Validation of real-time PCR for detection of six major pathogens in seafood products B. Taminiau, N. Korsak * , C. Lemaire, V. Delcenserie, G. Daube University of Liège, FARAH, Faculty of Veterinary Medicine, Department of Food Science, Sart-Tilman, B43bis, 4000 Liège, Belgium article info Article history: Received 7 November 2013 Received in revised form 12 March 2014 Accepted 22 March 2014 Available online 4 April 2014 Keywords: Seafood Real-time PCR Campylobacter EHEC Salmonella Vibrios abstract Seafood can pose a public health concern to consumers. It is often consumed raw and may be contaminated with several foodborne pathogens. In order to guarantee the safety of seafood, real-time polymerase chain reaction (PCR) protocols may be used as these enable results to be provided within 24 h. The first goal of our work was to develop real-time PCR protocols enabling the detection of six foodborne pathogens that may be present in seafood products (Campylobacter jejuni, Campylobacter coli, enterohemorrhagic Escherichia coli, Salmonella spp., Vibrio parahaemolyticus, and Vibrio vulnificus). The corresponding gene targets were: 50S/VS1 , rfbE, ttr, tlh, and vvp. A multiplex PCR was also developed to detect the virulence genes of V. parahaemolyticus: tdh and trh. A total of 420 bacterial strains belonging to four different genera/strains were used in this study. Sensitivity and specificity were always 100%, except in the case of Salmonella spp., where three strains were not detected by our PCR protocols. The second objective of our work was to assess the detection limit of our real-time PCR protocols on artificially contaminated seafood products (raw shrimps, cooked shrimps, and raw mussels), purchased in public stores. Six different levels of contamination were assayed in four replicates for each matrix. The real-time PCR protocols enabled a better level of detection than the ISO methods, except for Salmonella in raw shrimps and for V. vulnificus in shrimps (raw and cooked). The estimated level of detection was between 1 and 47 cfu/25 g sample for the ISO norms and between 1 and 315 cfu/25 g sample for the real- time PCR protocols tailored in our work. The real-time PCRs developed in our work allowed for good selectivity, sensitivity, and specificity. The sensitivity on seafood products was estimated at a level of 100%, except for Salmonella (97%). In the spiking assays, the levels of detection were lower with the real-time PCR protocol than those obtained with the ISO method. This was not the case for V. vulnificus in raw and cooked shrimps and for Salmonella in raw shrimps. These real-time PCR protocols appear to be good alternative methods for surveillance of seafood products to ensure the absence of foodborne pathogens. One additional conclusion is that laboratories have to use enrichment media that are compatible with those recommended by ISO standards. This may facilitate the isolation of the pathogen if the real-time PCR protocol gives a suspect positive signal during the first step of the seafood analysis. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Shellfish and crustaceans play an important role in foodborne diseases all over the world, including in developed countries. They can become contaminated by the environment owing to the fact that bivalve mollusks filter a significant volume of water. The latter explains the concentration of the pollutants in the tissues of mol- lusks. Additional reasons can be related to the pollution of water by human feces, the contamination of cropping areas due to flooding, and the importation of shellfish from foreign countries that do not apply good and reliable surveillance methods (Iwamoto, Ayers, Mahon, & Swerdlow, 2010). In developed countries, most in- fections result from the ingestion of raw or undercooked seafood (Chen et al., 2012). Our gastronomic culture favors raw seafood, facilitating the persistence and transmission of possible microbial contaminants. Moreover, the consumption of seafood is still increasing and represents a worldwide market. Following Iwamoto Abbreviations: BHI, Brain Heart Infusion; cfu, colonies forming units; ISO, In- ternational Organization for Standardization; PCR, Polymerase Chain Reaction. * Corresponding author. Tel.: þ32 4 366 40 40; fax: þ32 4 366 40 44. E-mail addresses: bernard.taminiau@ulg.ac.be (B. Taminiau), nkorsak@ulg.ac.be (N. Korsak), ce.lemaire@gmail.com (C. Lemaire), veronique.delcenserie@ulg.ac.be (V. Delcenserie), georges.daube@ulg.ac.be (G. Daube). Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont http://dx.doi.org/10.1016/j.foodcont.2014.03.031 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved. Food Control 44 (2014) 130e137