e. respectively an average of 2 and 3 colonies were counted example at dilution eight. For Cetrimide Broth (CB) the detection range was also comparable with that of MCA and CA, i.e. P. aeruginosa could be detected up to dilution eight, but the number of colonies was too high to be countable (Table (Table11). Table 1 Comparison of the sensitivity of different DNA-extraction protocols as assessed by means of conventional PCR combined with agarose gel electrophoresis and by real-time PCR on LightCycler using TaqMan probe Based on these results, the number of culturable cells in the original sputum preparation was calculated to be 1.6 log8 cfu/ml. Comparison of DNA-extraction protocols For each sputum dilution, DNA was extracted by four protocols using the bioM��rieux easyMAG Nuclisens semi-automated DNA-extractor and by the protocol for the manual High Pure PCR Template Preparation Kit (Roche).

Results are listed in Table Table1.1. In our hands, the BioM��rieux easyMAG Nuclisens protocol Generic 2.0.1, combined with proteinase K pretreatment, was the DNA-extraction protocol that enabled the most sensitive detection of P. aeruginosa from sputum of CF patients, both with conventional and with qualitative PCR, giving amplification of the P. aeruginosa oprL target gene up to dilutions 6 and 8, respectively. This DNA-extraction protocol was used further to compare a total of two different conventional PCR and four different (quantitative) real-time PCR formats.

Comparison of different PCR and real-time PCR formats Conventional PCR, using the Veriti 96-Well Thermal Cycler (Applied Biosystems), combined with visualisation of the PCR products by agarose gel electrophoresis and ethidium bromide staining respectively by capillary electrophoresis and fluorescence measurement, was compared with three different real-time PCR formats using the LightCycler 1.5 (Roche) and with a commercially available P. aeruginosa specific real-time PCR (TaqMan assay) using the ABI7000 (Applied Biosystems). One real-time PCR format used SybrGreen fluorescence as the detection method, whereas the other three real-time PCR formats relied on the fluorescence generated by probes for detection. Results are listed in Table Table2.2. For the conventional PCR, combined with agarose gel electrophoresis, P. aeruginosa DNA could be detected up to dilution 6, while with capillary electrophoresis amplified P.

aeruginosa DNA could be detected up to dilution 7. P. aeruginosa DNA could be detected up to dilution 7 with real-time PCR using SybrGreen, and up to dilution 8 with real-time PCR with the Hybprobes, with the TaqMan probe and with the commercial Pseudomonas aeruginosa TaqMan probe detection kit on the ABI7000. Anacetrapib In conclusion, the three probe based real-time PCR formats were the most sensitive molecular assays. Table 2 Comparison of the sensitivity of the different PCR formats for sputum dilutions extracted with easyMAG Generic 2.0.