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

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A singl...

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Bibliographic Details
Published in:Journal of fish diseases 2018-02, Vol.41 (2), p.247-254
Main Authors: Carraro, R, Dalla Rovere, G, Ferraresso, S, Carraro, L, Franch, R, Toffan, A, Pascoli, F, Patarnello, T, Bargelloni, L
Format: Article
Language:English
Subjects:
Animal tissues
Aquaculture
Assaying
Bacteria
Deoxyribonucleic acid
Detection
Diagnosis
Disease control
Dissociation
DNA
Dynamics
Fish
Fish diseases
Fish farms
Fish photobacteriosis
Geographical distribution
Methods
Nucleotide sequence
Outbreaks
Pasteurellosis
PCR
Photobacterium damselae
Photobacterium damselae subsp. damselae
Photobacterium damselae subsp. piscicida
Polymerase chain reaction
Real time
real‐time PCR
Sensitivity
Single-nucleotide polymorphism
Specificity
Tissue
Waterborne diseases
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Summary:The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, 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 P. damselae was designed targeting partial of the sequence of the bamB 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 Phdd‐Phdp 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 Phdp‐Phdd. This is the first time that a molecular method for P. damselae 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.
ISSN:0140-7775
1365-2761
DOI:10.1111/jfd.12703