Development of a rapid, real-time RT-RAA assay for sensitive detection of bovine enterovirus in fecal samples
Cuilan Wu1, Xixian Liu2, Lan Jia2
1Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530004, China; Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning 530001, China; Key Laboratory of China (Guangxi)-ASEAN Cross-Border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning 530001, China.
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.
