College of Veterinary Medicine, China Agricultural University, 100193 Beijing, China.
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Biosensors (Basel). 2022 Aug 13;12(8):636. doi: 10.3390/bios12080636.
Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are and . Wood's lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.
皮肤真菌病是一种由几种真菌引起的传染病，可影响毛发、指甲和/或皮肤的浅层，具有全球性意义。猫和狗中最常见的皮肤真菌是 和 。伍德灯检查、显微镜鉴定和真菌培养是常规的临床诊断方法，而 PCR（聚合酶链反应）和 qPCR（定量 PCR）在皮肤真菌鉴定中发挥着越来越重要的作用。然而，这些方法都不能应用于即时检测（POCT）。基于 CRISPR（成簇规律间隔短回文重复）的诊断平台的最新发展有望成为一种快速、准确、便携的诊断工具。在本文中，我们提出了一种基于 Cas12a 的荧光检测法，用于检测和区分临床样本中的主要皮肤真菌，具有很高的特异性和灵敏度。该基于 Cas12a 的检测法与重组酶聚合酶扩增（RPA）相结合。结果可以通过肉眼在蓝光下直接观察到，所有测试样本均与真菌培养和测序结果一致。与传统方法相比，RPA-Cas12a-荧光检测法所需时间（约 30 分钟）更短，所需设备更简单，肉眼可以清晰观察到视觉变化，适用于现场临床诊断。
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