An rpoB gene-based PCR-DGGE method for simultaneous detection of multiple Vibrio species

Using PCR-denaturing gradient gel electrophoresis (DGGE) targeting the RNA polymerase beta sub-unit (rpoB) gene,a simultaneous detection method for Vibrio species was established,rpoB gene-basedPCR-DGGE was carried out with eight Vibrio reference strains (each from different species),mixed sam-ple (including these Vibrio reference strains),two non Vibrio strains,four environmental Vibrio strains,and three unidentified environmental strains.For comparison,16S rRNA gene-based PCR-DGGE of theeight Vibrio reference strains was performed with universal primers.In addition,three unidentified strainswere identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accu-racy of rpoB gene-based PCR-DGGE detection.Results revealed that rpoB-based PCR-DGGE could welldiscriminate eight Vibrio reference strains and could not discriminate different strains within the samespecies.The bands derived from two non Vibrio strains could not match with any bands in referencemarker.Meanwhile,16S rRNA gene-based DGGE failed to distinguish these reference strains.Further-more,four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE.Se-quencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE.The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficientmethod for simultaneous detection of multiple Vibrio species,which can avoid the limitations inherent in16S rRNA gene-based PCR-DGGE.