Impact of Thermal Pretreatment of Saliva on the RT-PCR Detection of SARS-CoV-2

The use of saliva directly as a specimen to detect viral RNA by RT-PCR has been tested for a long time as its advantages are relevant in terms of convenience and costs. However, as other body fluids, its proven inhibition effect on the amplification reaction can be troublesome and compromise its use in the detection of viral particles. The aim of the present work is to demonstrate that saliva pretreatment may influence the RT-PCR amplification of three gene targets of SARS-CoV-2 significantly. A pool of RNA from confirmed COVID-19 patients was used to test the influence of heat pretreatment of saliva samples at 95°C for 5, 10, 15 and 20 min on the amplification performance of ORF1ab, E, and N SARS-CoV-2 genes. Prolonged heating at 95°C significantly improves the Ct value shift, usually observed in the presence of saliva, increasing the limit of detection of viral genes ORF1ab, E, and N. When tested using a cohort of COVID-19 patients' saliva, the increased time of heat pretreatment resulted in a significant increase in the detection sensitivity.