title: Identification of Pseudallescheria and Scedosporium species by three molecular methods.
abstract: The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.
authors.affiliation: Department of Dermatology, Peking University First Hospital and Research Center for Medical Mycology, Peking University, No. 8 Xishiku St., West District, 100034 Beijing, China.
authors.forename: Qiaoyun
authors.initials: Q
authors.lastname: Lu
authors.forename: A H G
authors.initials: AH
authors.lastname: Gerrits van den Ende
authors.forename: J M J E
authors.initials: JM
authors.lastname: Bakkers
authors.forename: Jiufeng
authors.initials: J
authors.lastname: Sun
authors.forename: M
authors.initials: M
authors.lastname: Lackner
authors.forename: M J
authors.initials: MJ
authors.lastname: Najafzadeh
authors.forename: W J G
authors.initials: WJ
authors.lastname: Melchers
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Li
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
mesh_terms: D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008271:Mycetoma / Q000175:diagnosis* / Q000382:microbiology*; D009172:Mycology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D011541:Pseudallescheria / Q000235:genetics / Q000302:isolation & purification*; D021681:Scedosporium / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D014404:Tubulin