Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.