A simple Magnetic-aided microfluidic screening approach for rabies virus via rolling circle amplification

Abstract

Rabies, being a fatal neurologic disease, is one of the neglected tropical diseases. Timely post-exposure prophylaxis is the key to healthcare, demanding an early diagnosis towards the much-envisaged ‘One Health’ measures. Despite being a disease with almost 100 % mortality cases, no significant progress has been made towards its early and accurate diagnosis. Herein, we report a simple microfluidic device based on magnetic trapping, without any complex design, in order to specifically screen rabies virus from biofluids. For this, we exploit Rolling Circle Amplification (RCA), a highly recommended isothermal nucleic acid amplification test (NAAT), with rapid and robust point-of-care diagnosis capabilities. This specific molecular assay was used for sensitive detection down to 2.69 fM and 14.5 fM for N- and L-gene targets, respectively. COMSOL simulations have accelerated experimental optimization time, resulting in effective capture and detection of magnetically-coupled amplicons. Fluorescently-tagged RCA products mixed with magnetic microparticles (M−RCPs) showed a clearly distinguishable signal between rabies vaccine strain and blood samples of healthy dogs. This simple technique presents a scalable proof-of-concept prototype for a highly specific ‘yes-or-no’ detection method for rabies virus, using a minimal sample volume and extendable for sensitive-cum-specific screening of other pathogens using tunable molecular probes.