title: Development of a high-resolution melting analysis assay for rapid and high-throughput identification of clinically important dermatophyte species.
abstract: Accurate identification of dermatophyte species is important both for epidemiological studies and for implementing antifungal treatment strategies. Although nucleic acid amplification-based assays have several advantages over conventional mycological methods, a major disadvantage is their high cost. The aim of this study was to develop a rapid and accurate real-time PCR-based high-resolution melting (HRM) assay for differentiation of the most common dermatophyte species. The oligonucleotide primers were designed to amplify highly conserved regions of the dermatophyte ribosomal DNA. Analysis of a panel containing potentially interfering fungi demonstrated no cross reactivity with the assay. To evaluate the performance characteristics of the method, a total of 250 clinical isolates were tested in comparison with the long-established PCR-RFLP method and the results were reassessed using DNA sequencing, as the reference standard method. The assay is able to type dermatophytes using normalised melting peak, difference plot analysis or electrophoresis on agarose gel methods. The results showed that, in comparison to PCR-RFLP, the developed HRM assay was able to differentiate at least 10 common dermatophytes species with a higher speed, throughput and accuracy. These results indicate that the HRM assay will be a useful sensitive, high throughput and cost-effective method for differentiating the most common dermatophyte species.
authors.affiliation: Department of Medical Mycology and Parasitology/Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran.|Department of Medical Mycology and Parasitology, Arak University of Medical Sciences, Arak, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Didehdar
authors.affiliation: Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran.
authors.forename: B
authors.initials: B
authors.lastname: Khansarinejad
authors.affiliation: Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran.|Department of Laboratory Sciences and Tropical Medicine Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: N
authors.initials: N
authors.lastname: Amirrajab
authors.affiliation: Department of Medical Mycology and Parasitology/Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, Iran.
authors.forename: T
authors.initials: T
authors.lastname: Shokohi
mesh_terms: D003883:Arthrodermataceae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D003881:Dermatomycoses / Q000175:diagnosis / Q000382:microbiology; D057166:High-Throughput Screening Assays / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal
keywords: Dermatophyte; HRM analysis; identification