Establishment of a Rapid and Efficient Method for the Detection of Avian Reovirus Based on RT-RAA-CRISPR/Cas12a...

Avian reovirus (ARV), a highly pathogenic agent in poultry, causes severe economic losses through immunosuppression and secondary infections. Traditional diagnostic methods like reverse transcription quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) face limitations in resource-limited settings due to equipment dependency and prolonged processing. To address this, we developed a rapid, portable detection method integrating reverse transcription-recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a. By targeting the conserved P17-coding region of the ARV S1 gene, this assay amplifies viral RNA isothermally (37 °C) within 20 min, followed by Cas12a-mediated collateral cleavage of fluorescent or lateral flow reporters for visual readout. The method achieved a sensitivity of 1 copy/μL, surpassing RT-qPCR (10 copies/μL), and completed detection in 40 min. Specificity tests against non-target pathogens confirmed zero cross-reactivity. Utilizing a portable incubator and low-cost visual tools, this platform eliminates reliance on thermocyclers and skilled personnel. Its field-deployable design enables on-site diagnosis, facilitating early ARV detection to mitigate outbreaks and economic losses in poultry farming. This study provides a paradigm shift in avian pathogen surveillance, combining speed, sensitivity, and accessibility for global agricultural and public health applications.