Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA–DNA hybridization on a microarray

Abstract

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.