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/microL was achieved within ~20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.