5 μM of primers PAO1 S Nutlin 3a and PAO1 A in combination with agarose gel electrophoresis and ethidium bromide staining and by oprL real-time PCR with 0.5 μM of primers PAO1 S and PAO1 A and 0.1 μM of TaqMan probe oprL TM (Table 3). Table 3 Sequences of primers and probes used Primer/Probe 5′-3′ Sequenced Amplicon size (bp) Reference or source PAO1 Sa PAO1 Aa ACC CGA ACG

CAG GCT ATG-TET CAG GTC GGA GCT GTC GTA CTC 92 TIB Molbiol oprL Fa oprL Ra ATG GAA ATG CTG AAA TTC GGC CTT CTT CAG CTC GAC GCG ACG 504 [13, 28] oprL-LC-FAMb TGC GAT CAC CAC CTT CTA CTT CGA GT-FAM / TIB Molbiol oprL-LC-ROXb ROX-CGA CAG CTC CGA CCT GAA G / TIB Molbiol oprL TMc FAM-AGAAGGTGGTGATCGCACGCAGA-BBQ / TIB Molbiol a Primers b HybProbes c TaqMan Probe. d TET, FAM and ROX are fluorescent labels. BBQ: BlackBerry quencher The DNA-extraction protocol, which enabled the most sensitive detection as assessed by these two PCR formats, was used to compare different PCR and real-time PCR formats. PCR and real-time PCR formats Depending on the type of PCR,

detection of P. aeruginosa was done using two primer sets (Table 2 and Table 3). Both primer sets are targeting the oprL gene because available sequences of different isolates show that this gene is highly conserved http://​www.​pseudomonas.​com/​related_​links.​jsp#alleles. A total of six PCR formats (incl. 4 real-time PCR formats) see more were compared. Conventional PCR, using the Veriti 96-Well Thermal Cycler (Applied www.selleckchem.com/products/azd0156-azd-0156.html Biosystems, Foster City, Ca.), was done with primers PAO1 S (TET-labeled) and PAO1 A, whereby PCR products

were subsequently visualized either with agarose gel electrophoresis and ethidium bromide staining or with fluorescent capillary electrophoresis. Agarose gel electrophoresis was carried out at 100 V on an agarose gel of 2.5% (w/v), containing 1 mg/ml ethidium bromide and visualized on a UV transilluminator at 540 nm. For capillary electrophoresis, 1 μL of PCR product was added to a mixture of 12 μL deionised formamide, 0.3 μL ROX-labeled GS-400 high-density size standard and 0.2 μL ROX labeled GS-500 size standard. This mixture was then electrophoresed on an ABI PRISM 310 (Applied Biosystems), as described 5 FU previously [35]. Of the four real-time PCR formats, three were carried out on the LightCycler 1.5 Instrument (Roche) using three different LightCycler real-time PCR kits, all with an optimized MgCl2 concentration, i.e. LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche), LightCycler FastStart DNA MasterPLUS HybProbe (Roche) and LightCycler Taqman Master (Roche) and one was carried out on the ABI7000 instrument, using the commercially available TaqMan Pseudomonas aeruginosa detection kit (Applied Biosystems). For all of these PCR formats, the PCR mixes were prepared as recommended by the manufacturer and also the PCR programs were carried out as prescribed by the manufacturer.