title: Combining ethidium monoazide treatment with real-time PCR selectively quantifies viable Batrachochytrium dendrobatidis cells.
abstract: Detection of the lethal amphibian fungus Batrachochytrium dendrobatidis relies on PCR-based techniques. Although highly accurate and sensitive, these methods fail to distinguish between viable and dead cells. In this study a novel approach combining the DNA intercalating dye ethidium monoazide (EMA) and real-time PCR is presented that allows quantification of viable B. dendrobatidis cells without the need for culturing. The developed method is able to suppress real-time PCR signals of heat-killed B. dendrobatidis zoospores by 99.9 % and is able to discriminate viable from heat-killed B. dendrobatidis zoospores in mixed samples. Furthermore, the novel approach was applied to assess the antifungal activity of the veterinary antiseptic F10(®) Antiseptic Solution. This disinfectant killed B. dendrobatidis zoospores effectively within 1 min at concentrations as low as 1:6400.
authors: Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Mark.Blooi@UGent.be
authors: Mark
authors: M
authors: Blooi
authors: An
authors: A
authors: Martel
authors: Francis
authors: F
authors: Vercammen
authors: Frank
authors: F
authors: Pasmans
mesh_terms: D001386:Azides / Q000737:chemistry*; D008411:Chytridiomycota / Q000737:chemistry / Q000235:genetics / Q000254:growth & development*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D007364:Intercalating Agents / Q000737:chemistry; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001386:Azides; D004271:DNA, Fungal; D007364:Intercalating Agents; C014535:8-azidoethidium