Use of Padlock Probes and Rolling Circle Amplification (RCA) for Rapid Identification of Trichophyton Species, Related to Human and Animal Disorder

Hamideh Zakeri; Tahereh Shokohi; Hamid Badali; Saba Mayahi; Mojtaba Didehdar

doi:10.5812/jjm.19107v2

Use of Padlock Probes and Rolling Circle Amplification (RCA) for Rapid Identification of Trichophyton Species, Related to Human and Animal Disorder

Jundishapur J Microbiol. 2015 Jul 27;8(7):e19107. doi: 10.5812/jjm.19107v2. eCollection 2015 Jul.

Authors

Hamideh Zakeri¹, Tahereh Shokohi², Hamid Badali², Saba Mayahi³, Mojtaba Didehdar⁴

Affiliations

¹ Student Research Committee, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, IR Iran.
² Invasive Fungi Research Center (IFRC), Mazandaran University of Medical Sciences, Sari, IR Iran ; Department of Medical Mycology and Parasitology, Mazandaran University of Medical Sciences, Sari, IR Iran.
³ Department of Medical Mycology and Parasitology, Mazandaran University of Medical Sciences, Sari, IR Iran.
⁴ Student Research Committee, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, IR Iran ; Arak Medical Faculty, Arak University of Medical Sciences, Arak, IR Iran.

Abstract

Background: The high degree of phenotypic similarity among Trichophyton species makes their identification difficult.

Objectives: The current study aims to establish the use of rolling circle amplification (RCA) based on internal transcribed spacer ribosomal DNA (ITS rDNA) as a powerful, simple, and rapid procedure for distinguishing closely related organisms, and specifically to identify Trichophyton species, which cause human and animal disorders.

Materials and methods: A total of sixty-one isolates belonging to three species of Trichophyton were identified to the species level based on microscopic and macroscopic examinations and their ITS rDNA regions were sequenced. Three specific circular oligonucleotide probes targeting the ITS1 and ITS2 regions were designed to differentiate Trichophyton rubrum, T. mentagrophytes, and T. tonsurans.

Results: Of the 61 putative Trichophyton clinical isolates, 52 were identified to the species level. The most common species was T. mentagrophytes var. interdigitale (31 isolates), followed by T. rubrum (11 isolates), T. tonsurans (9 isolates), and T. violaceum (1 isolates); moreover, 9 isolates were identified as non-Trichophyton species. The RCA method correctly identified four Trichophyton species and was 100% specific for each species. Neither cross-reaction between the examined species of Trichophyton nor false positive or false negative results were observed.

Conclusions: Species identification of Trichophyton is crucially important for epidemiological and phylogenetic purposes and for genotype delineation. RCA based on ITS polymorphisms can be used to generate identification barcodes and as an alternative to DNA sequencing; it is a very fast, specific, and economical tool for species identification.

Keywords: DNA, Ribosomal; Identification; Oligonucleotide Probes; Rolling Circle Amplification (RCA); Trichophyton.