Cuilan Wu^1,2,3#, Xixian Liu^1#, Lan Jia¹, Zhijie Mo¹, Yeheng Gao⁵, Shuhong Zhong^2,3, Shiwen Feng^2,3, Zhongwei Chen^2.3, Xian Li⁴, Xindong Wang¹, Xiongbiao Xuan^2,3, Huili He^2,3, Shuai Hu^2,3, Changting Li^2.3, Zuzhang Wei¹, Yongping Xie^2,3, Hao Peng^2,3*, Yingyi Wei^1*, Jun Li^2,3*

1. Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530004, China

2. Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China

3. Key Laboratory of China (Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning 530001, China

4. Guangxi Agricultural Engineering Vocational Technical College, Chongzuo 532100, China

5. Institute of Agricultural and Animal Husbandry Industry Development, Guangxi University, Nanning 530004, China

# Co-first authors, these authors contributed equally to this work.

         ABSTRACT：Bovine enterovirus (BEV) is a significant pathogen affecting bovine intestinal health, posing challenges to cattle production and public health. Existing detection methods, such as RT-PCR, RT-qPCR, and LAMP, are time-consuming and dependent on specialized laboratory facilities, highlighting the need for rapid and field-deployable diagnostic tools. In this study, we developed a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay designed for BEV detection, capable of delivering results within 20 min at 39℃. The real-time RT-RAA assay showed no cross-reactivity to other eight pathogens, including BCoV, BVDV, BKV, BRV, BAstV, STEC, FMDV, and PEV. The sensitivity assay revealed that the real-time RT-RAA method could reliably detect a minimum plasmid concentration of 5.50 × 10 ¹ copies/reaction. Clinical validation conducted with 780 field samples revealed performance comparable to those of conventional RT-PCR and RT-qPCR methods, achieving a positivity rate of 23.59 %. The RT-RAA method operates at low temperatures, requires minimal instrumentation, and reduces technical demands, making it ideal for resource-limited settings. Its portability, cost-effectiveness, and robustness position it as a transformative tool for BEV surveillance, enhancing outbreak control, livestock health management, and food safety..

        Keywords：bovine enterovirus; real- time; RT-RAA assay; rapid detection; clinical validation

        Journal of Virological Methods Available online 19 December 2025.

         https://doi.org/10.1016/j.jviromet.2025.115331