New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions.

Sebastian Gnat,Aneta Nowakiewicz,Dominik Łagowski,Mariusz Dyląg,Aleksandra Trościańczyk

doi:10.3390/pathogens10111361

Abstract

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

Highlights

Dermatophytes are a group of pathogenic fungi that can invade keratinized structures, leading to infection of skin, hair, and nails [1]
The cDNA was obtained using total RNA isolated in three independent repetitions from cells of T. verrucosum strains incubated in Sabouraud, potato dextrose, and MM-Cove medium
Slight variation in the Ct values was observed under the experimental conditions in various microbiological media between isolates obtained from humans, animals, and a reference strain (Figure 1)

Summary

Introduction

Dermatophytes are a group of pathogenic fungi that can invade keratinized structures, leading to infection of skin, hair, and nails [1]. Dermatophytes encompass more than 50 species belonging to the genera Trichophyton, Microsporum, Epidermophyton, Arthroderma, Nannizzia, Lophophyton, and Paraphyton [1,2]. An increasing number of dermatomycoses, including cases that are difficult to treat, are being documented in the scientific literature due to the renaissance of these diseases in developed countries [7,8,9,10]. One such dermatophyte species with an increasing prevalence of superficial infections in humans is Trichophyton verrucosum [9,11,12,13]. Traditional outdoor rearing with freely wandering and gathering animals may explain the acquisition and transmission of this dermatophyte species [11,12,14]. T. verrucosum dermatomycosis in humans can be regarded as an occupational disease [11]

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