Rapid Identification of Seven Waterborne <Emphasis Type="Italic">Exophiala</Emphasis> Species by RCA DNA Padlock Probes |
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Authors: | M J Najafzadeh V A Vicente Peiying Feng A Naseri Jiufeng Sun A Rezaei-Matehkolaei G S de Hoog |
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Institution: | 1.Department of Parasitology and Mycology, Faculty of Medicine,Mashhad University of Medical Sciences,Mashhad,Iran;2.Cancer Molecular Pathology Research Center,Mashhad University of Medical Sciences,Mashhad,Iran;3.Microbiology, Parasitology and Pathology Post-Graduation Program, Department of Basic Pathology,Federal University of Paraná,Curitiba,Brazil;4.Third Affiliated Hospital,Sun Yat-sen University,Guangzhou,China;5.Guangdong Provincial Institute of Public Health,Guangdong Provincial Center for Disease Control and Prevention,Guangzhou,China;6.Department of Medical Mycology, School of Medicine,Ahvaz Jundishapur University of Medical Sciences,Ahvaz,Iran;7.Westerdijk Fungal Biodiversity Institute,Utrecht,The Netherlands;8.Center of Expertise in Mycology Radboudumc/Canisius Wilhelmina Hospital,Nijmegen,The Netherlands |
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Abstract: | 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. |
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