Development of a Multiplex-PCR tool for the simultaneous detection of Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

Abstract

Event Abstract Back to Event Development of a Multiplex-PCR tool for the simultaneous detection of Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda Micaela Pinto1, Teresa M. Baptista2 and Clélia N. Afonso2* 1 Escola Superior de Turismo e Tecnologia do Mar, Instituto Politécnico de Leiria, Poland 2 MARE – Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria, Portugal Disease management and assessment of cultured aquatic animals is a major concern in commercial aquaculture. Disease outbreaks have direct effects on fish production, causing serious economic losses in this industry, which can be overcome by early detection through molecular high sensitivity tools such as PCR (Chain Reaction polymerase). One of the most critical steps in the study of bacterial fish diseases is the correct identification of the infectious agent. Considering the damage that some bacteria can cause to fish and humans, the development of a rapid detection method, simple, and low cost is the primary objective of this work. Edwardsiella tarda (ACC36.1), Vibrio harveyi (DSM19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521) were selected as development targets of a multiplex PCR tool. The standard strains of the four pathogenic microorganisms were grown in Tryptone Soy Broth (TSB) and the growth curves were established. The bacterial DNA was extracted with NZY Tissue gDNA isolation kit (Nzytech) and the supernatant was used directly in the PCR reactions. The multiplex PCR reactions were tested using a Taq polymerase and various reaction conditions were tested, including annealing temperatures (between 49°C and 55°C) and the change in the MgCl2 concentration from 2 mM to 8 mM. Best results are obtained at 51°C temperature and an MgCl2 concentration from 4 to 6 mM. The specificity of the primers was tested using purified DNA from the respective bacterial strains, yielding bands at expected sizes, which can be identified on agarose gels with clearness and without overlapping sizes. Results indicate that the multiplex PCR tool is suitable for the detection of target pathogens. Acknowledgements The strains Edwardsiella tarda (ACC36.1), Vibrio harveyi (DSM19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521)used in the present study were kindly provided by Professor Alicia E. Toranzo (Departamento de Microbiología y Parasitología, Facultad de Biología, University of Santiago de Compostela, Spain). This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE. Keywords: Disease Diagnostic, Multiplex-PCR, Vibrio alginolyticus, vibrio anguillarum, Vibrio harveyi, Edwardsiella tarda Conference: IMMR | International Meeting on Marine Research 2016, Peniche, Portugal, 14 Jul - 15 Jul, 2016. Presentation Type: Poster presentation Topic: Aquaculture Citation: Pinto M, Baptista TM and Afonso CN (2016). Development of a Multiplex-PCR tool for the simultaneous detection of Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2016. doi: 10.3389/conf.FMARS.2016.04.00028 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Apr 2016; Published Online: 13 Jul 2016. * Correspondence: Prof. Clélia N Afonso, MARE – Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria, Peniche, 2520-641, Portugal, clelia@ipleiria.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Micaela Pinto Teresa M Baptista Clélia N Afonso Google Micaela Pinto Teresa M Baptista Clélia N Afonso Google Scholar Micaela Pinto Teresa M Baptista Clélia N Afonso PubMed Micaela Pinto Teresa M Baptista Clélia N Afonso Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.