LAUSR

Abstract Columnaris disease, caused by Flavobacterium columnare, is an acute infection of gills and fins, that exists worldwide and causes remarkable losses in freshwater fish. The 16S rRNA gene is not a good candidate for primer design, since this gene has high identity among species in the same genus. In the present study, we developed a species-specific PCR for detection of Flavobacterium columnare based on the chondroitin AC lyase (cslA) gene. The cslA gene sequence from 13 F. columnare strains were aligned to design a specific primer set, Fcol-F and Fcol-R, based on conserved region of the gene. The new primer set produced a specific amplicon of 287 bp from 83 isolates of F. columnare but not from related or other bacterial pathogens. The PCR was sensitive and detected up to 3 pg of genomic DNA and as few as 7 colony-forming units of bacteria. The sensitivity was decreased tenfold when F. columnare gDNA was spiked into tissue samples. The designed primers precisely amplified F. columnare from skin and gills of infected fish, but not in those of clinically healthy fish, reflecting their possible use for diagnostic purposes.