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Rapid detection and enumeration of trh ‐carrying Vibrio parahaemolyticus with the alkaline phosphatase‐labelled oligonucleotide probe
Author(s) -
Raghunath Pendru,
Pradeep Balakrishnan,
Karunasagar Indrani,
Karunasagar Iddya
Publication year - 2007
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2006.01145.x
Subject(s) - vibrio parahaemolyticus , biology , enumeration , alkaline phosphatase , microbiology and biotechnology , vibrio , oligomer restriction , oligonucleotide , chromatography , bacteria , gene , enzyme , biochemistry , chemistry , genetics , mathematics , combinatorics
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 trh 1 or trh 2 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 × 10 2 to 3.4 × 10 3 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.