Use of random amplification to develop a PCR detection method for the causative agent of fish pasteurellosis, Photobacterium damselae subsp. piscicida (Vibrionaceae)

Abstract

Random amplification of polymorphic DNA (RAPD) was used to generate species-specific markers, which were exploited in the construction of a fast and convenient PCR-based diagnostic assay for the causative agent of fish pasteurellosis or pseudotuberculosis, the bacterium Photobacterium damselae subsp. piscicida, formerly known as Pasteurella piscicida. In RAPD genomic fingerprints of this bacterium, two distinctive fragments were observed. These fragments were cloned, sequenced and found to bear no significant homology with known DNA sequences. Two sets of primers were then synthesized to amplify, by PCR, fragments within the sequences between the terminal RAPD primers of the fragments. Another primer pair was designed to perform nested PCR internally to one of the amplified fragments. The species specificity of the PCR products was analyzed, by dot blotting and Southern hybridization, with DNA from 13 other bacterial species. Cross-reactivity was obtained only with isolates of P. damselae subsp. damselae (formerly Vibrio damselae) a bacterial species with a high degree of overall genomic homology with P. d. piscicida. The detection limit of the assay was 20 fg of template DNA by PCR and 2 fg by nested PCR. The assay was effective in revealing P. d. piscicida when applied to an aliquot of DNA extracted from 50-mg samples of kidney, spleen and liver tissues from naturally infected fish. In conclusion, these results demonstrate the utility of the RAPD plus PCR approach in the development of genetic diagnostic assays for fish pathogens.