Identification of Dermatophytes based on polymorphysms of nuclear and mitochondrial ribosomal DNA regions

本研究旨在评价分子技术在从人体皮损分离的皮肤真菌鉴定中的作用。采用线粒体(Mt)DNA核糖体(R)DNA区域的限制性片段长度多态性(RFLP)、基因组DNA间隔区的RFLP分析和基因组DNA中rDNA的内转录间隔区(ITS)序列分析等技术，对线粒体DNA rDNA区域的聚合酶链式反应扩增产物进行RFLP分析，结果表明，用限制性内切酶Hina III消化得到的图谱存在差异。但用Hae III和MSP I对扩增产物进行酶切后，两种菌的RFLP图谱没有差异。该技术可用于区分红色毛滴虫和精神毛滴虫，但所获得的信息量较小。方法…MORE具有高度的重复性和高度的敏感性。此外，一旦确定了序列，这些数据就可以在其他地方使用，并参考基因库中存储的数据。然而，该方法的成本较高，在实际应用中存在一定的缺陷。本研究采用聚合酶链式反应-限制性片断长度多态性分析方法，对灵芝复合体中的红色毛霉和绒毛毛霉的ITS区进行了分析。用MVA I或HINT对其1+2区(约700bp)进行酶切。我指出这些特征是物种特有的。该方法具有较高的灵敏度和重复性，可用于种水平鉴定。同时，该方法成本低廉、省时，适用于多种病原菌的同时处理。综上所述，笔者建议在实际应用中使用MVA I或Hinf I对其1+2区进行聚合酶链式反应-限制性片段长度多态性分析来鉴定皮肤真菌。如果结果不清楚或不可接受，则确定ITS 1的核苷酸序列，并将数据参考那些保存在基因库中的数据。较少

The present study was conducted to evaluate the efficacy of molecular techniques for the identification of dermatophytes isolated from human lesions. The techniques used in the study were, restriction fragment length polymorphisms (RFLP) of ribosomal (r) DNA regions in mitochondrial (mt) DNA, RFLP analysis of spacer regions of rDNA in genomic DNA, and sequence analysis of internal transcribed spacer (ITS) of rDNA in genomic DNA.Using RFLP of PCR amplicons of rDNA region of mtDNA, the profiles generated by digestion with restriction enzyme HinA III showed differences between T. rubrum and T. mentagrophytes. But there was no difference between the RFLP profiles between these two species when the amplicons were digested with Hae III and Msp I. The technique could be used for the differenciation between T. rubrum and T. mentagrophytes, but the information obtained by the technique was rather minimum.There were many advantages of sequence analysis of ITS1 of rDNA in genomic DNA. The method … More was highly reproducible and highly sensitive. Moreover, once the sequence is determined, the data could be used elsewhere and referred to data deposited in gene banks. The expense of the method was, however, the drawback in the application in practice.PCR-RFLP analysis of ITS regions were performed on several species in the T. mentagrophytes complex, T. rubrum and E. floccosum. Digestion of ITS 1+2 regions (ca. 700 bp) by Mva I or Hint. I indicated these profiles to be species specific. The sensitivity and reproducibility was enough for the species level identification. Also the technique was inexpensive and time saving, therefore, the method is also suitable for treatment of a number of isolate at the same time.In conclusion, I propose to use PCR-RFLP analysis of ITS 1+2 regions by Mva I or Hinf I for the identification of dermatophytes in practice. If the result is unclear or unacceptable, then determine the nucleotide sequence of ITS 1, and refer the data to those deposited in a genebanks. Less