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.

Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes

“…Accurate identification of dermatophyte species is important both for epidemiological studies and for implementing antifungal treatment strategies (Didehdar et al . ). A simple, rapid and specific method for the diagnosis of dermatophyte infections would obviously be a major improvement (Leibner‐Ciszak et al .…”

“…), high‐resolution melting (Didehdar et al . ), arbitrary primed PCR (Liu et al . ,b), and DNA sequencing (Martinez et al .…”

“…The diagnosis of superficial mycoses relies on a combination of clinical features and mycological identification of pathogenic fungi in dermatological specimens (Lasseter et al 2011;Ovren et al 2016). Accurate identification of dermatophyte species is important both for epidemiological studies and for implementing antifungal treatment strategies (Didehdar et al 2016). A simple, rapid and specific method for the diagnosis of dermatophyte infections would obviously be a major improvement (Leibner-Ciszak et al 2010;Petinataud et al 2016).…”

Molecular Epidemiology and In Vitro Antifungal Susceptibility of Candida Isolates from Women with Vulvovaginal Candidiasis in Northern Cities of Khuzestan Province, Iran

“…Nucleic acid-based methods have several advantages over conventional ones, including higher speed, sensitivity, and accuracy; these methods can also identify genera and species in a few hours. On the other hand, the disadvantage of these methods is their high cost, compared with other identification methods (17). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is one of the molecular techniques, used for the identification of Candida species (18).…”

New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

“…Melting curve analysis has been reported by several authors as a fast, reliable, and cost-effective method to identify fungal species while avoiding sequencing (16,17,18,19,20). Moreover, a combination of probe detection and high-resolution melting analysis has been already used for detecting and identifying Aspergillus spp.…”