Characterization of a cloned pR72H probe for Vibrio parahaemolyticus detection and development of a nonisotopic colony hybridization assay.
Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan. A rapid and efficient detection method is required to identify this foodborne pathogen. A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V. parahaemolyticus strain no. 93, designated as pR72H fragment, was used as a polynucleotide probe. It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method. The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay. Under stringent hybridization conditions, 122 of 124 isolates of V. parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth. The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms. The sensitivity and specificity of this DIG probe are 98% and 100%, respectively. This nonisotopic colony hybridization assay can be very useful for routine monitoring of V. parahaemolyticus in the food industry, environmental analysis and clinical laboratories.
Lee
C Y
CY
Graduate Institute, National Taiwan University, Taipei, Republic of China.
Chen
C H
CH
Chou
Y W
YW
D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000382:microbiology; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012680:Sensitivity and Specificity; D014735:Vibrio Infections / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification*
D015342:DNA Probes; D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin