Tokamani Maria, Figgou Eleftheria, Papamichail Lito, Sakka Eleni, Toros Athanasios, Bouchorikou Anastasia, Giannakakis Antonis, Matthaiou Efthymia Iliana, Sandaltzopoulos Raphael
Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece.
Department of Medicine, Division of Pulmonary Allergy and Critical Care Medicine, School of Medicine, Stanford University, Stanford, CA 94305, USA.
J Fungi (Basel). 2023 Aug 11;9(8):842. doi: 10.3390/jof9080842.
mold is a ubiquitously found, airborne pathogen that can cause a variety of diseases from mild to life-threatening in severity. Limitations in diagnostic methods combined with anti-fungal resistance render a global emerging pathogen. In industry, produce toxins, such as aflatoxins, which can cause food spoilage and pose public health risk issues. Here, we report a multiplex qPCR method for the detection and identification of the five most common pathogenic species, , , , , and Our approach exploits species-specific nucleotide polymorphisms within their ITS genomic regions. This novel assay combines multiplex single-color real time qPCR and melting curve analysis and provides a straight-forward, rapid, and cost-effective detection method that can identify five species simultaneously in a single reaction using only six unlabeled primers. Due to their unique fragment lengths, the resulting amplicons are directly linked to certain species like fingerprints, following either electrophoresis or melting curve analysis. Our method is characterized by high analytical sensitivity and specificity, so it may serve as a useful and inexpensive tool for diagnostic applications both in health care and the food industry.
霉菌是一种广泛存在的空气传播病原体，可引发从轻度到危及生命的各种疾病。诊断方法的局限性以及抗真菌耐药性使其成为一种全球新兴病原体。在工业中，霉菌会产生毒素，如黄曲霉毒素，可导致食物变质并引发公共卫生风险问题。在此，我们报告一种多重定量聚合酶链反应（qPCR）方法，用于检测和鉴定五种最常见的致病物种，即[物种名称1]、[物种名称2]、[物种名称3]、[物种名称4]和[物种名称5]。我们的方法利用了它们内部转录间隔区（ITS）基因组区域内的物种特异性核苷酸多态性。这种新颖的检测方法结合了多重单色实时定量PCR和熔解曲线分析，提供了一种简单、快速且经济高效的检测方法，仅使用六条未标记引物就能在单个反应中同时鉴定出五种物种。由于其独特的片段长度，经过电泳或熔解曲线分析后，所得扩增子就像指纹一样直接与某些物种相关联。我们的方法具有高分析灵敏度和特异性，因此可作为医疗保健和食品行业诊断应用中一种有用且廉价的工具。
