Development of a multiplex real-time PCR method for early diagnosis of three bacterial diseases in fish: A real-case study in trout aquaculture

Abstract

Lactococcus garvieae, Yersinia ruckeri and Flavobacterium psychrophilum are three of the most important pathogens in worldwide rainbow trout aquaculture (Oncorrhynchus mykiss Walbaum). In this work, a multiplex quantitative PCR (qPCR) method for the simultaneous detection of the three pathogens was developed and tested in a commercial trout farm. Fifty-four spleen and brain samples from a trout farm were analyzed using both multiplex qPCR and classic microbiological methods. When comparing qPCR results and classic plate diagnostic techniques no negative deviations were observed, which resulted in a 100% relative sensitivity of the multiplex qPCR method developed. In addition, efficiency of the qPCR method ranged between 97.5% and 108.8%. The specificity of the combination of primers and probes was successfully tested in 57 target and non-target bacterial strains Hence, the multiplex qPCR method developed in the present study could be used as a reliable diagnostic tool for the detection of L. garvieae, Y. ruckeri and F. psychrophilum in rainbow trout farms, enabling faster diagnostics (only a few hours) and contributing to a quick response and management of bacterial disease outbreaks.