Development of a real‐time PCR assay for rapid detection and quantification of <italic>Photobacterium damselae</italic> subsp. <italic>piscicida</italic> in fish tissues.

Abstract: The availability of a rapid and accurate method for the diagnosis of <italic>Photobacterium damselae</italic> subsp. <italic>piscicida</italic> (<italic>Phdp</italic>), able to discriminate its strictly correlated subsp. <italic>damselae</italic> (<italic>Phdd</italic>), formally known as <italic>Vibrio damsela</italic>, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single‐step, high‐sensitivity real‐time PCR assay for simultaneous detection and quantification of <italic>P. damselae</italic> was designed targeting partial of the sequence of the <italic>bamB</italic> gene and tested for specificity and sensitivity on laboratory‐generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non‐target bacterial species proved the assay was able to discriminate <italic>Phdd</italic>‐<italic>Phdp</italic> subspecies from diverse hosts/geographical origins and between non‐target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between <italic>Phdp</italic>‐<italic>Phdd</italic>. This is the first time that a molecular method for <italic>P. damselae</italic> diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease. [ABSTRACT FROM AUTHOR]