目的:在世界各地,人类皮肤癣菌病最常见的病因是红色毛癣菌,间型毛癣菌,和絮状表皮组织。这项研究的主要目的是设计和评估一种简单而直接的多重聚合酶链反应(PCR)测定法,以可靠地鉴定/区分临床分离株中的这些物种。
方法:利用几种皮肤癣菌分子靶标的可靠序列,设计了一种多重PCR方法,用于鉴定常见的致病性皮肤癣菌。在这项研究中检查的分离株和临床标本包括七个标准的皮肤癣菌菌株,已通过测序或PCR限制性片段长度多态性(RFLP)鉴定的101种皮肤癣菌和非皮肤癣菌霉菌/酵母分离株,以及155份怀疑有皮肤真菌病的病人的临床样本。
结果:红藻和T.interdigitale/T.的物种特异性引物对。根据翻译延伸因子1-α基因的序列数据设计了植叶植物,和针对絮状大肠杆菌的引物靶向内部转录间隔区(ITS)的特定序列。多重PCR成功检测出红毛虫,T.interdigitale/T.植叶植物,和通过测序或PCR-RFLP鉴定的絮状大肠杆菌菌株。然而,为T.interdigitale/T.选择的引物对植叶植物与扁桃体毛癣菌交叉反应。在直接测试临床样本的PCR系统时,多重PCR阳性比例高于阳性培养(68.1%vs.55.4%,分别)。
结论:多重检测可以检测到皮肤癣菌病的几种病因中的三种常见药物,即T.rubrum,T.interdigitale,和E.絮凝剂。因此,通过添加泛皮肤癣菌引物,它可以用作全面的检测/鉴定测试。
OBJECTIVE: The most common etiological agents of human dermatophytosis in various parts of the world are Trichophyton rubrum, Trichophyton interdigitale, and Epidermophyton floccosum. The main aim of this study was to design and evaluate a simple and straightforward multiplex polymerase chain reaction (PCR) assay for reliable identification/differentiation of these species in clinical isolates.
METHODS: The reliable sequences of several molecular targets of dermatophytes species were used to design a multiplex PCR for the identification of common pathogenic dermatophytes. The isolates and clinical specimens examined in this study included seven standard strains of dermatophytes, 101 isolates of dermatophytes and non-dermatophyte molds/yeasts which had already been identified by sequencing or PCR-restriction fragment length polymorphism (RFLP), and 155 clinical samples from patients suspected of cutaneous mycoses.
RESULTS: Species-specific primer pairs for T. rubrum and T. interdigitale/T. mentagrophytes were designed based on the sequence data of the translation elongation factor 1-alpha gene, and the primers for E. floccosum targeted the specific sequence of the internal transcribed spacer region (ITS). The multiplex PCR successfully detected T. rubrum, T. interdigitale/T. mentagrophytes, and E. floccosum strains that were identified by sequencing or PCR-RFLP. However, the primer pairs selected for T. interdigitale/T. mentagrophytes cross-reacted with Trichophyton tonsurans. In testing the PCR system directly for clinical samples, the proportion of positive multiplex PCR was higher than positive culture (68.1% vs. 55.4%, respectively).
CONCLUSIONS: The multiplex assay could detect three common agents out of several causal agents of dermatophytosis, namely T. rubrum, T. interdigitale, and E. floccosum. Therefore, by adding pan-dermatophyte primers it can be used as a comprehensive detection/identification test.