Flipse, J. | Search bioRxiv

bioRxiv2024

Direct and ultrasensitive bioluminescent detection of intact respiratory viruses

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of intact virus. Instead, specific detection of intact respiratory virus particles may better assess the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of free viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1 - 0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

bioengineering↗

bioRxiv2022

Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation

Nucleic acid detection methods based on isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or post-amplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. Whereas LUNAS itself features a detection limit of [~]1 pM for dsDNA targets, the LUNAS platform is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a single-pot assay. We designed a one-pot RT-RPA-LUNAS assay for detecting SARS-CoV-2 RNA without the need for RNA isolation and demonstrated the diagnostic performance for COVID-19 patient nasopharyngeal swab samples using a digital camera to record the ratiometric signal. Detection of SARS-CoV-2 from samples with viral RNA loads of [~]200 cp/L was achieved within [~]20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.

bioengineering↗