Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish.

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption. In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R(2) was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R(2)≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R(2)≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001). The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood.