Ultrasensitive and specific detection of rabies virus using microfluidic trapping of isothermal nucleic acid amplicons

Due to 100% mortality rate of rabies, the number of cases of this lethal and often unreported zoonotic disease has drawn attention for prompt diagnosis. The current detection methods, namely direct fluorescence antibody (DFA) assay which is the gold standard and Polymerase Chain Reaction (PCR) for rabies diagnosis, are limited by their sensitivity, application in remote settings and especially the former being restricted by sample type used for detection. Hence, a specific, sensitive and simplified detection platform for rabies diagnosis would be essentially beneficial for precise and timely detection of the virus. Here, we present a robust microfluidic affinity chromatography device with systematic investigations on technical parameters bordering both the fluidics (flow rate used for bead deposition) and bead-amplicon mixing modes ( ex-situ and in-situ ). The amplified products from rolling circle amplification (RCA) assay were specifically captured on affinity tagged beads packed in the device microchannel. A working calibration curve was then established using synthetic targets for estimating the dynamic range of the bioassay, and an LoD of ∼ 1 (0.87) fM was achieved. For platform validation, a total of 15 samples comprising both post-mortem as well as ante-mortem samples, with canine genomic DNA and rabies vaccine alongside, were analysed using a cocktail of two padlock probes (PLPs) for an enhanced detection of RCA products. The sensitive detection of ante-mortem samples opens the door for early diagnosis of rabies spanning over a range of sample sources, thereby finding use in both screening and molecular diagnostics as relevant to ‘One Health’ initiatives.