multiplex RPA based Nanopore sequencing for real-time detection of virus

The COVID19 pandemic is one of the most serious challenges to public health and global economy in modern history. Identification of new cases and surveillance of the spread and mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical public health measures to combat the pandemic. Here we describe a method for multiplexed isothermal amplification of the SARS-COV-2 genome in less than 20 minutes. Based on this, we developed NIRVANA (Nanopore sequencing of Isothermal Rapid Viral Amplification for Near real-time Analysis) to detect the presence of viral sequences and monitor mutations in multiple regions of the genome in up to 96 patients at a time. NIRVANA uses a newly developed algorithm termed RTNano for on-the-fly data analysis during Nanopore sequencing. The whole workflow can be completed in around 3.5 hours, and all reactions can be done in a simple heating block. NIRVANA provides a rapid field-deployable solution of detection of any viral pathogens, including SARS-COV-2, and surveillance of pandemic strains.