In this paper, we developed a rapid and accurate method for the detection of Vibrio
parahaemolyticus strains, using multiplex PCR and DNA-DNA hybridization. Multiplex
PCR was used to simultaneously amplify three diagnostic genes (tlh, tdh and fla) that
serve as molecular markers of V. parahaemolyticus. Biotinylated PCR products were
hybridized to primers immobilized on a microarray, and detected by chemiluminesce
with avidin-conjugated alkaline phosphatase. With this method, forty-five samples
were tested. Eight known virulent strains (tlh(+)/tdh(+)/fla(+)) and four known avirulent
strains (tlh(+)/tdh(-)/fla(+)) of the V. parahaemolyticus were successfully detected,
and no non-specific hybridization and cross-hybridization reaction were found from
fifteen closely-related strains (tlh(-)/tdh(-)/fla(+)) of the Vibrio spp. In addition,
all the other eighteen strains of non-Vibrio bacteria (tlh(-)/tdh(-)/fla(-)) gave
negative results. The DNA microarray successfully distinguished V. parahaemolyticus
from other Vibrio spp. The results demonstrated that this was an efficient and robust
method for identifying virulent strains of V. parahaemolyticus.
Copyright © 2011. Published by Elsevier Ltd.