Droplet digital PCR for the detection and monitoring of Legionella pneumophila

Abstract

Legionella pneumophila (L. pneumophila) is a harmful pathogen often found in water systems. In hospitals, the absence of L. pneumophila in water systems is mandatory by law, therefore, frequent and effective monitoring of water is of fundamental importance. Molecular methods based on reverse transcription-quantitative polymerase chain reaction (RT-qPCR) have been proposed for the detection of L. pneumophila, however, the sensitivity and accuracy of these methods have not been validated yet. Therefore, it is important to evaluate other strategies able to overcome the limits of culture-based and RT-qPCR methods. On these bases, we compared the sensitivity and accuracy of droplet digital PCR (ddPCR) and RT-qPCR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments. ddPCR showed a higher sensitivity rate and accuracy compared to RT-qPCR in detecting low bacterial load. In addition, ddPCR is not affected by the presence of fragmented DNA and showed higher accuracy than RT-qPCR in monitoring the efficacy of heat shock treatments. In conclusion, ddPCR represents an innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness, ddPCR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.