Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

• Published in: Journal of microbiological methods 2007, Vol.68 (1), p.137-144
• Main Authors: Behets, Jonas, Declerck, Priscilla, Delaedt, Yasmine, Creemers, Bart, Ollevier, Frans
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cooling circuit water; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Duplex real-time PCR; Exogenous internal positive control; Fundamental and applied biological sciences. Psychology; Legionella pneumophila; Legionella pneumophila - genetics; Legionella pneumophila - isolation & purification; Legionnaires' Disease - microbiology; Microbiology; Polymerase Chain Reaction - methods; Sensitive detection; Sensitivity and Specificity; Water Microbiology; Water Supply
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2006.07.002

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1–15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean™ Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.