parapsilosis ATCC 22019 and C. glabrata ATCC 39316, were from the [ATCC], Cryptococcus neoformans IFM 5844 and IFM 5855 were from IFM TPCA-1 price Quality Services

Pty Ltd [IFM], and Aspergillus fumigatus SzMC 2486, A. flavus SzMC 2536 and A. niger SzMC 2761 were from the Szeged Microbiological Collection [SzMC]. Furthermore, clinical strains of C. albicans (n = 14), C. glabrata (n = 5), C. tropicalis (n = 4), C. parapsilosis (n = 5), C. krusei (n = 4), C. quillermondii (n = 4), C. lusitaniae (n https://www.selleckchem.com/products/RO4929097.html = 3), C. norvegensis (n = 1), C. inconspicua (n = 2), C. dubliniensis (n = 2) and Cryptococcus neoformans (n = 2) from the Institute of Clinical Microbiology at the University of Szeged were also tested. Bacterial DNA purification The bacterial strains were grown on Columbia agar base under aerobic conditions, except that Bacteroides fragilis was grown under anaerobic conditions. The bacterial DNA was extracted with the QIAamp® DNA Blood Mini Kit (QuiaGene Inc, Chatsworth, Calif., USA), following the manufacturer’s instructions in “Protocols for Bacteria”. One millilitre of log-phase culture suspension, at a concentration of 107 CFU/mL, was used for the preparation. For determination of the sensitivity of the reaction, 100 μL of the serially diluted

S. aureus reference strain was used for DNA extraction. The number of bacterial cells was determined by plating aliquots of serially learn more diluted samples onto Columbia agar base. For lysis of the rigid multilayered G + bacterial cell wall, we used a pre-incubation step with 20 mg/mL lysozyme (in 20 mM Tris · HCl, pH 8.0, 2 mM EDTA, 1.2% TritonX100). The spin protocol for “DNA Purification from Tissues” was followed, after Adenosine incubation at 30°C for 30 min. The final concentration of DNA was 2.0-13.8 ng/μL, with a ratio A260/A280 = 1.6-1.8 after purification. Fungal DNA purification All the fungi were grown on Sabouraud medium. The fungal DNA was extracted from 1 mL of a log-phase culture suspension containing 9.6 × 107 of fungal cells. For determination of the sensitivity

of the reaction, 100 μL of the serially diluted C. albicans reference strain was used for DNA extraction. The number of fungal cells was determined by plating aliquots of serially diluted samples onto Sabouraud-glucose medium. We followed the QIAamp® DNA Mini Kit Protocol for Yeasts. In this case, additional reagents were required for elimination of the complex fungal cell-wall structure: sorbitol buffer (1 M sorbitol, 100 mM EDTA, 14 mM β-mercaptoethanol) [34] was used, and the samples were incubated with lyticase for 30 min at 30°C. Efficient and complete lysis was achieved in 1.5 hour in a shaking water-bath. This purification yielded 2.0–25 μg of DNA in 100 μL of water (2.0–13.8 ng/μL), with A260/A280 = 1.6–1.8. DNA preparation from infected blood Samples of 180 μL healthy donor bloods in EDTA vacutainer tubes were infected with 20 μL of log-phase culture suspension at a concentration of 108 CFU/mL bacterial and/or fungal suspensions.