A Robust, Safe and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Environmental Samples

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. MAVRICS is scalable and thus could become an enabling technology for widespread community testing and wastewater monitoring in the current and future pandemics.

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.