Department of Dermatology and Allergology, Ludwig Maximilians University of Munich, Munich, Germany.
Mycoses. 2011 Mar;54(2):137-45. doi: 10.1111/j.1439-0507.2009.01771.x.
Identification of dermatophytes is usually based on morphological characteristics determined by time-consuming microscopic and cultural examinations. An effective PCR-ELISA method has been developed for rapid detection of dermatophyte species directly from clinical specimens within 24 h. Isolated genomic DNA of skin scrapings and nail samples from patients with suspected dermatophyte infections is amplified with species-specific digoxigenin-labelled primers targeting the topoisomerase II gene. The subsequent ELISA procedure with biotin-labelled probes allows a sensitive and specific identification of the five common dermatophytes -Trichophyton rubrum, T. interdigitale, T. violaceum, Microsporum canis and Epidermophyton floccosum. PCR-ELISA, based on the new polyphasic species concept, was assessed using 204 microscopy-positive samples in two university mycological laboratories in Munich and Tübingen, and 316 consecutive specimens - regardless of mycological findings - in a dermatological practice laboratory in Neu-Ulm. One of the five dermatophytes was confirmed by PCR-ELISA in 163 of 204 (79.9%) of the clinical samples from the university hospitals found positive using microscopy. Culture was positive for dermatophytes in 59.8% of the same cases. A significant difference between these two methods could be demonstrated using the McNemar test (P < 0.005). Analysis of specimens from Neu-Ulm confirmed the results in a dermatological practice laboratory as 25.0% of the specimens had positive PCR results, whereas only 7.3% were positive according to culture. Direct DNA isolation from clinical specimens and the PCR-ELISA method employed in this study provide a rapid, reproducible and sensitive tool for detection and discrimination of five major dermatophytes at species level, independent of morphological and biochemical characteristics.
真菌的鉴定通常基于耗时的显微镜和培养检查来确定形态特征。已经开发了一种有效的 PCR-ELISA 方法，可直接从临床标本中在 24 小时内快速检测真菌种属。从疑似真菌感染的患者的皮肤刮屑和指甲样本中分离基因组 DNA，并用针对拓扑异构酶 II 基因的种特异性地高辛标记引物进行扩增。随后的 ELISA 程序与生物素标记的探针结合使用，可实现对五种常见真菌——红色毛癣菌、须癣毛癣菌、紫色毛癣菌、犬小孢子菌和絮状表皮癣菌的敏感和特异性鉴定。基于新的多相种概念的 PCR-ELISA 使用慕尼黑和图宾根的两所大学真菌学实验室的 204 个显微镜阳性样本以及纽伦堡皮肤科实践实验室的 316 个连续样本（无论真菌学发现如何）进行了评估。在使用显微镜检查发现阳性的 204 个临床样本中，PCR-ELISA 证实了其中的 163 个（79.9%）来自大学医院的五种真菌中的一种。在相同的情况下，培养法对真菌的阳性率为 59.8%。McNemar 检验表明这两种方法之间存在显著差异（P<0.005）。对纽伦堡标本的分析在皮肤科实践实验室中证实了结果，25.0%的标本 PCR 结果为阳性，而根据培养法只有 7.3%为阳性。直接从临床标本中分离 DNA 并采用本研究中使用的 PCR-ELISA 方法可提供一种快速、可重复和敏感的工具，用于在种属水平上检测和区分五种主要真菌，而不受形态和生化特征的影响。
