Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples

The COVID-19 pandemic has underscored the shortcomings in deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet growing testing needs, such often need samples collected through a swab, use RNA extraction kits, and expensive thermocyclers order successfully perform test. Isothermal amplification-based approaches also recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while reducing instrumentation complexity. In addition, there are limited reports saliva as source, some these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. this paper, we demonstrate an improved assay from using two-step RT-LAMP assay, where short 10 min RT step is performed only B3 backward inner primers before final reaction. We show that one-step demonstrates satisfactory results, optimized approach allows few molecules per performs significantly better than conventional all included step. control measurements RT-PCR, importantly, extraction-free RT-LAMP-based assays SARS-CoV-2 viral transport media clinical samples.