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Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams

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Abstract

Vibrio parahaemolyticus is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers (tlh species-specific gene and tdh virulence gene) of V. parahaemolyticus in bivalve mollusc. The analyses were performed on clams inoculated with V. parahaemolyticus ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other Vibrio species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for tdh and tlh genes, showed a detection limit of 104 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by tdh gene in real-time PCR reached the definitely highest sensitivity in shorter times, 101 CFU/g after 3 h of pre-enrichment, while the sensitivity for the tlh gene was not promising, detecting between 105 and 106 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of V. parahaemolyticus based on tdh gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases.

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Acknowledgements

The Authors would like to thank MARE. A s.r.l. (Cattolica, FC, Italy) to have provided the clam samples.

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Author notes

  1. Sara Federici and Diana I. Serrazanetti have contributed equally to this work.

Authors and Affiliations

  1. Department of Biomolecular Sciences, Division of Toxicological, Hygienic and Environmental Sciences, University of Urbino “Carlo Bo”, Via S. Chiara 27, 61029, Urbino, Italy

    Sara Federici, Raffaella Campana, Eleonora Ciandrini & Wally Baffone

  2. Inter-departmental Centre for Industrial Agri-Food Research, Alma Mater Studiorum, University of Bologna, Piazza Goidanich 60, 47521, Cesena, Italy

    Diana I. Serrazanetti & Andrea Gianotti

  3. Department of Food Science, Alma Mater Studiorum, University of Bologna, Viale Fanin 46, 40127, Bologna, Italy

    M. Elisabetta Guerzoni & Andrea Gianotti

  4. Dipartimento di Scienze e Tecnologie Agro-Alimentari (DISTAL), Department of Agricultural and Food Science, Alma Mater Studiorum, Università di Bologna, Via Fanin 50, 40127, Bologna, Italy

    Andrea Gianotti

  5. Unità Organizzativa di Cesena, Piazza Goidanich 60, Cesena, FC, Italy

    Andrea Gianotti

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  1. Sara Federici
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  2. Diana I. Serrazanetti
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  3. M. Elisabetta Guerzoni
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Correspondence to Andrea Gianotti.

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Federici, S., Serrazanetti, D.I., Guerzoni, M.E. et al. Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams. J Food Sci Technol 55, 749–759 (2018). https://doi.org/10.1007/s13197-017-2986-9

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