Optimization of Extraction-Free Protocols for SARS-CoV-2 Detection using a Commercial rRT-PCR Assay

Abstract The global combat against coronavirus disease 2019 (COVID-19) has primarily focused on developing innovative nucleic acid-based molecular diagnostic techniques and streamlining associated protocols. In this study, we investigated extraction-free approaches to simplify the process and enhance efficiency. We validated the effectiveness of extraction-free protocols incorporating heat treatment and sample dilution, along with Proteinase K and RNase inhibitors, to improve nucleic acid detection. The combination of sample dilution, heat treatment, and addition of Proteinase K and RNase inhibitors showed the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. The combination of sample dilution and heat treatment exhibited a sensitivity of 79.63%, which was a significant improvement of 38% compared to the use of heat treatment alone. The incorporation of proteinase K and RNase inhibitors in conjunction with sample dilution and heat treatment contributed to only a marginal improvement and did not yield statistically significant differences. Our study revealed that sample dilution significantly affected SARS-CoV-2 detection, and sample conditions played a crucial role in the efficiency of extraction-free methods. These findings will contribute to streamlining diagnostic testing, making it more accessible and cost-effective during the COVID-19 pandemics.