Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh

Vibrio parahaemolyticus is an important human pathogen which can cause gast roenteritis when consumed in raw or partially-cooked seafood. A multiplex P CR amplification-based detection of total and virulent strains of V. paraha emolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemo lysin-related trh genes. Following optimization using oligonucleotide prime rs targeting tl, tdh and trh genes, the multiplex PCR was applied to V. par ahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obt ained from various laboratories and 19 from oyster plants. All 111 V. parah aemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplificat ion of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggestin g both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approac h was successfully used to detect various strains of V. parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three ta rget gene segments was at least between 10(1)-10(2) cfu per 10 g of alkalin e peptone water enriched seeded oyster tissue homogenate. This high level o f sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventio nal microbiological culture methods, this multiplex PCR approach is rapid a nd reliable for accomplishing a comprehensive detection of V. parahaemolyti cus in shellfish. (C) 1999 Elsevier Science B.V. All rights reserved.