title: 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.
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.forename: Zhongsheng
authors.initials: Z
authors.lastname: Tong
authors.forename: Xiaoyou
authors.initials: X
authors.lastname: Chen
authors.forename: Tania
authors.initials: T
authors.lastname: Sorrell
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Qixuan
authors.initials: Q
authors.lastname: Wu
authors.forename: David
authors.initials: D
authors.lastname: Ellis
authors.forename: Sharon
authors.initials: S
authors.lastname: Chen
mesh_terms: D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016533:Mycological Typing Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013997:Time Factors; D014249:Trichophyton / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer