"Standardizing Free Extraction Methods And Comparing Efficiency With Automated Extraction In SARS-Cov-2 Detection Assays

Abstract

Background: The COVID-19 pandemic, caused by SARS-CoV-2, has necessitated rapid and reliable diagnostic methods. Real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for detection. However, RNA extraction, a critical step in this method, is time-consuming, costly, and heavily reliant on reagents and equipment often unavailable in low-resource settings.Objective: This study evaluates an extraction-free RT-qPCR method using a simplified protocol involving Proteinase K (PK) treatment and heat inactivation (HID) to enhance SARS-CoV-2 detection efficiency.Methods: A total of 294 nasopharyngeal swab samples were analyzed using the "PBS + PK + HID" method. Samples were heat-inactivated at 95°C for 10 minutes followed by 65°C for 10 minutes. CT (Cycle Threshold) values obtained were categorized into three groups: 18–20, 20–30, and 30–35, and analyzed for viral load distribution and amplification efficiency of SARS-CoV-2 target genes (E, ORF1ab, and N) using the COVIDsure Pro Multiplex RT-PCR kit. Results: Lower CT values (18–20) were associated with higher viral loads, indicating superior detection sensitivity under the simplified heating protocol. The heat treatment notably improved RNA accessibility by lysing virions and degrading inhibitory proteins. Comparative CT analysis showed consistent amplification of target genes, with minimal loss of sensitivity compared to standard extraction-based protocols.Conclusion: The PK + HID extraction-free RT-qPCR method is a viable, cost-effective alternative for SARS-CoV-2 detection, particularly in resource-limited settings. It reduces dependency on extraction reagents, minimizes processing time, and maintains diagnostic accuracy, thereby supporting high-throughput testing during pandemic surges.