Studies on the rapid detection of Vibrio parahaemolyticus in food
2008
Miyahara, M.(National Inst. of Health Sciences, Tokyo (Japan)) | Arakawa, E.
Vibrio parahaemolyticus is one of the major causative bacteria of foodborne illnesses in Japan. The most probable number (MPN) of V. parahaemolyticus in fresh or frozen (for raw eating) fish and shellfish is regulated as an allowable limit MPN=100/g in Japan. Alkaline peptone water is used as an enrichment culture medium, and TCBS, CHROMagar Vibrio and X-Vibrio agar are used as selective media for plating. V. alginolyticus has biological characteristics that are similar to that of V. parahaemolyticus. Fresh fish and shellfish are often contaminated with both bacteria. We attempted to develop a new detection method for V. parahaemolyticus in food by PCR. Sequencing of the toxRS gene of ten V. parahaemolyticus and thirteen V. alginolyticus strains was accomplished and the biological characteristics of these strains were determined. Analysis of the toxRS gene of these bacteria was carried out. Primers for the detection of toxR gene of V. parahaemolyticus were constructed and used for the detection of this species. Thirty-eight strains of V. parahaemolyticus and fifteen strains of V. alginolyticus were tested for their detection by PCR with a new primer set. All V. parahaemolyticus were detected whereas none of V. alginolyticus was detected by PCR using the new primer set. Other Vibrio species were also tested by the new primer-PCR. None of other Vibrio were detected. Inoculation of the squid sample with V. parahaemolyticus and excessive numbers of V. alginolyticus was carried out and then the template for PCR was prepared from this sample after cultivation. We were able to detect successfully only V. parahaemolyticus in the sample by PCR. We applied this PCR system for a rapid detection system in food. Levels over 100 cfu of V. parahaemolyticus/g could be detected in contaminated food in 4hrs enrichment and PCR.
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