Table 7 Architectural characteristics of the reference libraries Library number Number of raw spectra per RMS Number of RMS per strain Number of strains per species Library
characteristics B0 4 1 1 1 RMS4 x 30 strains B1 10 1 1 1 RMS10 x 30 strains B2 10 2 1 2 RMS10 x 30 strains B3 10 4 1 4 RMS10 x 30 strains B4 20 2 1 2 RMS20 x 30 strains B5 40 1 1 1 RMS40 x 30 strains B6 10 4 2 4 RMS10 x 60 strains B7 10 4 3 4 RMS10 x 90 strains Culture Each reference strain was subcultured on four Sabouraud Gentamicin Chloramphenicol agar plates (AES, France) at 30°C. The strains used to construct the reference libraries and the isolates obtained from clinical samples were analyzed as soon as a fungal colony grew on the agar (usually after 48–72 hours). The clinical isolates were identified via morphological assessment, DNA sequencing, and CA4P solubility dmso MALDI-TOF MS as described below. Clinical isolate identification All 200 clinical isolates were identified in parallel by two trained mycologists following the identification keys of the Atlas of Clinical Fungi [24]. If the morphological identification was impossible or conflicted with the MALDI-TOF MS-based identification results, the isolate was further analyzed using DNA sequencing.
DNA sequence-based identification was performed by analyzing the ITS 2 (primer ITS3: 17-DMAG (Alvespimycin) HCl CHIR-99021 cost GCA TCG ATG AAG AAC GCA GC and primer ITS4c: TCC TCC GCT TAT TGA TAT GC) and D1-D2 (primer D1: AAC TTA AGC ATA TCA ATA AGC GGA GGA and primer D2: GGT CCG TGT TTC AAG ACG G) variable regions of the 28S unit of the rRNA gene as described by de Hoog et al. [24]. DNA extraction was performed using a QIAmp DNA kit (QIAGEN, Courtaboeuf, France). The reaction mixture was subjected to 35 cycles
of 30 s denaturation at 94°C, 30 s primer annealing at 53°C, and 1 min primer extension at 72°C for the ITS 2 region and 40 cycles of 20 s denaturation at 94°C, 30 s primer annealing at 58°C, and 1 min primer extension at 72°C for the D1-D2 region. The sequencing reactions were performed using the same primers used for amplification. In both cases, the sequencing mixture was subjected to 25 cycles of 10 s denaturation at 96°C, 5 s primer annealing at 50°C, and 4 min primer extension at 60°C. Purification of the sequences was performed using BigDye® XTerminator™ (Applied Biosystems, Inc., Courtaboeuf, France), and the different reactions were STI571 purchase processed using a 3130 Genetic Analyzer (Applied Biosystems, Inc., Courtaboeuf, France). The resulting sequences were then compared using the Medical Fungi pairwise sequence alignment tool (http://www.cbs.knaw.nl/Medical/BioloMICSSequences.aspx).