title: Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.
abstract: Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.
authors.affiliation: Departments of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.|Departments of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Mirhendi
authors.affiliation: Departments of Medical Parasitology & Mycology, School of Public Health, National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Marjan
authors.initials: M
authors.lastname: Motamedi
authors.affiliation: Teikyo University Institute of Medical Mycology, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Makimura
authors.affiliation: Teikyo University Institute of Medical Mycology, Tokyo, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Satoh
mesh_terms: D003883:Arthrodermataceae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal / Q000235:genetics; D006801:Humans; D057230:Limit of Detection; D009262:Nails / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology; D014005:Tinea / Q000175:diagnosis* / Q000382:microbiology; D014404:Tubulin / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D014404:Tubulin
keywords: Pan-dermatophyte; TaqMan probe; beta tubulin gene; real-time PCR