Identification ofFlavobacteriumandFlexibacterSpecies by Species-Specific Polymerase Chain Reaction Primers to the 16S Ribosomal RNA Gene

Abstract

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare (Flexibacter columnaris) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum (Flexibacter psychrophila), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter maritimus (400 bp). These primers were shown to correctly speciate field isolates in double-blind experiments (P = 0.01).