Department of Parasitology and Mycology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Cancer Molecular Pathology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Mycopathologia. 2018 Aug;183(4):669-677. doi: 10.1007/s11046-018-0256-7. Epub 2018 Mar 5.
The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.
黑酵母属外瓶霉属包括许多潜在的机会性物种，这些物种可能在免疫功能正常的个体中引起系统性和播散性感染。引起系统性疾病的物种能够在 37-40°C 下生长，而其他物种则始终缺乏耐热性，它们参与冷血、水生脊椎动物的疾病，偶尔也参与无脊椎动物的疾病。我们解释了一种快速而敏感的检测方法，用于识别和鉴定无需测序的水生外瓶霉属物种。外瓶霉属的七个物种（E. equina、E. salmonis、E. opportunistica、E. pisciphila、E. aquamarina、E. angulospora 和 E. castellanii）的 ITS rDNA 区域以及近亲 Veronaea botryosa 被测序并进行了比对，以设计用于检测特征性单核苷酸多态性的特定套锁探针。该检测方法成功地扩增了目标真菌的 DNA，允许在种水平上进行检测。扩增产物在 1%琼脂糖凝胶上可视化，以确认探针-模板结合的特异性。减少了试剂的用量，以防止产生假阳性结果。该方法的简单性、温和性、稳健性和低费用使得套锁探针检测（RCA）在 DNA 诊断的等温方法中具有明确的地位，是一种非常实用的方法。
