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Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish

Ana R. Ferreira1, Teresa M. Baptista2 and Clélia N. Afonso2*
  • 1 Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, Portugal
  • 2 Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, MARE – Marine and Environmental Sciences Centre, Portugal

The aquaculture sector has registered a significant growth in the last years; however, the presence of infectious diseases is a serious threat, leading to huge losses in this sector. Bacteria belonging to the genus Vibrio (Toranzo et al., 2005) cause some of these diseases. It is of uttermost importance to create methods to detect precociously the presence of these bacteria in aquaculture, in order to prevent potential outbreaks that may arise and minimize the consequences (Martins et al., 2015; Shi et al., 2012; Sorroza et al., 2012). A critical step in the study of bacterial fish diseases is the correct identification of the infectious agent (Avendaño-Herrera et al., 2004), but classical microbiological methods does not allow distinguishing the different species of Vibrio and are not expedite enough (Kim & Lee, 2014). Furthermore, it is known that the precise identification of different species of the genus Vibrio is problematic, especially when dealing with related species (Kwok et al., 2002), namely due to the variability of the biochemical characteristics, which complicates the phenotypical identification of these bacteria (Nhung et al., 2007). The conventional polymerase chain reaction (PCR) tool provides rapid, specific and sensitive analysis of Vibrio species, but it time consuming and expensive, when dealing with a large number of targets. Multiplex polymerase chain reaction, commonly referred to as multiplex-PCR (m-PCR), is a type of PCR in which two or more target sequences can be amplified simultaneously, by including more than one pair of oligonucleotides of amplification (primers) in the reaction. This type of m-PCR is advantageous when looking at time and resources in the laboratory (Markoulatos et al., 2002), as well as to reduce expenses in reagents, without compromising the results. The aim of this study is to develop and validate a molecular tool that detects the presence of Vibrio anguillarum, Vibrio harveyi and Vibrio alginolyticus, three of the pathogens that cause more losses in aquaculture, using a multiplex-PCR tool. The standard bacterial strains were grown in Tryptone Soya Broth (TSB) and the growth curves were established. The bacterial DNA was extracted with NZY Tissue gDNA isolation kit (Nzytech) and compared to the boiling method. Different conditions for the multiplex-PCR were tested in order to establish the optimum conditions to be used. The multiplex PCR reactions were tested using a Taq polymerase and varying concentrations of MgCl2, being the ideal concentration of MgCl2 of 7.5 mM. Better results were obtained with 35 cycles of amplification and optimum annealing temperature at 58°C. The specificity of the primers was tested using purified DNA from the respective bacterial strains, yielding clear bands at the expected sizes. With respect to the sensitivity of the tool, there is a gradient of band intensity, which can be correlated with the concentration of DNA present. These results are in agreement with that described by Pinto et al. (2017), indicating a band intensity gradient, with m-PCR efficiently amplifying up to the concentration of 1 ng.μl-1. Results indicate that the multiplex PCR tool is suitable for the detection of the target pathogens and that this molecular tool is important especially when it is suspected that there are multiple infections derived from different pathogens in commercial aquaculture. The results indicate that this method allows a diagnosis of vibriosis in one working day and thus, it appears more convenient than the classical microbiological methods, which are time consuming and not always accurate.

Acknowledgements

This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE.

References

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Kim J.Y. & Lee J-L (2014) Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction. J Sci Food Agric 94: 2807–2817.


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Markoulatos P., Siafakas N., Moncany M. (2002) Multiplex Polymerase Chain Reaction: A Practical Approach. Journal of Clinical Laboratory Analysis 16: 47–51

Martins, P., R.V.V. Navarro, F.J.R.C. Coelho e N.C.M. Gomes. 2015. Development of a molecular methodology for fast detection of Photobacterium damselae subspecies in water samples. Aquaculture. 435: 137-142.

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Pinto M.P., Baptista T., Afonso C.C.A. (2017) Development of a new multiplex-PCR for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda. Aquatic Living Resources 30: 1-6

Shi, Y.-H., Chen, J., Li, C.H., Lu, X.-J., Zhang, D.-M. et al. (2012). Detection of bacterial pathogens in aquaculture samples by DNA microarray analysis. Aquaculture, 338-341: 29-35.

Sorroza L., Padilla D., Acosta F., Roman L., Grasso V., Veja J., Real F. (2012) Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum. Veterinary Microbiology 155: 369–373

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Keywords: Vibriosis, Fish pathology, Aquaculture, Disease Diagnostic, Multiplex-PCR

Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Blue Biotech

Citation: Ferreira AR, Baptista TM and Afonso CN (2019). Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00001

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Received: 27 Apr 2018; Published Online: 07 Jan 2019.

* Correspondence: Prof. Clélia N Afonso, Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, MARE – Marine and Environmental Sciences Centre, Peniche, 2520-641, Portugal, clelia@ipleiria.pt