Characterization of a Cloned pR72H Probe for Vibrio parahaemolyticus Detection and Development of a Nonisotopic Colony Hybridization Assay

Abstract

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.

Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR

“…These results indicate that the oligonucleotide primers VP33 and VP32, as well as the PCR procedure, were adequate for specific detection of V. parahaemolyticus. These results are also in agreement with our previous DNA hybridization results (28), which indicated that the reported nucleotide sequence of the insert of V. parahaemolyticus 93, pR72H, is conserved in all V. parahaemolyticus strains.…”

“…DNA sequence determination. Plasmid pR72 is a pUC119 derivative containing a 0.76-kb HindIII insert of V. parahaemolyticus 93, and the insert DNA fragment is designated pR72H (28). Large-scale plasmid preparation and purification were performed by a modified alkaline lysis procedure and polyethylene glycol precipitation protocol (39).…”

“…This cloned DNA fragment, designated pR72H, can be used as a species-specific DNA probe for V. parahaemolyticus. Although the function of this DNA fragment is still unknown, the pR72H DNA fragment is conservative in all strains of V. parahaemolyticus tested (28). In the present study, we determined the sequence of pR72H and developed a successful approach for detection of V. parahaemolyticus in shellfish by using enrichment culturing with a PCR assay.…”

Fatty acid profiles of Vibrio parahaemolyticus and its changes with environment

“…All strains were identified by 16S rDNA using a pair of common primers (27F/1492R). The specific primer pair VP33/VP32, derived from a pR72H fragment, which is conserved in all strains of V. parahaemolyticus was also used to identify V. parahaemolyticus .…”

Identification of Vibrio parahaemolyticus Strains at the Species Level by PCR Targeted to the toxR Gene

“…These genes appear to be fairly specific to V. parahaemolyticus. Lee et al (6,7) cloned a 0.76-kb nucleotide sequence of unknown function and claimed that the nucleotide sequence is specific to V. parahaemolyticus by hybridization and PCR methods (6,7). However, only limited numbers of strains were examined in those studies, and the relationships of the hemolysin genes and the 0.76-kb sequence with the phylogeny of V. parahaemolyticus are not known.…”