16S RIBOSOMAL-RNA-TARGETED RT-PCR FOR THE DETECTION OF VIBRIO-PENAEICIDA, THE PATHOGEN OF CULTURED KURUMA PRAWN PENAEUS-JAPONICUS

Vibrio penaeicida is the causative bacterium of vibriosis in cultured kuruma prawn Penaeus japonicus in Japan. To develop a specific and sen sitive method for the detection of the pathogen, a species-specific se quence in the 16S rRNA of V. penaeicida was determined and a polymeras e chain reaction (PCR)-based method was devised on the basis of the se quence. Prior to sequencing, a part of the variable regions of the 16S rRNA was amplified by using primers designed from 2 conserved regions according to previously reported data on Vibrionaceae. The region of the 16S rRNA (nucleotide numbers 440 to 490 in Escherichia coli 16S rR NA) obtained by this procedure was found to be species-specific for V. penaeicida. It was confirmed that PCR and RT (reverse transcription)- PCR amplifications with a sense primer designed from the V. penaeicida -specific sequence were both able to differentiate V. penaeicida from other prawn-pathogenic vibrios. 16S rRNA-targeted RT-PCR was demonstra ted to have 100 times higher sensitivity than 16S rDNA-targeted PCR an d 10 fg of total nucleic acids extracted from cultured bacterial cells was sufficient to yield the visible fragment in gel electrophoresis. These results indicate that RT-PCR amplification with this primer is u seful for specific and sensitive detection of V. penaeicida.