Development of a <i>mreB</i>-targeted real-time PCR method for the quantitative detection of <i>Vibrio harveyi</i> in seawater and biofilm from aquaculture systems
2020
Mougin, Julia | Roquigny, Roxane | Travers, Marie-Agnès | Grard, Thierry | Bonnin-Jusserand, Maryse | Le Bris, Cédric | BioEcoAgro - UMR transfrontalière INRAe - UMRT1158 ; Université d'Artois (UA)-Université de Liège = University of Liège = Universiteit van Luik = Universität Lüttich (ULiège)-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA) ; Université catholique de Lille (UCL)-Université catholique de Lille (UCL) | BioEcoAgro - Equipe 8 - Food and Digestive Microbial Ecosystems: Interactions - Dynamics - Application(s) ; BioEcoAgro - UMR transfrontalière INRAe - UMRT1158 ; Université d'Artois (UA)-Université de Liège = University of Liège = Universiteit van Luik = Universität Lüttich (ULiège)-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA) ; Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Université d'Artois (UA)-Université de Liège = University of Liège = Universiteit van Luik = Universität Lüttich (ULiège)-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA) ; Université catholique de Lille (UCL)-Université catholique de Lille (UCL) | Université du Littoral Côte d'Opale (ULCO) | Interactions Hôtes-Pathogènes-Environnements (IHPE) ; Université de Perpignan Via Domitia (UPVD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM) | Unité Santé, Génétique et Microbiologie des Mollusques (SGMM) ; Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER) | Hauts-de-France regional council | ULCO | French government | Ifremer | Region Hauts-de-France | FEAMP as part of the LUVIBAR project PFEA470017FA1000006
International audience
Afficher plus [+] Moins [-]anglais. Vibrio harveyi is a particularly problematic Gram-negative bacterium because it can form biofilms on aquaculture facility surfaces, leading to resistance of bacteria against antibiotics and water sanitizers. A SYBR Green I quantitative real-time PCR method was developed to detect V. harveyi directly from environmental samples, including seawater and biofilm. Specific primers targeting the mreB gene were designed. The exclusivity and inclusivity of the newly designed primers were evaluated using a panel of 85 bacteria: 58 V. harveyi from multiples origins and 27 non-V. harveyi isolates, and compared with two pairs of primers targeting the topA and toxR genes that were designed previously. All sets of primers were able to distinguish V. harveyi from closely related species belonging to the Harveyi clade. However, the mreB primers showed better inclusivity and were thus used to develop the real-time PCR assay. A quantification curve was obtained from pure culture of V. harveyi and exhibited excellent efficacy with detection levels as low as 5 genome copies in the PCR reaction. After selection of the extraction kit allowing the best DNA quantity and purity, validation was performed on both seawater and biofilm samples collected from a fish farm. The presence of inhibitors in the DNA templates was evaluated and a 10-fold dilution of template DNA was recommended in order to avoid their effects. The assay was able to detect V. harveyi from environmental samples, confirming the validity of the method. This real-time PCR method will help to evaluate the dynamics of V. harveyi in aquaculture facilities. Suitable prophylactic control measures could be designed using this method, instead of the use of curative methods such as antibiotics
Afficher plus [+] Moins [-]Mots clés AGROVOC
Informations bibliographiques
Cette notice bibliographique a été fournie par Institut national de la recherche agronomique
Découvrez la collection de ce fournisseur de données dans AGRIS