Differential Identification of Flavobacterium Species by Sequence Analysis of Genus-Specific Hypervariable 16S-23S rDNA Intergenic Spacer Target

The aim of the current study was to develop a molecular system for differential identification of various fish diseases caused by Flavobacterium species. The system uses the hypervariability of the 16S-23S rDNA Intergenic spacer region (ISR) to develop PCR-based sequence analysis assay. For this purpose, eight different 16S-23S rDNA ISR sequences of six different Flavobacterium species were aligned and compared to detecta hypervariable region with conserved flanking sequences as a target for amplification. The conserved flanking regions were used to design primers that target the selected hypervariable ISR sequence from all Flavobacterium species. American Type Collection of Cultures and other reference strains were used to assess the precision and specificity of the system. The results revealed the ability of the described molecular assay to accurately identify all the ATCC and reference Flavobacterium species to the strain level. In addition, two clinical isolates that were conventionally identified in the current study as Flavobacterium psychrophilum and Flavobacterium columnare were re-identified, using the molecular assay as F. columnare and Flavobacterium johnsoniae, respectively. The currently described ISR sequence analysis-based differential identification assay provides rapid and accurate identification of the different diseases causing Flavobacterium species and represent a useful tool for successful epidemiological studies and management of Flavobacterium speciescaused fish diseases.