Extraction-Free Rapid Cycle Quantitative RT-PCR and Extreme RT-PCR for SARS-CoV-2 Virus Detection

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. This study explored the kinetic limitations of extraction-free rapid cycle quantitative real-time RT-PCR for SARS-CoV-2 virus detection using the commercially available capillary-based LightCycler. After optimizing for time and reaction conditions, a protocol for sensitive and specific quantitative RT-PCR of SARS-CoV-2 RNA from nasopharyngeal swabs in <20 minutes was developed, with minimal hands-on time requirements. This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n = 40/41), and negative patient samples was 100% (40/40). The study further demonstrates that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR, and product verification by melting can be completed in <3 minutes. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.