Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and co-infections of multiple respiratory viruses by Rapid field-deployable sequencing

Strategies for monitoring the COVID-19 infection are crucial for combating the pandemic. Here we describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2 and co-infecting respiratory viruses, and monitor mutations for up to 96 samples in real time. The method, termed NIRVANA for <underline>N</underline>anopore sequencing of <underline>I</underline>sothermal <underline>R</underline>apid <underline>V</underline>iral <underline>A</underline>mplification for <underline>N</underline>ear real-time <underline>A</underline>nalysis, showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples. It also simultaneously detected other viral pathogens (e.g. influenza A) in clinical and municipal wastewater samples. It provides a rapid field-deployable solution of COVID-19 and cos-infection detection and surveillance of the evolution of pandemic strains.