Abstract：Abstract Listonella anguillarum has emerged as a serious pathogen causing signi?cant mortalities in various ?sh and shell?sh species over a wide geographical area. With the large-scale application of intensive farming model in aquaculture, the speed of the transmission of aquatic pathogens has been greatly improved. Cultivation of Listonella anguillarum is seen as the gold standard for detection, although it is very time consuming and labour intensive. Accordingly, the establishment of practical and effective diagnostic methods has long been desired. In this study, several pairs of primers and an exo-probe targeting the zinc metalloprotease gene (empA) of Listonella anguillarum was designed and a rapid real-time detection method for Listonella anguillarum based on recombinase polymerase amplification (RPA) was established by optimizing the reaction system and conditions. The results showed that the established Real-time RPA technique could detect Listonella anguillarum directly within 20 min and did not cross-react with other seven common aquatic pathogenic bacteria. The detection limit of Real-time RPA for gDNA of Listonella anguillarum was 1 pg/μL, and reproducibility was good. However, the lowest detection limit of artificial pollution sample was 3.4×102 CFU/mL. It is expected to be the routine which can provide scientific basis and technical reference for the development of rapid diagnostic kit and commercial aquaculture Listonella anguillarum.
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