Mirhendi Hossein, Motamedi Marjan, Makimura Koichi, Satoh Kazuo
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.
Mycoses. 2016 Aug;59(8):520-7. doi: 10.1111/myc.12502. Epub 2016 Apr 13.
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.
尽早将皮肤癣菌病与其他皮肤真菌病区分开来对于避免不恰当的治疗至关重要。包括实时PCR在内的基于DNA的技术越来越多地被用于检测临床标本中的真菌成分。在本研究中，对13种常见和罕见的致病性皮肤癣菌进行β微管蛋白（BT2）基因的部分序列分析后，设计了一种基于TaqMan探针的PCR格式的泛皮肤癣菌引物和探针组。用22株皮肤癣菌参考菌株、234份阳性临床标本、从正常指甲提取的32份DNA样本、几种非皮肤癣菌的真菌和人类DNA对该系统的敏感性和特异性进行了测试。对制备的重组质粒的系列稀释DNA进行设计的PCR分析检测限表明，每个样本仅五个分子是该检测方法可靠检测的最小数量。从临床样本中提取的234份DNA中有226份（96.5%）呈PCR阳性，但来自健康志愿者的32份指甲样本均无阳性。本研究建立的靶向β微管蛋白基因的实时PCR可能是一种敏感的诊断工具，比传统培养方法显著更快，在临床环境、大规模流行病学研究和抗真菌治疗临床试验中应具有实用价值。
