RESUMO
PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) 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.
Assuntos
Proteínas Hemolisinas/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Alimentos Marinhos/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Proteínas de Bactérias , DNA Bacteriano/análise , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Amplificação de Genes , Genes Bacterianos , Proteínas Hemolisinas/genética , Humanos , Sensibilidade e Especificidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidadeRESUMO
We report here the first analysis of Erysipelothrix spp. using pulsed-field gel electrophoresis (PFGE). Seventy strains of Erysipelothrix spp. were analyzed. SmaI, AscI, and NotI were tested for the ability to cleave the DNA extracted from those strains, and among them, SmaI was the most reliable enzyme. Sixty-three distinct PFGE patterns were produced, and no DNA degradation was observed, allowing the identification of all of the strains. Based on these results and on those of a previous analysis using randomly amplified polymorphic DNA and ribotyping, PFGE with SmaI might be considered to be more sensitive than those methods and to be the best method for epidemiological studies of strains of this genus.
