Location: Aquatic Animal Health ResearchTitle: Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene
|MABROK, M - Chulalongkorn University|
|PUTITA, C - Chulalongkorn University|
|KAYANSAMRUAJ, P - Kasetsart University|
|DONG, HT - Chulalongkorn University|
|RODKHUM, C - Chulalongkorn University|
Submitted to: Aquaculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/11/2019
Publication Date: 3/20/2020
Citation: Mabrok, M., Putita, C., Lafrentz, B.R., Kayansamruaj, P., Dong, H., Rodkhum, C. 2020. Development of a species-specific polymerase chain reaction for highly sensitive detection of Flavobacterium columnare targeting chondroitin AC lyase gene. Aquaculture. 521(2020):734597. https://doi.org/10.1016/j.aquaculture.2019.734597.
Interpretive Summary: Flavobacterium columnare, the causative agent of columnaris disease is considered one of the most threatening pathogens limiting the culture of many fish species. Several polymerase chain reaction assays (PCR) have been developed for the detection of F. columnare using primers specific for the 16S rRNA gene. However, many of these assays lack specificity due to variability in the sequences of this gene among isolates representative of diverse genetic backgrounds. The objective of this research was to develop a new PCR assay for the specific detection of F. columnare. PCR primers were developed based on the sequence of the chondroitin AC lyase gene (cslA). Optimization and testing of the assay determined that the assay was specific to F. columnare isolates of diverse genetic backgrounds and was highly sensitive. This new PCR assay provides a specific and sensitive tool for the detection and confirmation of isolates as F. columnare.
Technical 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.