Method versatility in RNA extraction-free PCR detection of SARS-CoV-2 in saliva samples

• Published in: Progress in biophysics and molecular biology Vol. 182; pp. 103 - 108
• Main Authors: Allicock, Orchid M., Yolda-Carr, Devyn, Earnest, Rebecca, Breban, Mallery I., Vega, Noel, Ott, Isabel M., Kalinich, Chaney, Alpert, Tara, Petrone, Mary E, Wyllie, Anne L.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.09.2023; Published by Elsevier Ltd
• Subjects: COVID-19; COVID-19 - diagnosis; COVID-19 Testing; Endopeptidase K; Heat inactivation; Humans; Polymerase Chain Reaction; RNA; Saliva; SARS-CoV-2; SARS-CoV-2 - genetics; Sensitivity and Specificity; Testing; COVID-19; Heat inactivation; SARS-CoV-2; Testing
• ISSN: 0079-6107; 1873-1732
• DOI: 10.1016/j.pbiomolbio.2023.06.004

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95 °C for 30 min, 95 °C for 5 min or 65 °C for 15 min) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.