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Development a diagnostic pan‐dermatophyte TaqMan probe real‐time PCR assay based on beta tubulin gene
Author(s) -
Mirhendi Hossein,
Motamedi Marjan,
Makimura Koichi,
Satoh Kazuo
Publication year - 2016
Publication title -
mycoses
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.13
H-Index - 69
eISSN - 1439-0507
pISSN - 0933-7407
DOI - 10.1111/myc.12502
Subject(s) - dermatophyte , taqman , primer (cosmetics) , biology , polymerase chain reaction , real time polymerase chain reaction , microbiology and biotechnology , dna , recombinant dna , plasmid , dna sequencing , gene , genetics , chemistry , organic chemistry
Summary 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 ( BT 2 ) 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 DNA s. Analytical detection limit of the designed PCR on serially diluted DNA s 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%) DNA s 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.