Objective Early diagnosis of invasive aspergillosis is essential for positive patient outcome. and genotyping of strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management. Introduction is the major filamentous fungal pathogen in severely ill patients causing disseminated infections [1]. Invasive aspergillosis (IA) is associated with mortality above 50% [2]. Early and reliable diagnosis of invasive fungal infections (IFIs) can lower patients’ mortality and reduce treatment expenses due to an early targeted therapy but this is often difficult and might be in part also due to the limitations of conventional diagnostic techniques (culture and histology) which often lack sensitivity promptness and efficiency [3]-[5]. As an example detection of circulating galactomannan (GM) has become commonly used but false-positive and false-negative results are still a major limitation to this auxiliary method for an earlier IA diagnosis [6]. Nucleic acid-based assays have the capacity to diagnose fungal diseases in an early stage. False-positive results represent drawbacks as a direct consequence of the high sensitivity of molecular assays and to ubiquitous presence of in the environment [3] [7]. Molecular assays are presently recommended for correct identification of within section and evaluation of its genetic diversity a critical issue for outbreak controlling [8]-[11]. Current gold standard in microbial genotyping is Multilocus sequence typing (MLST) developed for by Bain et al. [12]. The present MLST panel combines seven housekeeping genes showing a discriminatory power of 0.93 not as high as microsatellite genotyping but with the advantage of providing a free online database which improves data transferability and genetic analyses. MLST proved to be specific for this fungal species within section presenting a discriminatory power of 0.9993 comparing with MLST [19]. The aim of this study was to design an assay that combines detection identification and genotyping in a single reaction and that performs directly on DNA extracts gained from clinical specimens. To this aim we selected a panel of discriminatory-informative SNPs that are variable enough to accurately detect identify and genotype isolates and combine those markers in a single multiplex reaction named assay as this assay is built on SNPs located in MLST genes. A set of seven housekeeping genes was studied in 20 clinical and unrelated isolates as suggested by Bain et al. [12]. These strains were obtained from sputum samples collected from patients admitted at Hospital Rabbit Polyclonal to NUSAP1. S. Jo?o (Oporto Portugal) and had been previously genotyped by microsatellite-based strategy [8] [9]. Singleplex amplification was performed as previously described [12]. These primers were found unsuitable for a multiplex reaction as they formed dimers. Therefore new primers were designed using Primer 3 (http://frodo.wi.mit.edu/) and the absence of hairpins and dimers verified in silico using Autodimer software (http://www.cstl.nist.gov/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm); newly selected primers are listed in Table 1. Multiplex PCR amplification reaction consisted of: 0.5 μL of genomic DNA (50 ng-250 ng) 2.5 μL of My Taq? HS MIX (Bioline) and 0.5 μL of primer mix (each primer at 2.0 μM) in a final volume of 5.0 μL. The multiplex PCR reaction was run at the KN-62 following conditions: initial denaturation step at 95°C KN-62 for 15 min followed by 35 KN-62 cycles of denaturation at 95°C for 30 s annealing at 61°C for 1 min and extension at 70°C for 1 min followed by a final KN-62 extension step at 70°C for 10 min. Amplification products of multiplex PCR products KN-62 were confirmed on polyacrylamide gel and visualized by silver staining. Table 1 Primers used for MLST genotyping of in a multiplex reaction. Sequencing analysis Amplification products were purified with ExoSAP-IT (USB Corporation); 1.0 μL of ExoSAP-IT was added to 1.5 μL of amplification product. After purification sequencing reaction was conducted as previously described [9]. Sequencing data were processed and analyzed with Sequencing 5.2 analysis software (Applied Biosystems). assay The methodic strategy used for the assay is based on targeting only SNPs present in MLST genes.