Establishment of methods for visual and rapid detection of Nocardia seriolae based on isothermal recombinase polymerase amplification

Nocardia seriolae is the main pathogenic bacterium that causes nocardiosis in fish, seriously endangering the healthy and green development of aquaculture. There is an urgent demand in the market for rapid detection methods of N. seriolae. Therefore, in this study, we developed methods for rapid and visual detection of N. seriolae by isothermal recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time fluorescence RPA based on the conserved sequence of 16S-23S rRNA internal transcribed spacer (ITS). The RPA-LFD and real-time fluorescence RPA methods achieved rapid detection of N. seriolae within 20 min. The minimum detection limit of the two methods for the plasmid pMD18-ITS was 103 copies/mu L, and the minimum detection concentration for the genomic DNA of N. seriolae was 100 pg/mu L. The sensitivity was comparable to that of polymerase chain reaction (PCR), and a high specificity was noted, which can enable the early detection of N. seriolae. In addition, RPA-LFD and real-time fluorescence RPA methods were used to analyze 40 largemouth bass (Micropterus salmoides) samples from fish farms, and the results were consistent with those obtained with conventional PCR. In summary, the methods established in this study are easy to use and exhibit high sensitivity, laying an important foundation for rapid diagnosis of N. seriolae infection.