The record of rodA sequences at GenBank has been improved by the addition
of the information on A. novofumigatus. Conclusions As molecular diagnosis is being increasingly employed in clinical labs [21, 22] and some labs can only detect fungal DNA (culture of the fungal agent cannot be obtained), it will become increasingly important to possess molecular protocols for the identification of moulds while avoiding misidentification of fungal species. Thus, a multiplex PCR strategy is now available that can selleck screening library easily differentiate A. fumigatus and N. udagawae from other fumigatus-related species. In addition, www.selleckchem.com/products/qnz-evp4593.html the proposed methodology can be used in cases of low sporulating fungal isolates frequently detected in culture, as in the case of two isolates from our collection. Pathogenic species of section Fumigati could be identified by sequencing βtub and rodA fragments by following the list of polymorphic sites provided in this work. Molecular identification is at present recommended for the correct identification of species within the A. fumigatus complex group of species. The assay described in the present study proved to be specific and highly reproducible, representing a fast and economic way of targeting molecular identification of A. fumigatus in clinical PRI-724 research buy laboratories. Methods Fungal strains and culture conditions A set of 35 clinical isolates of A. fumigatus from the Department of Microbiology, Faculty of Medicine, University of Porto, PtdIns(3,4)P2 were used
in this study; the reference strain, A. fumigatus ATCC 46645, was also included. The isolates were identified based on macroscopic and microscopic morphological characteristics, and standard mycological procedures were followed [23]. The genotype of this set of A. fumigatus isolates was unique, as established by a previously standardized microsatellite based multiplex PCR specially designed for this mould [24]. A second group of fungal strains of the section Fumigati was obtained from Centraalbureau voor Schimmelcultures (CBS): pathogenic moulds including Aspergillus fumigatiaffinis (CBS 117186), Aspergillus lentulus (CBS 116880 and CBS 117180), Neosartorya hiratsukae (CBS
124073), Neosartorya pseudofischeri (CBS 208.92), and Neosartorya udagawae (CBS 114217), and two non-pathogenic moulds of section Fumigati, Aspergillus novofumigatus (CBS 117519) and Aspergillus unilateralis (CBS 126.56). In addition, a third set of 12 isolates that included strains of other Aspergillus sections (Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus and Aspergillus glaucus) and two low sporulating Aspergillus species from our collection were included in this study. Single colonies of all fungal isolates were cultured on Sabouraud dextrose agar for 5 days at 30°C. A sodium-hydroxide-based method was used to extract DNA from fungal conidia (the protocol is available at http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols).