title: Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers.
abstract: Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.
authors.affiliation: Department of Parasitology and Mycology and Cancer Molecular Pathology Research Center, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: M J
authors.initials: MJ
authors.lastname: Najafzadeh
authors.forename: S
authors.initials: S
authors.lastname: Dolatabadi
authors.forename: M
authors.initials: M
authors.lastname: Saradeghi Keisari
authors.forename: A
authors.initials: A
authors.lastname: Naseri
authors.forename: P
authors.initials: P
authors.lastname: Feng
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
mesh_terms: D015342:DNA Probes; D004271:DNA, Fungal / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005093:Exophiala / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016533:Mycological Typing Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D060446:Phaeohyphomycosis / Q000382:microbiology; D017422:Sequence Analysis, DNA
chemical_list: D015342:DNA Probes; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer
keywords: Black yeasts; Exophiala; Identification; Rolling circle amplification