Differentiation of <i>Aeromonas</i> genomospecies using random amplified polymorphic DNA polymerase chain reaction (RAPD‐PCR)

Oakey, H.J.; Ellis, John; Gibson, L. F.

doi:10.1111/j.1365-2672.1996.tb03235.x

Cited by 33 publications

(37 citation statements)

References 28 publications

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“…Several bacterial species have also been differentiated from closely related species using particular RAPD primers. Examples among fish-pathogenic bacteria include primers generating fingerprints which clearly differentiated Vibrio salmonicida, V. anguillarum, and V. fisheri (Martinez et al 1994) as well as different Aeromonas species isolated from fish (Miyata et al 1995) or from widely different clinical and environmental sources (Oakey et al 1996). Because very few template DNA and no previous knowledge of nucleotide sequence are required, and because the whole genome is analyzed (Oakey et al 1996), RAPD has several advantages over the other techniques that have been used alone or in various combinations for identifying Flavobactenum psychrophilun?, such a s phenotypical investigations (Holt 1987), DNA/DNA hybridization (Bernardet & Grirnont 1989), serological tests revealing specific antigens (Pacha 1968, Holt 1987, Cipriano et al 1996, wholecell protein (Bernardet et al 1996, Cipriano et al 1996 or fatty acid analyses (Bernardet et al 1996), a n d 1 6 s rDNA-targeted PCR (Toyama e t al.…”

Section: Identification Of F Psychrophilum With This Techniquementioning

confidence: 99%

Random Amplified Polymorphic DNA analysis provides rapid differentiation among isolates of the fish pathogen Flavobacterium psychrophilum and among Flavobacterium species

Chakroun¹,

Urdaci²,

Faure³

et al. 1997

Dis. Aquat. Org.

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Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish. Originally isolated in North Amenca only, the bacterium is now also responsible for severe mortalities in many salmonid hatcheries in Europe, as well as in Japan, Chile and Tasmania. The random amplified polymorphic DNA (RAPD) technique was used to analyze the genetic diversity among a collection of 177 F. psychrophilum strains isolated from different fish species and in different geographical areas. Forty 10-mer primers were tested and 5 of them were selected for further analysis of the bacterial DNAs. The primers OPH 06, OPH 08, OPG 08, OPG 14, and OPG 16 generated several reproducible profiles during a preliminary screening of the whole collection of stralns. Based on these results, the polyrnerase chain reaction (PCR) products of a selection of 60 bacterial DNAs were submitted to slow agarose gel electrophoresis for numerical analysis of the DNA fingerprintings. No correlation occurred between the combined RAPD profiles of the primers and the geographical origin of the strains, while some profiles were clearly associated with the fish species from which the strains were isolated. Another primer, OPG 10, yielded a unique RAPD profile common to all F. psychrophilum strains whereas the 9 other valld Flavobactenum species, several of which coexist in freshwater environments and may also be isolated from fish, displayed other profiles. Thus, depending on the primer used, both the typing of F. psychrophilum strains for epidemiology studies as well as the identification of this fish pathogen and its differentiation from related bacterial species could be achieved by using RAPD.

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Section: Identification Of F Psychrophilum With This Techniquementioning

confidence: 99%

Random Amplified Polymorphic DNA analysis provides rapid differentiation among isolates of the fish pathogen Flavobacterium psychrophilum and among Flavobacterium species

Chakroun¹,

Urdaci²,

Faure³

et al. 1997

Dis. Aquat. Org.

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“…The RAPD method involves the use of short random sequence primers, usually 9 to 10 nucleotides long, and low-stringency primer annealing conditions to amplify arbitrary fragments of template DNA. The single primer anneals anywhere on the genome where a near-complementary sequence exists, and if two priming sites are sufficiently close, PCR then amplifies the fragment between them (27). The following primers were used for RAPD typing: OPB-1 5Ј-GTTTCGCTCC-3Ј (27), OPB-6 5Ј-TGCTCTGCCC-3Ј (27), OPB-7 5Ј-GGT GACGCAG-3Ј (27), AP3 5Ј-TCACGATGCA-3Ј (35), AP5 5Ј-TCACGCTGC G-3Ј (35), AK1 5Ј-ATCACTATGA-3Ј, AK2 5Ј-GATCCTGCAG-3Ј, and AK3 5Ј-TAAGGTTCGG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…The following primers were used for RAPD typing: OPB-1 5Ј-GTTTCGCTCC-3Ј (27), OPB-6 5Ј-TGCTCTGCCC-3Ј (27), OPB-7 5Ј-GGT GACGCAG-3Ј (27), AP3 5Ј-TCACGATGCA-3Ј (35), AP5 5Ј-TCACGCTGC G-3Ј (35), AK1 5Ј-ATCACTATGA-3Ј, AK2 5Ј-GATCCTGCAG-3Ј, and AK3 5Ј-TAAGGTTCGG-3Ј. RAPD PCR mixtures were prepared as previously described (27). The reaction mixture consisted of 4 l of 10ϫ reaction buffer [750 mM Tris-HCl (pH 8.8), 200 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20], a 250 M concentration of each deoxynucleoside triphosphate (dNTP), 50 pmol of a primer, 3.75 mM MgCl 2 , 35 ng of template DNA, and 2 U of Taq polymerase (MBI Fermentas) made up to 40 l with sterile distilled water.…”

Section: Methodsmentioning

confidence: 99%

Typing of Clinical and Environmental Aeromonas sp. Strains by Random Amplified Polymorphic DNA PCR, Repetitive Extragenic Palindromic PCR, and Enterobacterial Repetitive Intergenic Consensus Sequence PCR

Szczuka

Kaznowski

2004

J Clin Microbiol

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A collection of 120 strains isolated from stool specimens collected from humans suffering from gastroenteritis and from environmental samples were analyzed by random amplified polymorphic DNA PCR (RAPD), repetitive extragenic palindromic PCR (REP-PCR), and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). Species of Aeromonoas hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, and A. veronii revealed clonal structure. There was no dominant clone causing gastroenteritis in humans. Moreover, there was no genetic similarity between clinical and environmental strains of Aeromonas sp. isolated from different geographical areas as well as from the same geographical area. Some clones colonized specific ecosystems, e.g., drinking water distribution systems. RAPD and ERIC-PCR methods had the same discriminatory power and proved to be useful for epidemiological investigation and population genetic analysis of Aeromonas spp., whereas REP-PCR was less effective for differentiating the isolates of Aeromonas spp.Bacteria of Aeromonas sp. are gram-negative, straight cells (rod-shaped to coccoid) with rounded ends. They are oxidase and catalase positive, reduce nitrate to nitrite, and ferment D-glucose. These bacteria are widely spread in the environment, especially in surface water and sewage; they also occur in untreated and treated drinking water (1, 2, 4, 18). In humans, Aeromonas spp. are responsible for gastroenteritis, chronic diarrhea, wound infections, respiratory tract infections, peritonitis, urinary tract infections, and septicemia (2, 17). Among Aeromonas-associated infections of humans A. hydrophila, A. caviae, and A. veronii are the predominating species, whereas A. eucrenophila, A. popoffii, (2), and A. culicicola (28) have never been found in clinical samples. Some Aeromonas species are associated with a wide variety of diseases in cold-and warm-blooded animals, including fish, frogs, water buffaloes, reptiles, birds, and cattle (16, 28). A. veronii, A. hydrophila, and A. salmonicida are capable of causing septicemia in freshwater and marine fish (3, 9). Clinical and environmental Aeromonas sp. isolates secrete many extracellular products, such as hemolysins, enterotoxins, and proteases. Studies conducted by Kühn et al. (22) showed that some isolates of a given species produce virulence factors more frequently than others. These findings indicate that the virulence within the genus Aeromonas might be a clonal property and only some clones may be responsible for progressive disease. However, there have been no studies that would have determined clonal structure within Aeromonas spp. and the spread of specific clones in human population and in the environment. Our study was undertaken to recognize the clonal relatedness of strains derived from diarrheal stool specimens collected from humans living in different geographical areas. We also compared genetic similarities of clones recovered from stool and the environment. In addition, we attempted to determine the genetic relati...

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“…Methods like randomly ampli¢ed polymorphic DNA (RAPD) analysis [5,6] and pulsed-¢eld gel electrophoresis (PFGE) [7,8] are very useful for individual typing of Aeromonas isolates but are either too sensitive to be used in genomic species discrimination or have not yet been tested throughout the entire genus. Analysis of sequence polymorphisms in ribosomal DNA (ribotyping) has been successfully used for both epidemiological and taxonomic investigations in Aeromonas [9,10].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

Huys

1999

FEMS Microbiology Letters

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A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radioisotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLPgenerated data with DNA-DNA homology data. ß

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Differentiation of Aeromonas genomospecies using random amplified polymorphic DNA polymerase chain reaction (RAPD‐PCR)

Cited by 33 publications

References 28 publications

Random Amplified Polymorphic DNA analysis provides rapid differentiation among isolates of the fish pathogen Flavobacterium psychrophilum and among Flavobacterium species

Random Amplified Polymorphic DNA analysis provides rapid differentiation among isolates of the fish pathogen Flavobacterium psychrophilum and among Flavobacterium species

Typing of Clinical and Environmental Aeromonas sp. Strains by Random Amplified Polymorphic DNA PCR, Repetitive Extragenic Palindromic PCR, and Enterobacterial Repetitive Intergenic Consensus Sequence PCR

Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

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