Development of Two Multienzyme Isothermal Rapid Amplification Techniques for the Rapid Visual Detection of <i>Micropterus salmoides</i> rhabdovirus

Largemouth bass (Micropterus salmoides) is an important freshwater-cultured species in China. M. salmoides rhabdovirus (MSRV) is a devastating pathogen that causes high mortality (exceeding 90% in acute outbreaks) in largemouth bass fry, posing a severe threat to the global largemouth bass aquaculture industry. To address the urgent demand for rapid, on-site MSRV detection, two multienzyme isothermal rapid amplification (MIRA)-derived methods-multienzyme isothermal rapid amplification with fluorescence detection (MIRA-FLU) and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD)-were established and optimized targeting the conserved nucleoprotein (N) gene of MSRV. The performance of both methods was systematically evaluated, and their practical applicability was verified using clinical samples collected from diseased fish farms. The results demonstrated that both methods exhibited high sensitivity, with a detection limit of 9 copies/μL for the standard plasmid. Both detection methods demonstrated high specificity and showed no cross-reactivity with other common aquatic viruses, indicating their high specificity for MSRV detection. MIRA-FLU completed amplification within 25 min at 42°C, while MIRA-LFD achieved amplification within 12 min at 37°C. MIRA-FLU is used for real-time fluorescent quantitative detection, whereas MIRA-LFD enables visual, instrument-free reading via lateral flow test strips. In comparative analyses of clinical samples, the diagnostic results of MIRA-FLU and MIRA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our MIRA-FLU and MIRA-LFD detection methods demonstrate excellent specificity and sensitivity, providing two simple, rapid and reliable approaches for large-scale field surveys of MSRV in resource-limited settings.