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Table S1 Quantitative PCR primer and probe sequences. Primer Primer type Sequence (5' to 3') Reference EubF EubR EubPR PaerF PaerR PaerPR Total bacterial forward Total bacterial reverse Total bacterial Taqman probe P. aeruginosa forward P. aeruginosa reverse P. aeruginosaTaqman probe TCCTACGGGAGGCAGCAGT GGACTACCAGGGTATCTAATCCTGTT FAM-CGTATTACCGCGGCTGCTGGCAC-TAMRA TGCTGGTGGCACAGGACAT TTGTTGGTGCAGTTCCTCATTG FAM-CAGATGCTTTGCCTCAA-TAMRA Nadkarni, 2002 Nadkarni, 2002 Nadkarni, 2002 Shannon, 2007 Shannon, 2007 Shannon, 2007 All quantitative (Q)PCR analyses were performed in triplicate. Total bacterial density was determined using a Taqman assay, in which a 466 bp fragment of the 16S ribosomal RNA gene was amplified, as described previously (Nadkarni et al 2002). P. aeruginosa density was determined using a Taqman assay which amplified a 65 bp fragment of the regA gene, as described previously (Rogers et al 2010, Shannon et al 2007). Details of the relevant primers and probes used are shown in the Table above. Bacterial primers and probe were used at a concentration of 100 nM each, whereas P. aeruginosa-specific primers were used at a concentration of 1000 nM each, and the probe at a concentration of 250 nM. 1 μl (~800 ng) of mixed template DNA (human and microbial) was used in the P. aeruginosa assay. 1 μl of a 100 fold dilution (~8 ng) was used in the total bacterial assay. All PCR reactions were carried out in a total volume of 25 μl in Taqman® Universal PCR Mastermix (Applied Biosystems, Warrington, UK). Quantitative PCR assays were carried out using the Rotorgene 6000 (Qiagen,Crawley,UK) with a temperature profile of 50 °C for 2 min, 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s. For both total bacterial and P. aeruginosa-specific quantitative PCR assays, densities (cfu/ml) were determined by comparison with standard curves generated from bacterial isolates. Nutrient broth cultures of P. aeruginosa (NCTC 12934/ ATCC 27853) were incubated at 37 °C for 16 h, with cfu ml −1 estimated by incubation of dilutions (n=4) on Columbia Blood Agar at 37° for 24 h, followed by colony counts. DNA was extracted from tenfold dilutions of these broth cultures in the same way as for the sputum samples, and RT-PCR was carried out as above on the DNA extracts. The standard curve generated using P. aeruginosa was used for both P. aeruginosa and total bacterial enumeration to allow direct comparisons to be made. Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002). Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology148: 257-266. Shannon KE, Lee DY, Trevors JT, Beaudette LA (2007). Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment.Sci Total Environ382: 121-129.
