Yang Guoling, Zhang Mingli, Li Wenli, An Lijia
Department of Dermatology, The 1st Affiliated Hospital of Dalian Medical University, 116011 Dalian, China.
Mycopathologia. 2008 Oct;166(4):203-8. doi: 10.1007/s11046-008-9130-3. Epub 2008 Jun 4.
To seek a rapid and reliable molecular biology method to identify the common pathogenic dermatophyte fungi from clinical samples.
The genome DNA was extracted from cultured strains of seven common dermatophyte fungi species and part of each positive clinical specimen by microscopy. Intergenic spacer regions of ribosomal DNA (ITS) were amplified by semi-nested PCR (snPCR) with three universal primers (NS5, ITS1, and ITS4) for fungi. The amplified products were digested with two restriction endonucleases (BciT130 I, Dde I), the Restriction Fragment Length Polymorphism(RFLP). The rest of each clinical specimen was cultured in Sabouraud's Agar medium. Then the results of RFLP were compared with the traditional culture results.
The digestion of seven common dermatophyte fungi produced seven different restriction profiles. Restriction profiles of 17 clinical specimens matched, respectively, to that of the cultured strains, and 14 profiles of the 17 ones matched the culture result completely. The coincidence was 100.0%.
snPCR-RFLP analysis of intergenic spacer regions of ribosomal DNA is a valuable method of exactness and clarity for species identification of common dermatophyte fungi from clinical specimens.
寻找一种快速可靠的分子生物学方法，用于从临床样本中鉴定常见致病性皮肤癣菌。
从七种常见皮肤癣菌的培养菌株以及通过显微镜检查的部分阳性临床标本中提取基因组DNA。使用三种真菌通用引物（NS5、ITS1和ITS4）通过半巢式PCR（snPCR）扩增核糖体DNA（ITS）的基因间隔区。扩增产物用两种限制性内切酶（BciT130 I、Dde I）进行消化，即限制性片段长度多态性分析（RFLP）。每个临床标本的其余部分在沙氏琼脂培养基中培养。然后将RFLP结果与传统培养结果进行比较。
七种常见皮肤癣菌的消化产生了七种不同的限制性图谱。17份临床标本的限制性图谱分别与培养菌株的图谱匹配，其中17份中的14份图谱与培养结果完全匹配。符合率为100.0%。
核糖体DNA基因间隔区的snPCR-RFLP分析是一种从临床标本中准确清晰地鉴定常见皮肤癣菌种类的有价值方法。
