Kamiya Atsushi, Kikuchi Akihiko, Tomita Yasushi, Kanbe Toshio
Department of Dermatology, Center for Neural Disease and Cancer, Nagoya Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, Aichi,466-8550, Japan.
J Dermatol Sci. 2004 Feb;34(1):35-48. doi: 10.1016/j.jdermsci.2003.10.007.
We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes.
To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR-RFLP methods for stability and reproducibility.
Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA).
In this study, six dermatophyte species (T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR-RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region.
The PCR and PCR-RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.
我们聚焦于几种致病真菌的DNA拓扑异构酶II基因，并开发了针对该基因的聚合酶链反应（PCR）及PCR-限制性片段长度多态性（RFLP）方法用于皮肤癣菌的鉴定。
通过检测这些PCR及PCR-RFLP方法的稳定性和可重复性，评估基于PCR的鉴定方法在皮肤癣菌病病因学研究中的可用性。
从305例癣病患者中分离出356株皮肤癣菌菌株，其基因组DNA用作PCR模板，使用由用于将皮肤癣菌鉴定到种水平的基因特异性引物组成的引物混合物（PsT、PsME、dPsD1或dPsD2）。对红色毛癣菌的基因组DNA进一步进行核糖体DNA（rDNA）非转录间隔区（NTS）的亚重复元件分析。
在本研究中，获得了6种皮肤癣菌（红色毛癣菌、须癣毛癣菌、断发毛癣菌、犬小孢子菌、石膏样小孢子菌和絮状表皮癣菌）。在所有病例中，基于DNA拓扑异构酶II基因的PCR及PCR-RFLP鉴定结果与基于传统形态学特征的鉴定技术结果一致。基于PCR的鉴定的敏感性为直径约3mm的菌落。此外，根据TRS-1区域产生的产物大小和数量，红色毛癣菌被分为三组（17种类型），根据TRS-2区域分为三种类型。
针对DNA拓扑异构酶II基因的PCR及PCR-RFLP在皮肤癣菌的种属鉴定中快速、稳定且可重复，因此是皮肤癣菌病病因学研究的便捷工具。
