Location: Aquatic Animal Health ResearchTitle: Multiplex PCR for rapid genotyping of Flavobacterium columnare Author
Submitted to: International Aquaculture Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 9/21/2018
Publication Date: 11/25/2018
Citation: LaFrentz, B.R., Garcia, J.C., Shelley, J.P. 2018. Multiplex PCR for rapid genotyping of Flavobacterium columnare [abstract]. 5th International Conference of Members of the Genus Flavobacterium 2018. p. 24.
Technical Abstract: Columnaris disease, caused by the Gram-negative bacterium Flavobacterium columnare, is one of the leading causes of disease losses to the catfish industry in the Southeast USA. Recent research in our laboratory has identified four distinct genetic groups among isolates of F. columnare; however, there is a lack of knowledge of which genetic group(s) is most prevalent in the industry. The objective of this research was to develop a multiplex PCR to rapidly genotype F. columnare and use the assay to determine the predominant genotype(s) in the catfish industry. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group to exploit for developing the assay. PCR primers were designed to amplify different sized amplicons specific for each genetic group. The designed primers were multiplexed and PCR conditions were optimized using purified gDNA as template. Once optimized, the multiplex PCR was tested on gDNA from a panel of F. columnare isolates of unknown genetic group and DNA sequencing was conducted in parallel. The results demonstrated 100% accordance between multiplex PCR results and assignment to genetic group. The multiplex PCR reported here provides a useful tool for rapidly assigning an unknown isolate to genetic group. Currently we are collaborating with various diagnostic laboratories in the Southeast USA, to determine which genetic groups of F. columnare are circulating and most predominant in the catfish industry. An understanding of this will allow for a more focused effort to develop efficacious control and treatment measures for columnaris disease.