The relevant pathogens of section Fumigati, such as A fumigatiaf

The relevant pathogens of section Fumigati, such as A. fumigatiaffinis, N. fischeri and N. udagawae, were

easily identified, however, testing this strategy in a broader range of species and isolates would better support identification of species within Aspergillus section Fumigati. This strategy has been successfully tested before in the identification of microsatellite transferability in close related species [29]. Furthermore, the genotyping strategies of less studied species of section Fumigati can now be better approached, as new microsatellite markers have now been proposed for A. unilateralis and N. fischeri. Wide application of typing methodologies can give pertinent information regarding microbial epidemiology, chronic Mdivi1 cost colonization for several patients and effectiveness of antibiotic treatments [11–14]. The initial question on the real specificity of the microsatellite markers selected for A. fumigatus genotyping was answered in the present work and it represents a selleck genuine and required improvement for PCI-34051 applicability of the methodology. We proved that the proposed panel with eight microsatellites [11] is highly appropriate for genotyping A. fumigatus. Besides genotyping, microsatellite-based multiplex PCR allows the

identification of A. fumigatus and a slight modification of PCR conditions also allow identifying other pathogenic species within section Fumigati, particularly A. fumigatiaffinis N. fischeri, and N. udagawae. Sequence analysis of marker MC6b showed the that A. lentulus and A. viridinutans were different from all

the other tested species. Methods Fungal strains and culture conditions A set of fungal isolates described as belonging to Aspergillus section Fumigati was obtained from Centraalbureau voor Schimmelcultures (CBS): the pathogenic moulds Aspergillus fumigatiaffinis (CBS 117186), Aspergillus lentulus (CBS 116880, 117180, 117182, and 117885), Aspergillus viridinutans (CBS 121595), Neosartorya fischeri (CBS 316.89), Neosartorya hiratsukae (CBS 124073), Neosartorya pseudofischeri (CBS 208.92 and 110899), and Neosartorya udagawae (CBS 114217), and two non-pathogenic moulds Aspergillus novofumigatus (CBS 117519) and Aspergillus unilateralis (CBS 126.56). The reference strain A. fumigatus ATCC 46645 was also included in the present work, as well as ten different strains of A. fumigatus from our collection. Monospore isolates from all the fungal strains 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 (protocol at http://​www.​aspergillus.​org.​uk/​indexhome.​htm?​secure/​laboratory_​protocols). Fungal DNA was suspended in 50 μl of sterile water and frozen at -20°C. Control of the DNA quality was carried out by amplifying and sequencing the β-tubulin region in all tested fungi, using previously selected primers [10].

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