Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibrio parahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibrio parahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio parahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibrio parahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. parahaemolyticus. This PCR protocol clearly identified TDH-producing strains of V. parahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.
FeinbergA.P., VogelsteinB.1984; A technique for radio-labelling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 137:266–267
HondaT., NishibuchiM., MiwataniT., KaperJ.B.1986; Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-Ol strains. Applied and Environmental Microbiology 52:1218–1220
HondaT., NiY., YohM., MiwataniT.1989; Production of monoclonal antibodies against thermostable direct hemolysin of Vibrio parahaemolyticus and application of the antibodies for enzyme-linked immunosorbent assay. Medical Microbiology and Immunology 178:245–253
LeeC.Y., ChenL.H., LiuM.L., SuY.C.1992; Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters. Applied and Environmental Microbiology 58:3419–3422
MiyamotoY., KatoT., ObaraY., AkiyamaS., TakizawaK., YamaiS.1969; In vitro hemolytic characteristics of Vibrio parahaemolyticus: its close correlation with human pathogenicity. Journal of Bacteriology 100:1147–1149
MiyamotoT., MiwaH., HatanoS.1990; Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods. Applied and Environmental Microbiology 56:1480–1484
NakajimaH., InoueM., MoriT., ItohK.I., ArakawaE., WatanabeH.1992; Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method. Journal of Clinical Microbiology 30:2484–2486
NishibuchiM., KaperJ.B.1985; Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus. Journal of Bacteriology 162:558–564
NishibuchiM., IshibashiM., TakedaY., KaperJ.B.1985; Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other Vibrio species by the DNA colony hybridization test. Infection and Immunity 49:481–486
NishibuchiM., DokeS., ToizumuS., UmedaT., YohM., MiwataniT.1988; Isolation from a coastal fish of Vibrio hollisae capable of producing a hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus. Applied and Environmental Microbiology 54:2144–2146
NishibuchiM., TaniguchiT., MisawaT., Khaeomanee-I.V., HondaT., MiwataniT.1989; Cloning and nucleotide sequence of the gene (trh) encoding the hemolysin related to the thermostable direct hemolysin of Vibrio parahaemolyticus. Infection and Immunity 57:2691–2697
NishibuchiM., FasanoA., RussellR.G., KaperJ.B.1992; Enterotoxigenicity of Vibrio parahaemolyticus with and without genes encoding thermostable direct hemolysin. Infection and Immunity 60:3539–3545
PollardD.R., JohnsonW.M., LiorH., TylerS.D., RozeeK.R.1990; Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. Journal of Clinical Microbiology 28:2477–2481
SarkarB.L., KumarR., DeS.P., PalS.C.1987; Hemolytic activity of and lethal toxin production by environmental strains of Vibrio parahaemolyticus. Applied and Environmental Microbiology 53:2696–2698
ShiraiH., ItoH., HirayamaT., NakamotoY., NakabayashiN., KumagaiK., TakedaY., NishibuchiM.1990; Molecular epidemiologic evidence for association of thermostable direct hemolysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis. Infection and Immunity 58:3568–3573
ShiraiH., NishibuchiM., RamamurthyT., BhattacharyaS.K., PalS.C., TakedaY.1991; Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. Journal of Clinical Microbiology 29:2517–2521
SteinA., RaoultD.1992; Detection of Coxiella burnetii by DNA amplification using polymerase chain reaction. Journal of Clinical Microbiology 30:2462–2466
TadaJ., OhashiT., NishimuraN., ShirasakiY., OzakiH., FukushimaS., TakanoJ., NishibuchiM., TakedaY.1992; Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Molecular and Cellular Probes 6:477–487
TaniguchiH., OhtaH., OgawaM., MizuguchiY.1985; Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct haemolysin and thermolabile hemolysin genes. Journal of Bacteriology 162:510–515
TaniguchiH., KubomuraS., HiranoH., MizueK., OgawaM., MizuguchiY.1990; Cloning and characterization of a gene encoding a new thermostable haemolysin from Vibrio parahaemolyticus. FEMS Microbiology Letters 67:339–346
TomoyasuT.1992; Development of the immunomagnetic enrichment method selective for Vibrio parahaemolyticus serotype K and its application to food poisoning study. Applied and Environmental Microbiology 58:2679–2682
VictorT., Du ToitR., VanzylJ., BesterA.J., Van HeldenP.D.1991; Improved method for the routine identification of toxigenic Escherichia coli by DNA amplification of a conserved region of the heat-labile toxin A subunit. Journal of Clinical Microbiology 29:158–161
WidjojoatmodjoM.N., FluitA.C., TorensmaR., VerdonkG.P.H.T., VerhoefJ.1992; The magnetic immuno polymerase chain reaction assay for direct detection of Salmonellae in fecal samples. Journal of Clinical Microbiology 30:3195–3199
YamamotoK., HondaT., MiwataniT.1992; Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Canadian Journal of Microbiology 38:410–416
YohM., HondaT., MiwataniT.1986; Purification and partial characterization of a Non-Ol Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus. Infection and Immunity 52:319–322
YohM., HondaT., MiwataniT., TsunasawaS., SakiyamaF.1989; Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus. Journal of Bacteriology 171:6859–6861