Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.
OBJECTIVES
A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.


METHODS
Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.


RESULTS
Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 10(4) colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.


CONCLUSION
This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.
['Park|Jie Yeun|JY|', 'Jeon|Semi|S|', 'Kim|Jun Young|JY|', 'Park|Misun|M|', 'Kim|Seonghan|S|']
Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus TaqMan probe multiplex real-time PCR