A FokI-driven signal amplification platform for the simultaneous detection of multiple viral RNA pathogens

Abstract

Accurate signal quantification is a critical parameter for precise pathogen diagnosis. Furthermore, the development of novel diagnostic methods amenable for portable or scalable purposes may facilitate the real-time surveillance of emerging pathogenic threats. Here, we extrapolated the use of a FokI-driven signal amplification approach, powered by the nickase activity of the FokI restriction endonuclease, to enhance the signal detection of multiple respiratory viral pathogens relevant to human health. This approach utilises a set of dumbbell-like fluorescent oligonucleotides against three families of human single-stranded RNA respiratory viruses (Coronaviridae, Paramyxoviridae, and Pneumoviridae), including human betacoronaviruses (OC43/HKU1), human betacoronavirus SARS-CoV-2, human parainfluenza viruses (HPIV1/HPIV3/HPIV2/HPIV4), and human metapneumovirus (HMPV)/respiratory syncytial virus (HRSV). FokI-assisted digestion of these dumbbell-like fluorescent probes in the presence of their respective viral targets enhanced their detectable signal in a highly specific manner. In addition, this technique exhibited high multiplex potential to detect multiple viral targets in a single assay. A molecular coupling between the FokI-assisted reaction and the isothermal rolling circle amplification technique significantly improved the limit of detection in samples from infected patients, ensuring adequate specificity and sensitivity. These results highlight the diagnostic potential of this methodology, representing a cost-effective alternative for the identification of respiratory viral pathogens of interest to the public healthcare system.