title: Use of rolling circle amplification to rapidly identify species of Cladophialophora potentially causing human infection.
abstract: The genus Cladophialophora comprises etiologic agents of disease in immunocompetent patients, ranging from mild cutaneous colonization to cerebral encephalitis, in addition to saprobic species. Due to the high degree of phenotypic similarity between closely related species of the genus, identification problems are imminent. In the present study, we described rapid and sensitive rolling circle amplification (RCA) method based on species-specific padlock probes targeted for the internal transcribed spacer regions of rDNA. ITS regions of 12 Cladophialophora species were sequenced, and subsequently, 10 specific padlock probes were designed for the detection of single nucleotide polymorphisms. The majority of circularizable padlock probes were designed based on single nucleotide polymorphisms (SNPs), while for C. bantiana, C. immunda and C. devriesii were characterized by two or more nucleotides. Individual species-specific probes correctly identified in all ten Cladophialophora species correctly by visualization on 1.2 % agarose gels used to verify specificity of probe-template binding; no cross-reactivity was observed. Simplicity, sensitivity, robustness and low costs provide RCA a distinct place among isothermal techniques for DNA diagnostics. However, restriction and specificity and sensitivity should be lowered and increased, respectively, to be useful for a wide variety of clinical applications.
authors.affiliation: Laboratory Research Center, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Hamzehei
authors.forename: Seyed Amir
authors.initials: SA
authors.lastname: Yazdanparast
authors.forename: Mehrnaz Mohammad
authors.initials: MM
authors.lastname: Davoudi
authors.forename: Sadegh
authors.initials: S
authors.lastname: Khodavaisy
authors.forename: Mitra
authors.initials: M
authors.lastname: Golehkheyli
authors.forename: Saham
authors.initials: S
authors.lastname: Ansari
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
authors.forename: Hamid
authors.initials: H
authors.lastname: Badali
mesh_terms: D001203:Ascomycota / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics; D021901:DNA, Intergenic / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009181:Mycoses / Q000175:diagnosis*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D021901:DNA, Intergenic; D015345:Oligonucleotide Probes