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Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labelled oligonucleotide probe

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*E-mail mircen@sancharnet.in; or karuna8sagar@yahoo.com; Tel. (+91) 824 2246384; Fax (+91) 824 2246384.

Summary

An alkaline phosphatase (AP)-labelled oligonucleotide probe was developed to detect and enumerate trh+Vibrio parahaemolyticus in seafood. The probe was evaluated using 40 isolates of V. parahaemolyticus, 45 isolates of other vibrios and 55 non-vibrio isolates. The probe reacted specifically with V. parahaemolyticus possessing either the trh1 or trh2 variant of the trh gene and was found to be 100% specific for trh+V. parahaemolyticus. Using the trh probe, V. parahaemolyticus carrying trh gene was targeted in 34 seafood samples by direct plating and colony hybridization procedure. The trh+V. parahaemolyticus could be detected in five of 34 (14.7%) samples and the levels ranged from 5.0 × 102 to 3.4 × 103 cfu g−1. Colonies of trh+V.parahaemolyticus were isolated from the five positive samples. Forty seafood samples were analysed for trh+V. parahaemolyticus by colony hybridization following enrichment in alkaline peptone water. 16 samples (40%) were positive for trh gene and trh+V. parahaemolyticus was isolated from 15 samples (37.5%). To assess the sensitivity of the trh probe, seafood homogenates spiked with known concentrations of trh-positive V. parahaemolyticus were plated and hybridized. Counts obtained using the probe were similar to those of inocula. The results suggest that the AP-labelled trh probe is useful for the detection and enumeration of trh+V. parahaemolyticus in seafood.

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