Integrated Enzyme-Mediated One-Step Sample Processing and Duplex Amplification System for Rapid Detection of Carpione rhabdovirus in Aquaculture-Derived Food Products

Golden pompano (<i>Trachinotus ovatus</i>) is the largest-scale marine aquaculture fish species in China, with a significant economic and nutritional value as a high-quality seafood product. The recent outbreak of an epidemic caused by a novel <i>Carpione rhabdovirus</i> (CAPRV) occurred in cultured golden pompano. To address it, a CAPRV enzyme-mediated one-step sample processing-reverse transcription-enzyme-mediated duplex exponential amplification (EmOSP-RT-EmDEA) detection system was developed. This innovative molecular diagnostic tool integrates enzyme-mediated one-step sample processing (EmOSP) with enzyme-mediated duplex exponential amplification (EmDEA) technology. Unlike traditional RPA-Cas12a detection methods, this system directly incorporates fluorophores into RNA components, eliminating the need for exogenous fluorescent probes while maintaining high sensitivity. It enables rapid, sensitive, and specific detection of CAPRV2023 across various sample types, including clinical, invasive, minimally invasive, and environmental specimens. Performance evaluation of the CAPRV2023 EmOSP-RT-EmDEA detection system against conventional diagnostic methods, such as TaqMan qPCR and traditional PCR, demonstrated superior sensitivity, with a detection limit as low as 4 copies/μL, and exceptional specificity. The optimized EmOSP protocol for nucleic acid extraction from fecal, hepatic, and water samples provided robust and reproducible results. The EmOSP-RT-EmDEA system achieved a detection rate of 68.14% in fecal samples, matching the performance of the gold-standard TaqMan qPCR assay.