Abstract
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).
Highlights
The emergence of COVID-19 in the city of Wuhan, China in December 2019 has rapidly evolved into a pandemic
Devices collected in Zymo, Spectrum, and Omni devices, the amplifiFor the saliva samples
Omni vialsN(which were devoid any was media), whereas noinamplifisamples collected in Omni vials, whereas noThus, amplification was observed in saliva samples in Spectrum or Zymo devices
Summary
The emergence of COVID-19 in the city of Wuhan, China in December 2019 has rapidly evolved into a pandemic. The high transmission rate, along with the high percentage of asymptomatic infected individuals, have been identified as the major reason for the spread of the disease. Under these circumstances, diagnostic testing for COVID-19 remains the most rational approach for containing the virus, and is of unprecedented importance, because if infected individuals are detected early in the course of their infection, globally implemented strategies such as quarantine and contact tracing can be more effective [1,2]. The emergence of a second upsurge in infections in countries previously showing a decline in cases highlights the need for a rapid, sensitive, cost-effective, and mass population testing methodology that can be implemented on a global scale [4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
