Microsatellite-based PCR multiplex for identification of fungal species We have confirmed the specificity of the microsatellite multiplex for A. fumigatus within section Fumigati with a single exception observed in A. unilateralis (marker MC6b). However, it could not be discarded the detection of few other markers in species belonging to section Fumigati if less stringent PCR conditions were employed, as some markers were found in the genome of N. fischeri NRRL 181. Therefore, we had tested distinct amplification temperatures

(from 48 to 60°C) in the group of species belonging to section Fumigati. Few markers could be amplified after decreasing the PCR annealing temperature from 60°C to 55°C (see Table 1). Eight peaks previously observed in A. fumigatus were similarly found when testing less stringent Selleck GS 1101 PCR conditions. Sequencing analysis

buy Ensartinib of those amplicons revealed genomic similarities to A. fumigatus (see Additional file Table A 1; a single exception was MC3 primers that amplified an unspecific region). Remarkably, distinct electrophoretic profiles were obtained for all tested species based on the amplification of the microsatellite multiplex panel at 55°C, as seen in Table 1. The relevant pathogens of section Fumigati, A. fumigatiaffinis, N. fischeri and N. udagawae, were clearly distinguished from A. fumigatus and from all the other species within this section. In addition, A. novofumigatus was also identified. Besides A. fumigatus isolate, MC6a was uniquely amplified with N. fischeri isolate, while MC8 was obtained exclusively with N. udagawae. The marker MC5 was amplified with A. fumigatiaffinis and A. novofumigatus (Table 1). Few microsatellites showed more than three repeat motifs, as it was the case of MC6a in A. lentulus and MC6b in A. unilateralis (sequence analysis of the amplified markers was added as supplementary Table A 1). Sequence analysis of marker MC6b showed that A. lentulus and A. viridinutans (the most relevant species in clinics besides A. fumigatus) were different from

all the other tested species. Table 1 List of markers amplified at 55°C annealing Amobarbital temperature in the group of species belonging to section  Fumigati    MC3 MC1 MC8 MC5 MC2 MC6a MC7 MC6b Aspergillus fumigatus ATCC 46645 √ √ √ √ √ √ √ √ Aspergillus fumigatiaffinis CBS 117186 √ a     √       √ Aspergillus lentulus CBS 116880b √ a             √ Aspergillus novofumigatus CBS 117519 √ a     √         Aspergillus unilateralis CBS 126.56 √ a             √ Aspergillus viridinutans CBS 121595 √ a             √ Neosartoryafischeri CBS 316.89 √ a     √   √   √ Neosartoryahiratsukae CBS 124073 √ a             √ Neosartoryapseudofischeri CBS 208.92b √ a             √ Neosartoryaudagawae CBS 114217 √ a   √         √ a) Unspecific amplification with MC3 primers (confirmed after sequence analysis). b) Similar results were observed with other tested reference strains. Discussion Species such as A. lentulus, A.