# 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

## TL;DR

A new rapid and portable test for detecting Muscovy duck reovirus was developed, enabling on-site diagnosis without needing advanced lab equipment.

## Contribution

The first lyophilized RT-RAA assay for MDRV detection, enabling point-of-care testing without cold-chain dependence.

## Key findings

- The assay detected MDRV with a limit of 1.03 × 10¹ copies/μL within 20 minutes.
- The lyophilized format showed 95.8% sensitivity and 100% specificity compared to RT-qPCR.
- A portable blue light imager allowed visual detection without specialized instruments.

## Abstract

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.