A few other techniques, such as random amplified polymorphic DNA [10, 11], restriction fragment length polymorphisms [12] and a new proposed microsphere-based Luminex assay [13], may enable molecular identification of A. fumigatus without sequencing. However, these methodologies are quite time consuming and labour demanding and are thus impractical in most clinical labs. In addition, they can be very expensive when employed to study collections of large numbers of isolates. Thus, a rapid, practical and cheap alternative method for the molecular identification of A. fumigatus and the

distinction of the species within the section Fumigati is required. In this study, a multiplex PCR was developed using prior information Bucladesine based on βtub and

rodA partial gene sequences. We propose a single PCR to target the molecular recognition of the A. fumigatus fungus, avoiding the use of restriction enzymes. Additional sequencing of fragments of βtub and rodA allowed the identification of several A. fumigatus related species. Results Multiplex optimization The present progestogen antagonist strategy was proposed to simultaneously target βtub and rodA gene fragments that are specific to a single species (A. fumigatus) and other gene fragments that are common to a group of species (all species of section Fumigati). A similar strategy was attempted with calmodulin sequences from species within Entinostat the section Fumigati, but we could not obtain primers that were specific for A. fumigatus (data not shown). Thus, pairs of primers were selected based on the information on polymorphic and conserved regions of βtub and rodA genes among fungal species, as shown in Table 1 (for primer design criteria see the Methods section). As primer specificity could be improved by increasing the amplification temperature, a range from 60°C to 72°C was tested with our multiplex; highly specific primers work C-X-C chemokine receptor type 7 (CXCR-7) at high temperatures (Figure 1),

whereas the amplification of some regions (e.g., the rodA region of 313 bp) could only be observed in non-fumigatus species at 60°C. A region of the βtub gene of 198 bp was observed only in A. fumigatus even when low amplification temperatures were tested. The electrophoretic profile obtained for each fungal species was very clear, revealing few secondary and/or minor bands as a consequence of primer combinations in the multiplex PCR (four nonspecific bands in the case of A. fumigatus and occasionally two bands in the case of non-fumigatus species). Those secondary bands did not reduce the performance of the multiplex PCR, as shown in Figure 1. Table 1 Forward (F) and reverse (R) PCR primers employed for molecular identification of all Aspergillus species of section Fumigati and for Aspergillus fumigatus.