Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing Vibrio parahaemolyticus in Seafood

Abstract

ABSTRACT A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene ( tdh ) and TRH gene ( trh ) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh - or trh -positive ( tdh + trh + ) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh + trh + V. parahaemolyticus and dense populations of a viable strain of tdh- and trh- negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh + trh + strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh + trh + strain together with dense populations of the tdh- and trh- negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.