Detection of Vibrio parahaemolyticus hemolysin genes in frozen shrimps using multiplex polymerase chain reaction

Abstract

The detection of the lecithin dependent hemolysin gene (ldh), thermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in frozen shrimp samples by multiplex PCR. One hundred frozen shrimp samples were obtained from frozen seafood factories in Bangkok and Samutsakorn province from July 2002 to February 2004. According to The Thai Food and Environmental Hygiene organization, 89% (89/100) of samples were acceptable in all of the microbiological criteria including aerobic total viable count, V. parahaemolyticus, coliforms and Escherichia coli. The lowest genomic DNA of three genes (ldh, tdh and trh) of V. parahaemolyticus type strain VP156-9-7-1993 was detected at 1 ng/μl by multiplex PCR. Examining 19 strains of Vibrio spp. and enteric bacteria with these 3 genes by multiplex PCR resulted in 100% specificity. In 14% (14/100) of frozen shrimp enrichment samples V. parahaemolyticus was detected by both the conventional method and multiplex PCR. In addition, in 16% (16/100) of samples V. parahaemolyticus was detected by the combination of both methods. All frozen shrimp samples tested negative for tdh and trh by multiplex PCR and for TDH by modified Elek test and TRH by urease activity test. Enrichment samples cultured with V. parahaemolyticus were positive for ldh gene in 85.7% (12/14). In contrast, enrichment samples not cultured with V. parahaemolyticus were positive for ldh in 3% (2/65). Similarly, 70 isolates of V. parahaemolyticus collected from 14 enrichment samples cultured with V. parahaemolyticus were positive for ldh gene and negative for tdh and trh genes. The multiplex PCR detecting for ldh, tdh and trh of V. parahaemolyticus directly from the enrichment samples showed sensitivity, specificity and efficiency that compared well with the conventional method of 85.7%, 97.7% and 96%, respectively. Thus, the multiplex PCR could be an alternative method as a sensitive, specific, and less time consuming method to detect V. parahaemolyticus and ;"การตรวจหายีนที่กำหนดการผลิตสารพิษ ldh (lecithin dependent hemolysin)