Rezaei-Matehkolaei A, Makimura K, Shidfar Mr, Zaini F, Eshraghian Mr, Jalalizand N, Nouripour-Sisakht S, Hosseinpour L, Mirhendi H
Dept. of Medical Parasitology & Mycology, School of Public Health, National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran.
Iran J Public Health. 2012;41(3):82-94. Epub 2012 Mar 31.
Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes.
The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis.
The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum) and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings.
It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.
皮肤癣菌是浅表真菌病最常见的病原体。从治疗和流行病学角度来看，对这些真菌进行物种鉴定很重要。传统的基于菌落宏观和微观特征在物种水平上鉴定皮肤癣菌的方法，在很多情况下通常既耗时又不可靠。最近，多种快速准确的基于DNA的技术成功用于皮肤癣菌的物种划分。
使用通用真菌引物ITS1和ITS4扩增皮肤癣菌各参考菌株rDNA的ITS1-5.8S-ITS2区域。PCR产物用单一限制性内切酶MvaI消化。在计算机模拟和实际PCR-RFLP分析中对该酶进行评估，以找到每个物种确切的鉴别限制性图谱。为验证标准化的PCR-RFLP系统，对所有测试菌株进行测序和序列分析。
获得的RFLP模式对许多物种具有特异性，包括指间毛癣菌、红色毛癣菌、紫色毛癣菌、变色毛癣菌、奥杜盎小孢子菌、断发毛癣菌（钝顶棒状杆菌）和絮状表皮癣菌，但对一些密切相关的物种如犬小孢子菌/铁锈色小孢子菌则相似。对所有模式菌株的ITS1-5.8S-ITS2片段进行测序证实了RFLP结果。
实际表明，ITS-PCR后进行MvaI-RFLP是一种用于鉴定和区分几种致病物种的有用且可靠的方案，可用于临床和流行病学环境中对甚至密切相关的皮肤癣菌物种进行快速筛查。
