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首页> 外文期刊>Journal of Clinical and Diagnostic Research >Optimization of PCR?RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions
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Optimization of PCR?RFLP Directly from the Skin and Nails in Cases of Dermatophytosis, Targeting the ITS and the 18S Ribosomal DNA Regions

机译：直接针对皮肤和指甲的ITS和18S核糖体DNA区域直接从皮肤和指甲PCR-RFLP的优化

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Purpose: 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.Materials and Methods: 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.Results: 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.Conclusion: 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.
机译：目的：进行针对内部转录间隔区（ITS）区域的泛真菌引物，并使用针对18S核糖体DNA（rDNA）区域的皮肤真菌特异性引物对PCR-RFLP进行优化，以直接从临床标本中鉴定皮肤真菌物种和菌株。材料与方法：从临床怀疑的皮肤癣菌病病例中收集了138份标本（129张皮肤碎片和9张指甲剪），并进行直接显微镜检查和培养。其中使用Mva I，Hae III和Dde I限制性内切酶进行PCR-RFLP分析，对66个皮肤刮擦和3个指甲剪进行基因分型。结果：在138个标本中，有81个标本为皮肤癣菌阳性一个是红毛癣菌（47），然后是薄荷毛癣菌（25）和絮状表皮细菌（9）。在47株红曲霉菌株中，有10株为红曲霉。在表型上被鉴定为脲酶阳性并且通过DNA测序被鉴定为raubitschekii。由于它们表现出较小的形态和生理特征，因此它们目前已被称为T. rubrum。在25种棉铃虫分离株中，有3种为叉毛癣菌，通过DNA测序鉴定。在66例皮肤标本涂片中，培养和PCR显示分别有36例（54.54％），42例（63.63％）和47例（71.21％）存在皮肤癣菌。在三个指甲样本中，通过涂片，培养和PCR检测发现只有一个皮肤癣菌阳性。结论：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。通过使用Mva I和Dde I酶靶向ITS区域的PCR对RFLP分析同样有效。但是，通过使用上述三种限制酶，在红球菌和薄荷茶的菌株中未检测到菌株变异。

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