Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places where good vaccination coverage has been achieved. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing implementation in resource-limited settings. It is therefore important to develop testing protocols that facilitate implementation and are inexpensive, fast, and sufficiently sensitive. In this work, we optimized the composition of a buffer (PKTP) containing a protease, a detergent, and an RNase inhibitor, that is compatible with the RT-qPCR chemistry, allowing for direct testing of SARS-CoV-2 from saliva in an RNA extraction-independent manner. This buffer is compatible with heat-inactivation reducing the biohazard risk of handling the samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (82.2% for the N1 and 84.4% for the N2 target, respectively) and correlations (R=0.77, p<0.001 for N1, and R=0.78, p<0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to reduce the number of PCR reactions per patient and further reduce overall costs while maintaining diagnostic standards in favor of massive testing.