100% ethanol (RNA grade) were added into the extracted RNA supernatant, vortexed, and incubated on a shaker at room temperature for 10 min.…”

“…The TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific) and CDC N1 primer-probes from the 2019-nCoV RUO kit (IDT) were used according to the manufacturers’ instructions. The RT-qPCR assay for the N1 segment of the SARS-CoV-2 RNA was optimized as described previously ( Liu et al, 2022 ). RT-qPCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) using the QuantStudio Design and Analysis Software v1.5.1.…”

Efficacy Validation of SARS-CoV-2-Inactivation and Viral Genome Stability in Saliva by a Guanidine Hydrochloride and Surfactant-Based Virus Lysis/Transport Buffer

“…Although the presence of high organic substance content appears to slightly reduce the virucidal activity of Prep Buffer A, these results indicate that Prep Buffer A enhances biosafety during the collection, packaging, transportation, and handling of saliva samples for diagnosing SARS-CoV-2 infection by RT-PCR. In several guanidine-based lysis buffers, GuHCl or GTC is routinely used at a working concentration of 4–6 M (before mixing with virus-containing samples) [ 28 , 36 , 41 , 42 ]. The final concentration for virus inactivation depends on prior dilution with other reagents, if any, as well as the virus-containing sample-to-buffer ratio.…”