Comparison of the sensitivity of culture, PCR and quantitative real-time PCR for the detection of Pseudomonas aeruginosa in sputum of cystic fibrosis patients
Pseudomonas aeruginosa is the major pathogen involved in the decline of lung function in cystic fibrosis (CF) patients. Early aggressive antibiotic therapy has been shown to be effective in preventing chronic colonization.
Therefore, early detection is important and sensitive detection methods are warranted. In this study, we used a dilution series of P.
aeruginosa positive sputa, diluted in a pool of P. aeruginosa negative sputa, all from CF patients - to mimick as closely as possible the sputa sent to routine laboratories - to compare the sensitivity of three culture techniques versus that of two conventional PCR formats and four real-time PCR formats, each targeting the P.
aeruginosa oprL gene. In addition, we compared five DNA-extraction protocols.
Results: In our hands, all three culture methods and the bioMerieux easyMAG Nuclisens protocol Generic 2.0.1, preceded by proteinase K pretreatment and followed by any of the 3 real-time PCR formats with probes were most sensitive and able to detect P.
aeruginosa up to 50 cfu/ml, i.e. the theoretical minimum of one cell per PCR mixture, when taking into account the volumes used in this study of sample for DNA-extraction, of DNA-elution and of DNA-extract in the PCR mixture.
Conclusion: In this study no difference in sensitivity could be found for the detection of P.
aeruginosa from sputum between microbiological culture and optimized DNA-extraction and real-time PCR. The results also indicate the importance of the optimization of the DNA-extraction protocol and the PCR format.
Author: Pieter DeschaghtThierry De BaereLeen Van SimaeySabine Van daeleFrans De BaetsDaniel De VosJean-Paul PirnayMario Vaneechoutte
Credits/Source: BMC Microbiology 2009, 9:244
Published on: 2009-11-29
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
make sure to read our disclaimer prior to contacting 7thSpace Interactive. To contact our editors, visit our online helpdesk. If you wish submit your own press release, click here.
Social Bookmarking
RETWEET This! | Digg this! | Post to del.icio.us | Post to Furl | Add to Netscape | Add to Yahoo! | Rojo
|
|
