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

## TL;DR

A new rapid and sensitive method for detecting avian reovirus using CRISPR technology was developed, offering a portable and efficient solution for poultry disease control.

## Contribution

The first rapid detection system for avian reovirus combining RT-RAA and CRISPR/Cas12a, achieving high sensitivity and portability.

## Key findings

- The assay detects avian reovirus with a sensitivity of 1 copy/μL, surpassing traditional qPCR methods.
- The method is specific and does not cross-react with other common avian viruses.
- Results can be visualized under blue light, enabling field-deployable and on-site diagnosis.

## Abstract

Avian reovirus (ARV) is a significant arthrogenic virus in the poultry industry that is capable of infecting various avian species and leads to substantial economic losses. In this study, we developed for the first time a rapid detection system for ARV based on reverse transcription–recombinase-aided amplification (RT-RAA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) technology. Using the recombinant plasmid pMD18T-ARVS1 as a template, the assay achieved high sensitivity at 37 °C within 40 min, with a detection limit as low as 1 copy/μL—10 times more sensitive than a comparable qPCR method. Specificity testing demonstrated that the RT-RAA-CRISPR/Cas12a assay does not cross-react with other common avian viruses that significantly impact poultry farming. Furthermore, the results can be visualized under blue light, making this accurate and portable detection method highly promising for ARV control in the poultry industry.

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.