Integrated antigenic and nucleic acid detection in single virions and virion-infected host-derived extracellular vesicles

Abstract

The coronavirus disease of 2019 (COVID-19) led to the rapid development of novel assays to improve sensitivities for detecting the virion responsible for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite that, there have been over 767 million reported cases and over 6.9 million deaths worldwide. Therefore, tunable, sensitive, and high-throughput assays are warranted to control future outbreaks. Herein, we developed a tunablein situassay to selectively sort virions and infected host-derived extracellular vesicles (IHD-EVs) and simultaneously detect their antigens and nucleic acid cargo at a single-particle resolution. The Biochip Antigen and RNA Assay (BARATM) integrates positive immunoselection and infection dynamics to sort particles, immunofluorescence to detect antigens, and fluorescencein situhybridization to detect nucleic acids. BARATMenhanced sensitivities by detecting biomolecular signatures at the single-particle level, enabling the detection of virions in asymptomatic patients, and genetic mutations in single SARS-CoV-2 virions. Furthermore, BARATMrevealed the continued long-term expression of virion-RNA in the IHD-EVs of post-acute sequelae SARS-CoV-2 infection (PASC) patients. BARATMwas validated on saliva (30 healthy donors and 33 symptomatic and 20 asymptomatic patients) and nasopharyngeal swabs (19 healthy donors and 40 patients), revealing a highly accurate diagnosis by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA on single SARS-CoV-2 virions with sensitivities of 100 % and 95 %, respectively, and specificities of 100 % for both biofluids. Altogether, the single-particle detection of antigens and virion-RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks.Significance StatementViral outbreaks are imminent and rapidly translatable assays are necessary to keep contagion at bay. The Biochip Antigen and RNA Assay (BARATM) enables the simultaneous detection of antigens and nucleic acid cargo in virions and extracellular vesicles (EVs) from infected host cells at a single-particle resolution. Detecting single particles enhances sensitivities and specificities, enables long-term disease, and latent state monitoring, and provides a unique perspective into genetic mutations and virion lineages. Our work provides evidence for the utility of single-particle technologies as multifaceted diagnostic assays, providing more comprehensive information than traditional diagnostics.ClassificationBiological Sciences (Applied Biological Sciences)