Development of a multiplex real-time PCR method for pathogenic Vibrio parahaemolyticus detection (tdh+ and trh+)

A multiplex real-time PCR (RT-PCR) method was developed in this work for pathogenic Vibrio parahaemolyticus detection in water and food samples. This bacterium is found in the environment and it is considered a major foodborne pathogen that poses a considerable public health risk; in fact, last year the World Health Organization (WHO) organized an expert meeting to review and recommend microbiological methods in order to monitor the levels of pathogenic Vibrio spp. in seafood and water. Our method was tested with different pathogenic strains of V. parahaemolyticus containing tdh and/or trh pathogenicity genes and results were compared with the cultured-based method ISO 21872-1:2007. The limit of detection (LOD) for pathogenic V. parahaemolyticus was established at 6 cfu/25 g for tdh, 11 cfu/25 g for trh1 and 8 cfu/25 g for trh2 with both methods studied. However, with ISO 21872-1:2007 only the detection of total V. parahaemolyticus is possible and this takes at least 3 days followed by complementary techniques to identify pathogenic serotypes. With multiplex real-time PCR developed in this work identification of pathogenic V. parahaemolyticus takes only 24 .

► We developed a multiplex RT-PCR for pathogenic Vibrio parahaemolyticus detection. ► We compared the RT-PCR method developed with ISO/TS 21872-1:2007. ► We designed a RT- PCR primer and probe specific for trh gene. ► We compared two chromogenic culture media with ISO standard TCBS medium.