Rapid identification and differentiation of Trichophyton species, based on sequence polymorphisms of the ribosomal internal transcribed spacer regions, by rolling-circle amplification

Abstract

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two "group-specific" probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp.

FIG. 1.

RCA results monitored by the RotorGene 3000 real-time PCR machine (Corbett Research). The accumulation of double-stranded DNA was detected by staining with Sybr green I. Positive signals (labeled as “positive signal”) are shown as exponential increases in fluorescence signal. The experiment was conducted using the T. mentagrophytes-specific RCA probe (Tm-ITS1) and tested on eight T. mentagrophytes and other dermatophyte isolates. Ligation-mediated RCA with matched templates of T. mentagrophytes containing the targeted SNPs produced positive signals. In contrast, other species (e.g., T. tonsurans, T. rubrum, T. violaceum, E. floccosum, and M. canis) showed an absence of signal (labeled as “negative signal”).

FIG. 2.

Algorithm for identification of Trichophyton spp. using a padlock probe RCA assay.