Rapid and Visual Detection of Muscovy Duck Reovirus Using a Reverse Transcription Recombinase-Aided Amplification Assay in a Lyophilized Format for Point-of-Care Applications.

Muscovy duck reovirus (MDRV) causes substantial economic losses in the waterfowl industry, necessitating rapid and field-deployable diagnostic tools. In this study, we developed, for the first time, a lyophilized reagent-based reverse transcription recombinase-aided amplification (RT-RAA) assay targeting the σC gene of MDRV for point-of-care testing (POCT). The assay was optimized to operate at 39°C with a probe concentration of 100 nM. It achieved a detection limit of 1.03 × 101 copies/μL within 20 min, which is comparable to RT-quantitative polymerase chain reaction (qPCR) and superior to existing methods. It exhibited high specificity for MDRV with no cross-reactivity against other common waterfowl pathogens. Particularly, the assay was successfully lyophilized into a ready-to-use format, representing the first reported RT-RAA-based method for MDRV detection in such a format. The lyophilized reagents retained the key analytical performance characteristics without cold-chain dependance. When evaluated on 42 clinical samples, the lyophilized RT-RAA demonstrated 95.8% diagnostic sensitivity and 100% specificity compared to RT-qPCR, with almost perfect agreement (κ = 0.952). A visual readout using a portable blue light imager further enabled naked-eye interpretation without instrumentation. This robust, rapid, and field-deployable RT-RAA assay provides a practical solution for on-site MDRV surveillance, particularly in low-resource settings.