5 mmol/L and that the elongation time Pitavastatin datasheet was 40 s instead of 1 min. All primers and

probes were obtained from Thermo Hybaid, Interactiva Division (Ulm, Germany) except the Spn9802 FAM probe which was obtained from Eurogentec, Seraing, Belgium. The real-time PCR assay was performed in a Rotor-Gene 3000 instrument (Qiagen, Hilden, Germany). The optimized real-time PCR amplifications were performed in 25-μL reactions containing 0.3 μmol/L of each primer, 0.2 μmol/L of the Spn9802 FAM probe, 0.1μmol/L of the P6 JOE probe and ctrA ROX probe, 3.5 mmol/L MgCl2, 0.2 mmol/L deoxynucleoside triphosphate, and 1 U HotStar Taq polymerase (Qiagen) in PCR buffer. A total of 5 μL of target DNA was used in the assay. The qmPCR was performed according to the following program: 15 min of enzyme activation at 95°C, followed by 45 cycles of 95°C for 15 s and 60°C for 40 s. Reproducibility of analytical sensitivity and quantification The analytical sensitivity of the Spn9802, P6 and ctrA PCRs was determined LCZ696 supplier by serial dilutions of target DNA in carrier tRNA (1μg/mL). Two experiments were performed with 5 to 600 genome copies per reaction tube and 2 to 4 tubes of each dilution. The reproducibility of quantification was evaluated by testing DNA

preparations with known concentrations (duplicates of 500, 2,000 and 10,000 genome copies per PCR reaction) in five consecutive runs and also in 73 BAL samples and in 8 CSF samples. PCRs with primer/probe reagents in both monoplex and selleck products multiplex were tested in parallel. In addition we tested the reproducibility of quantification with positive control DNA of S. pneumoniae, H. influenzae and N. meningitidis in separate tubes and combined in a single tube. fucK PCR The fucK PCR was used as previously described [28], to confirm the presence of H. influenzae in samples which proved negative by culture but positive by qmPCR. lytA PCR For respiratory samples the lytA PCR was tested in a gel based PCR for S. pneumoniae as previously described [29].

In short, extracted DNA (10 μL) was Protein tyrosine phosphatase added to a PCR mixture, and after 40 cycles, PCR products were detected on ethidium bromide-stained agarose gels. By serial dilution of bacterial strains, the detection level of lytA PCR has been shown to be 102 colony forming units (CFU)/mL sample [29]. 16 S rRNA PCR for CSF samples The primers and other PCR conditions used to amplify the 5′-half of the 16 S rRNA gene were previously described [24]. The PCR product was visualized in an agarosegel and DNA bands of expected size were cut from the gel, purified with a Qiaquick Gel Extraction kit (Qiagen) and subjected to cycle sequencing using the ABI prism Big Dye Terminator Sequencing Ready Reaction kit, v.1.1 (Applied Biosystems). The sequencing reaction products were analyzed using an ABI PRISM 310 Genetic Analyser (Applied Biosystems). After DNA sequence editing, the GenBank BLAST program was used for sequence comparisons.