Kanbe Toshio, Suzuki Yasuhiro, Kamiya Atsushi, Mochizuki Takashi, Kawasaki Masako, Fujihiro Machiko, Kikuchi Akihiko
Division of Molecular Mycology and Medicine, Department of Advanced Medical Science, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
J Dermatol Sci. 2003 Oct;33(1):41-54. doi: 10.1016/s0923-1811(03)00150-6.
We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species.
To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence.
The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed.
Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum.
PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.
我们一直专注于致病真菌的DNA拓扑异构酶II基因，并且之前已经应用基于聚合酶链反应（PCR）的方法对包括一些主要皮肤癣菌物种在内的多个物种进行了鉴定。
通过PCR和PCR-限制性片段长度多态性（RFLP）技术，在不测定核苷酸序列的情况下，将皮肤癣菌（18个物种）鉴定到种水平。
使用针对皮肤癣菌的通用引物组（dPsD1）通过PCR扩增皮肤癣菌（10种毛癣菌、7种小孢子菌和絮状表皮癣菌）的基因组DNA，随后使用其他引物组（dPsD2、PsT和PsME）进行巢式PCR，这些引物组包含针对皮肤癣菌DNA拓扑异构酶II基因的特异性引物。使用PsT和PsME进行的PCR用于毛癣菌、小孢子菌和絮状表皮癣菌的种鉴定。用限制性内切酶（Hinc II、Hinf、Afl II和PflM I）消化dPsD2产生的PCR产物，并分析限制性图谱。
在18种皮肤癣菌中，5种（红色毛癣菌、紫色毛癣菌、犬小孢子菌、石膏样小孢子菌和絮状表皮癣菌）通过使用PsT和PsME的PCR被特异性鉴定到种水平，其余物种通过PCR-RFLP分析中每个物种独特的限制性图谱进行鉴定，但指间型须癣毛癣菌的限制性图谱与昆克氏须癣毛癣菌的相同。
针对DNA拓扑异构酶II基因的PCR和PCR-RFLP技术简单快速，作为将皮肤癣菌鉴定到种水平的工具非常有用。
