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Multiplex Isothermal Amplification Coupled with Nanopore Sequencing for Rapid Detection and Mutation Surveillance of SARS-CoV-2

Molecular testing and surveillance of the spread and mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critical public health measures to combat the pandemic. There is an urgent need for methods that can rapidly detect and sequence SARS-CoV-2 simultaneously. Here we describe a method for multiplex isothermal amplification of the SARS-CoV-2 genome in 20 minutes. Based on this, we developed NIRVANA (Nanopore sequencing of Isothermal Rapid Viral Amplification for Near real-time Analysis) to detect viral sequences and monitor mutations in multiple regions of SARS-CoV-2 genome for up to 96 patients at a time. NIRVANA uses a newly developed algorithm for on-the-fly data analysis during Nanopore sequencing. The whole workflow can be completed in as short as 3.5 hours, and all reactions can be done in a simple heating block. NIRVANA provides a rapid field-deployable solution of SARS-CoV-2 detection and surveillance of pandemic strains.