Control, containment, and mitigation of the coronavirus disease 2019 (COVID-19) pandemic require accessible tools and assays for the timely detection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from patient samples. Because saliva and gargle samples are non-invasive, easy to collect, and abundant, they emerge as favorable alternative sampling options to the conventional nasopharyngeal swab (NPS). However, saliva and gargle are more complicated, and viral particles are often present at lower levels. The objective of this research is to develop a rapid, efficient, and simple method for extracting, preserving, and concentrating SARS-CoV-2 viral RNA from saliva and gargle samples.<br/>The SARS-CoV-2 viral RNA contains a 3' polyadenylated tail similar to messenger RNA. By taking advantage of this unique 33-residue poly-A tail, we developed a rapid, efficient, and simple viral RNA extraction method using poly-T conjugated magnetic beads. We also developed an extraction buffer enabling efficient lysis of viral particles and preservation of the released viral RNA in saliva and gargle samples. The released viral RNA remains stable at ambient temperature for more than 2-weeks in our buffer. The viral RNA extraction can be completed within 15 minutes under room temperature.<br/>Using this extraction method, we achieved near 100% recovery of SARS-CoV-2 viral RNA and were able to detect as few as 10 viral particles in 100 µL of saliva or gargle samples using reverse transcription quantitative polymerase chain reaction (RT-qPCR). We successfully coupled this method to a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for point-of-care detection of SARS-CoV-2 in saliva and gargle.

Extraction, preservation, and concentration of SARS-CoV-2 viral RNA from saliva and gargle

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ACS Fall 2022

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