Multiplex PCR for genotypingFlavobacterium columnare

Abstract

Recent research has identified four distinct genetic groups among isolates of Flavobacterium columnare through multilocus phylogenetic analyses; however, there are no quick methods to determine the genotype of an isolate. The objective of this research was to develop a multiplex PCR to rapidly genotype F. columnare to genetic group. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group, and primers were designed to specifically amplify different sized amplicons for each genetic group. The optimized assay was demonstrated to be specific for each genetic group and F. columnare, and no specific amplicons were generated using gDNA from a panel of other Flavobacterium spp. and bacterial fish pathogens. The analytical sensitivity of the assay ranged from 209 to 883 genome equivalents depending on the genetic group. The multiplex PCR was evaluated by genotyping a panel of 22 unknown F. columnare isolates and performing DNA sequencing of the dnaK gene in parallel. The results demonstrated 100% accordance between multiplex PCR results and assignment to genetic group via phylogenetic analysis. The multiplex PCR provides a useful tool for assigning an unknown isolate to genetic group and may be used to determine which genetic groups of F. columnare are circulating and most predominant in different aquaculture industries.

The Infection Dynamics of Experimental Edwardsiella ictaluri and Flavobacterium covae Coinfection in Channel Catfish (Ictalurus punctatus)

“…Presumptive F. covae recovered from dead fish were confirmed by multiplex PCR as described by [ 7 , 37 ]. Each 25 μL reaction contained a 12.5 µL 2× hot-start PCR-to-gel-master mix (Amresco LLC, Solon, OH, USA), 2 µL of the primer cocktail (0.5 µM GG-forward, 0.1 µM GG1-reverse, 0.45 µM GG2-reverse, 0.45 µM GG3-reverse, 0.3 µM GG4-reverse), 9.5 µL of nuclease-free water, and 1.0 µL of template DNA.…”

Ultraviolet light differentially reduces viability of fish‐ and fish farm‐associated flavobacteria (families Flavobacteriaceae and Weeksellaceae)

“…: C. aahli ( n = 1; Loch and Faisal 2014b), C. aquaticum ( n = 1; Kim et al 2008), and C. scophthalmum ( n = 1; VanDamme et al 1994). The remaining 30 isolates were newly identified as flavobacteria or F. columnare using previously published protocols (Loch et al 2013; LaFrentz et al 2019).…”

Coinfection by Group C Streptococcus dysgalactiae subsp. equisimilis and Flavobacterium davisii in Nile Tilapia from the United States

“…Briefly, gDNA (20 ng) from isolate 2K was used as template in the multiplex PCR, an appropriate positive control (consisting of a mixture of 10 ng gDNA each of F. columnare ATCC 23463 T , F. covae AL-02-36 T , F. davisii 90-106 T , and F. oreochromis Costa Rica 04-02-TN T ), and a no template negative control was included. PCR was performed as described by LaFrentz, García, and Shelley (2019). Following PCR, amplified products were resolved with 1.5% agarose gel electrophoreses and the size of the product was used for identification, with F. columnare, F. covae, F. davisii, and F. oreochromis yielding products with size of 415, 320, 894, and 659 bp, respectively.…”