Validation of real-time PCR for detection of six major pathogens in seafood products

Seafood can pose a public health concern to consumers. It is often consumed rawand 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 realtime
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.