Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for visual detection of largemouth bass virus

Largemouth bass (Micropterus salmoides) has become one of the important economical freshwater fish species in China in recent years. However, the frequent outbreak of viral diseases induced by largemouth bass virus (LMBV) always caused high mortality and led to heavy economic losses in the largemouth bass aquaculture industry. Notably, LMBV was also found to infect other species of Percidae, such as smallmouth bass (Micropterus dolomieu) and bluegill (Lepomis macrochirus). However, rapid, specific, and visual methods for LMBV detection were limited up to now. In this study, a rapid and convenient technique combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was established to detect LMBV. The specific, sensitive primers and probes targeting major capsid protein (MCP) of LMBV were screened. The assay of RPA-LFD took approximately 10 min at 38℃ using a water bath, and exhibited no cross-reactivity with several other aquatic pathogens. The detection limit of RPA-LFD was 10 copies/μL, which was 10 times more sensitive than that from routine PCR. Moreover, 36 tissue samples from largemouth bass were detected to validate the application of RPA-LFD. The coincidence rate of the positive test samples was 97.2% between RPA-LFD and PCR. Taken together, the established RPA-LFD assay here provided a rapid, convenient, specific and sensitive method for field diagnosis of LMBV without special equipment.