Rapid visual detection of <i>Micropterus salmoides</i> rhabdovirus by reverse transcription multienzyme isothermal rapid amplification combined with lateral flow dipstick

ABSTRACT Objective The larvae death caused by the infection of Micropterus salmoides rhabdovirus (MSRV) has seriously affected the development of the Largemouth Bass Micropterus nigricans industry. Therefore, it is necessary to establish a fast, convenient, and sensitive detection method to detect MSRV-positive fish quickly and control virus transmission. Methods In this study, primers and probes were designed based on the G gene of MSRV. Through primer screening, specificity, sensitivity, and repeatability tests, a reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) combined with the lateral flow dipstick (LFD) method was established. Additionally, we applied this method and the published reverse transcription-polymerase chain reaction method to simultaneously detect clinical samples. Results The reaction temperature was 42°C, and the total duration of the entire detection process was 35 min. The specificity test revealed that the MSRV could be specifically detected and had no cross-reaction with other common susceptible pathogens of fish. The sensitivity test indicated that the detection limit for recombinant plasmid standard was 102 copies/μL. The repetitive test showed that this method had good reproducibility. There was no significant difference between the results of RT-MIRA-LFD and RT-PCR. Conclusions The RT-MIRA-LFD established in this study for detecting MSRV is fast, simple, highly sensitive, and highly specific, without the need for precise or expensive equipment. Therefore, it is suitable for resource-limited laboratories and on-site detection.