SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

Sahajpal, Nikhil S.; Mondal, Ashis K.; Ananth, Sudha; Njau, Allan; Ahluwalia, Pankaj; Newnam, Gary; Lozoya-Colinas, Adriana; Hud, Nicholas V.; Kota, Vamsi; Ross, Ted M.; Reid, Michelle D.; Fulzele, Sadanand; Chaubey, Alka; Hegde, Madhuri; Rojiani, Amyn M.; Kolhe, Ravindra

Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).