ABSTRACT
With the persistence of the COVID-19 pandemic caused primarily by constant viral mutations, rapid identification of different lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by large-scale screening at the point-of-care could be key to monitoring and assessing viral evolutions. Herein, we developed a Fluorescence Enhanced Microarray for Multiplex Analysis of Nucleic acids (FEMMAN) for detecting 8 SARS-CoV-2 variants simultaneously in ~ 3 hours without the need of RNA extraction, opening the possibility of point-of-care testing of multiple SARS-CoV-2 variants while reducing the cost significantly to ~ $ 7 per sample from ~ $100 by Next-Generation Sequencing (NGS). Combined with isothermal amplification, the multiplexed RNA assay achieved single-copy detection sensitivity and single nucleotide variant (SNV) distinction owing to the nanotechnology based plasmonic gold (pGOLD) near-infrared fluorescence enhancing platform. Probing 10 targets of three mutational hotspots in S gene, we differentiated 8 viral lineages (Wild type, Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron) of SARS-CoV-2, validated using nasopharyngeal swabs obtained from 127 individuals, achieving a 100% sensitivity and 100% specificity in SARS-CoV-2 detection, and a 91.1% concordance with NGS in variant identification. The scalable, multiplexed FEMMAN assay could shift the paradigm of COVID-19 diagnostic and surveillance from positive/negative assessments to simultaneous lineage identification in large-scale screening, greatly facilitating the global monitoring of SARS-CoV-2 variants.