Elavarashi Elangovan, Kindo Anupma Jyoti, Kalyani Jagannathan
PhD Research Scholar, Department of Microbiology, Sri Ramachandra University , Chennai, India .
J Clin Diagn Res. 2013 Apr;7(4):646-51. doi: 10.7860/JCDR/2013/5363.2873. Epub 2013 Feb 12.
A pan fungal primer targeting the Internal Transcribed Spacer (ITS) region and optimization of PCR-RFLP using a dermatophyte specific primer targeted the 18S ribosomal DNA (rDNA) region were performed for the identification of dermatophyte species and strains directly from clinical specimens.
One hundred and thirty eight specimens (129 skin scrapings and 9 nail clippings) from clinically suspected cases of dermatophytosis were collected and subjected to direct microscopy and culture. Among them, 66 skin scrapings and 3 nail clippings were processed for genotyping by PCR-RFLP analysis using the Mva I, Hae III and the Dde I restriction enzymes.
Of the 138 specimens, 81 specimens were positive for dermatophytosis, the most common one being Trichophyton rubrum (47), followed by Trichophyton mentagrophytes (25) and Epidermophyton floccosum (9). Of the 47 T. rubrum isolates, 10 were T. rubrum var. raubitschekii which were identified phenotypically as urease positive and by DNA sequencing. Since they exhibited minor morphological and physiological features, they have currently been synonymized with T. rubrum. Of the 25 T. mentagrophytes isolates, three were Trichophyton interdigitale, which were identified by DNA sequencing. Among the 66 skin specimens smear, culture and PCR showed the presence of dermatophytes in 36 (54.54%), 42 (63.63%) and 47 (71.21%) cases respectively. Among the three nail specimens, only one was found to be positive for dermatophytosis by smear, culture and PCR.
Amplification of the dermatophyte specific primer is appropriate in the identification of dermatophytes directly from the clinical material. PCR targeting the ITS region by using the Mva I and the Dde I enzymes was equally good for the RFLP analysis. However, by using the above three restriction enzymes, no strain variations were detected among the T. rubrum and the T. mentagrophytes strains.
使用靶向内部转录间隔区（ITS）的泛真菌引物以及针对18S核糖体DNA（rDNA）区域的皮肤癣菌特异性引物对PCR-RFLP进行优化，以直接从临床标本中鉴定皮肤癣菌的种类和菌株。
收集138份来自临床疑似皮肤癣菌病病例的标本（129份皮肤刮屑和9份指甲剪屑），进行直接显微镜检查和培养。其中，66份皮肤刮屑和3份指甲剪屑通过使用Mva I、Hae III和Dde I限制性内切酶进行PCR-RFLP分析来进行基因分型。
在138份标本中，81份皮肤癣菌病呈阳性，最常见的是红色毛癣菌（47份），其次是须癣毛癣菌（25份）和絮状表皮癣菌（9份）。在47株红色毛癣菌分离株中，有10株是红色毛癣菌变种raubitschekii，通过表型鉴定为脲酶阳性并经DNA测序。由于它们表现出微小的形态和生理特征，目前已与红色毛癣菌同义。在25株须癣毛癣菌分离株中，有3株是指间毛癣菌，通过DNA测序鉴定。在66份皮肤标本涂片、培养和PCR检测中，分别有36例（54.54%）、42例（63.63%）和47例（71.21%）检测到皮肤癣菌。在3份指甲标本中，涂片、培养和PCR检测仅发现1份皮肤癣菌病呈阳性。
皮肤癣菌特异性引物的扩增适用于直接从临床材料中鉴定皮肤癣菌。使用Mva I和Dde I酶靶向ITS区域的PCR对RFLP分析同样适用。然而，使用上述三种限制性内切酶，在红色毛癣菌和须癣毛癣菌菌株中未检测到菌株变异。
