Optimized reverse primer for 16S‐<scp>RFLP</scp> analysis and genomovar assignment of <i>Flavobacterium columnare</i>

LaFrentz, Benjamin R.; García, Julio C.; Dong, Ha Thanh; Waldbieser, Geoffrey C.; Wong, F S; Chang, Shan‐Chwen

doi:10.1111/jfd.12583

Cited by 17 publications

(15 citation statements)

References 15 publications

(26 reference statements)

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“…Strains from the different genomovars affect geographically distributed fish species at different virulence levels (Arias et al, 2004; Darwish and Ismaiel, 2005; Shoemaker et al, 2008), Genomovar II strains tend to be more virulent than genomovar I strains in most fish species (Olivares-Fuster et al, 2011; Lafrentz et al, 2012), but there is considerable strain variation in virulence within the genomovars. Recently, a standard and optimized protocol was developed to distinguish F. columnare isolates using expected restriction patterns for genomovars I, II, II-B, and III (Lafrentz et al, 2014, 2016). …”

Section: Introductionmentioning

confidence: 99%

Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512

et al. 2017

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Flavobacterium columnare is a Gram-negative fish pathogen causing columnaris disease in wild and cultured fish species. Although the pathogen is widespread in aquatic environments and fish worldwide, little is known about biology of F. columnare and mechanisms of columnaris disease pathogenesis. Previously we presented the complete genome sequence of F. columnare strain ATCC 49512. Here we present a comparison of the strain ATCC 49512 genome to four other Flavobacterium genomes. In this analysis, we identified predicted proteins whose functions indicate F. columnare is capable of denitrification, which would enable anaerobic growth in aquatic pond sediments. Anaerobic growth of F. columnare ATCC 49512 with nitrate supplementation was detected experimentally. F. columnare ATCC 49512 had a relatively high number of insertion sequences and genomic islands compared to the other Flavobacterium species, suggesting a larger degree of horizontal gene exchange and genome plasticity. A type VI subtype III secretion system was encoded in F. columnare along with F. johnsoniae and F. branchiophilum. RNA sequencing proved to be a valuable technique to improve annotation quality; 41 novel protein coding regions were identified, 16 of which had a non-traditional start site (TTG, GTG, and CTT). Candidate small noncoding RNAs were also identified. Our results improve our understanding of F. columnare ATCC 49512 biology, and our results support the use of RNA sequencing to improve annotation of bacterial genomes, particularly for type strains.

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Section: Introductionmentioning

confidence: 99%

Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512

et al. 2017

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“…Triyanto and Wakabayashi () developed a 16S rRNA restriction fragment length polymorphism (RFLP) assay and established three genetic types or genomovars (genomovar I, II and III). The RFLP assay has been standardized (LaFrentz, Waldbieser, Welch, & Shoemaker, ) and optimized (LaFrentz et al., ), and currently, five genomovars have been described (I, I/II, II, II‐B and III).…”

Section: Description Of Flavobacterium Columnare Isolates Used In Thimentioning

confidence: 99%

“…LaFrentz et al. () reported similar PCR failure for some isolates, which was attributed to nucleotide diversity at the reverse primer binding site, and an optimized new reverse primer (1500R‐1) was designed. The PCR was repeated using the optimized reverse primer as described by LaFrentz et al.…”

Section: Description Of Flavobacterium Columnare Isolates Used In Thimentioning

confidence: 99%

“…The PCR was repeated using the optimized reverse primer as described by LaFrentz et al. (), and a robust amplicon of the appropriate size (~1,250 bp) was obtained for TI982 and the other isolates. Following digestion of the amplicons with HaeIII and electrophoresis, isolates TI982 and EE923 generated a restriction pattern comprised of DNA bands with size of 616, 291, 276 and 71 bp, corresponding to genomovar II (Figure ).…”

Section: Description Of Flavobacterium Columnare Isolates Used In Thimentioning

confidence: 99%

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Characterization of atypical Flavobacterium columnare and identification of a new genomovar

García

LaFrentz

Waldbieser³

et al. 2018

Journal of Fish Diseases

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“…In the original description of the assay, three different genotypes (referred to as genomovars) were described (Triyanto & Wakabayashi, ). Subsequent research identified additional genotypes (Olivares‐Fuster, Shoemaker, Klesius, & Arias, ), and following standardization of the technique and examination of additional isolates, six different genotypes were formally described (García, LaFrentz, Waldbieser, Wong, & Chang, ; LaFrentz et al, ; LaFrentz, Waldbieser, Welch, & Shoemaker, ). This genotyping method was the foundation for numerous studies investigating host associations, virulence, etc., among isolates from the different genetic groups (Evenhuis & LaFrentz, ; LaFrentz, LaPatra, Shoemaker, & Klesius, ; Olivares‐Fuster, Baker, et al, ; Olivares‐Fuster, Bullard, McElwain, Llosa, & Arias, ; Shoemaker & LaFrentz, ; Shoemaker, Olivares‐Fuster, Arias, & Klesius, ).…”

Section: Introductionmentioning

confidence: 99%

Multiplex PCR for genotypingFlavobacterium columnare

LaFrentz

García

Shelley

2019

Journal of Fish Diseases

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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.

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Optimized reverse primer for 16S‐RFLP analysis and genomovar assignment of Flavobacterium columnare

Cited by 17 publications

References 15 publications

Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512

Comparative Genomics and Transcriptional Analysis of Flavobacterium columnare Strain ATCC 49512

Characterization of atypical Flavobacterium columnare and identification of a new genomovar

Multiplex PCR for genotypingFlavobacterium columnare

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