Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea agents of chromoblastomycosis

Abstract

Loop-mediated isothermal amplification (LAMP) was developed for rapid detection of pathogenic or allergenic fungal in the environment. Primers applied were derived from the rDNA Internal Transcribed Spacer and the 5.8S rRNA gene. The assay enabled amplification of target fungi at the level of genus or closely related species using pure cultures after 1h reaction at 65 degrees C in a water bath. No cross-reactivity to related species was observed. The DNA detection limit was 0.2fg. The method also proved to work well with fungi on non-sterile adhesive tape. Amplification products were detected by visual inspection using SYBR Green I as well as by electrophoresis on agarose gels. As a model organism we selected Fonsecaea, a fungal genus containing etiologic agents of chromoblastomycosis, a widely distributed tropical and subtropical skin disease in otherwise healthy patients and supposed to be acquired by environmental inoculation. It is suggested that LAMP can also be used for rapid clinical diagnosis, for environmental detection, and for retrospective studies in archived clinical samples.