in combination with optimized Pitavastatin order DNA-extraction methods and used in addition real-time PCR to increase PCR sensitivity further. However, using a sputum dilution series of P. aeruginosa, and in accordance to most studies, we found no difference in sensitivity between any of the three culture methods and the most sensitive molecular method, i.e. DNA-extraction with easyMAG protocol Generic 2.0.1 and proteinase K pretreatment combined with any of the three probe-based real-time PCRs. In our hands, culture was more sensitive https://www.selleckchem.com/products/lcz696.html than PCR and SybrGreen based real-time PCR and the difference was even more pronounced when not optimal DNA-extraction methods were used. It
should be noticed that we found no difference between selective and nonselective culture methods, but this may be due to the fact that no bacteria, other than P. aeruginosa in the two P. aeruginosa positive patients, could be cultured from the sputa of the 8 CF patients. As shown
in other studies and confirmed here, the pretreatment of the sample and the DNA-extraction protocol strongly influence the sensitivity of the PCR [27, 28]. The most sensitive molecular detection method was obtained using the easyMAG Generic 2.0.1 protocol with proteinase K pretreatment in combination with real-time PCR with the TaqMan probe or the HybProbes. Previous studies showed already that the easyMAG extractor
is one of the most sensitive and reliable JNK-IN-8 cost methods for DNA-extraction [29–31]. An additional advantage of automated DNA-extraction like easyMAG might be the lower sample processing variability . Because both approaches, i.e. culture and (real-time) PCR, have important advantages as well as drawbacks [14, 20, Protein tyrosine phosphatase 32, 33], in our opinion, both should be or can be combined. PCR technology has the potential to detect the fastidious P.aeruginosa variants, which are not detected by the routinely used classical culture procedures [9, 10], whereas culture yields a complete genome that can be used for e.g. phenotypic susceptibility testing and whole genome based genotyping techniques like RAPD, PFGE and AFLP . Indeed, several of the published studies indicate that there are instances of culture positive PCR negative samples [11, 12, 15] as well as culture negative PCR positive samples [11–13, 18, 19], whereby P. aeruginosa infection can only be reliably demonstrated when both approaches are combined. Conclusion In summary, we showed, by testing P. aeruginosa positive sputum dilution series, that there is no difference in sensitivity for the detection of P. aeruginosa in sputum by selective and non-selective culture and by the most efficient DNA-extraction method combined with the most efficient real-time PCR formats, i.e. the probe-based ones.