Multi-Probe-Fluorescence in situ Hybridization for the Rapid Enumeration of Viable Vibrio parahaemolyticus

A one-step multi-probe FISH method of detecting viable Vibrio parahaemolyticus was developed. Three candidate regions, corresponding to Helix 440+441, Helix 588, and Helix 1241 in 16S rRNA, were selected for detection, the thermodynamic parameters (ΔG(overall)) of the probes were optimized, and VP437, VP612 and VP1253, whose fluorescence were 1.7 to 11.3 times that of ΔG(overall)-unadjusted sequences, were designed. The addition of competitive oligonucleotides to reactions with VP612 and VP1253 strengthened the specificity of the probes. The three probes were labeled with FITC, TAMRA, and Cy5, respectively, and using a mixture of the probes and six competitive oligonucleotides, one-step FISH was applied to the species-specific detection of V. parahaemolyticus including epidemic strains of O3:K6 and O4:K68 serotypes. V. alginolyticus, V. rotiferianus, and V. campbellii were not detected in the reaction. Microcolonies (30-80 μm in diameter) of V. parahaemolyticus were observed within 6 hours at 37°C on seawater agar plates in both fresh and heat-damaged V. parahaemolyticus. Viable bacterial counts based on the proposed method were significantly different from those measured with typical vibrio selective media (CHROMagar Vibrio and TCBS).