Rapid and low-level toxic PCR-based method for routine identification of Flavobacterium psychrophilum.
We describe a rapid, low-toxicity and simple method for the detection of the bacterial fish pathogen Flavobacterium psychrophilum. The method, based on the polymerase chain reaction (PCR), combined the electrophoresis of PCR products in a vertical agarose gel and a modified methylene blue stain. DNA was amplified directly either from bacterial suspensions or from tissues experimentally infected with F. psychrophilum, using different non-toxic commercial DNA extraction kits. The protocol allowed to detect 15 to 150 cells of the pathogen in bacterial suspension, without prior DNA extraction, and 7500 to 75,000 cells in seeded spleen tissue and ovarian fluid using Dynabeads DNA DIRECT extraction system. This method, which has the advantage of not using hazardous products, is proposed as a fast tool for routine identification of F. psychrophilum.
Cepeda
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Department of Microbiology and Parasitology, Faculty of Biology, University of Santiago de Compostela, Spain. mpcatuxa@usc.es
Santos
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D000818:Animals; D004269:DNA, Bacterial / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005393:Fish Diseases / Q000382:microbiology*; D005417:Flavobacterium / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D008751:Methylene Blue / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D008751:Methylene Blue