Increased Sensitivity in PCR Detection of tdh-Positive Vibrio parahaemolyticus in Seafood with Purified Template DNA
Abstract:PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysinproducing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V. parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V. parahaemolyticus. In this study, the use of PCR to detect the tdh gene of V. parahaemolyticus in various seafoods artificially contaminated with tdh-positive V. parahaemolyticus was examined. PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp. To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template. For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity. The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V. parahaemolyticus in contaminated seafood.
Document Type: Research Article
Affiliations: 1: Department of Microbiology, National Institute of Health Sciences, 1-1-8-1 Kamiyouga, Stagaya-ku, 158-8501 Tokyo, Japan 2: Department of Veterinary Microbiology, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara-shi, 229-8501 Kanagawa, Japan 3: Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, 112-8681, Tokyo, Japan 4: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan
Publication date: September 1, 2003
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