Chip-based duplex real-time PCR for water quality monitoring concerning Legionella pneumophila and Legionella spp.
in: Water Research (2020)
Based on biomolecular methods, rapid and selective identification of human pathogenic water organisms becomes an important issue. Legionella spp, are gram negative pathogenic water bacteria with worldwide significance. Prevalent detection methods for these microorganisms are time and/or cost intensive. We describe a detection setup and relating DNA assay which has the advantage of lower amount of reagents and rapid analysis. A miniaturized real-time polymerase chain reaction (real-time PCR) for direct on-line discrimination of Legionella pneumophila and Legionella spp. was established and integrated into a real-time PCR-chip-system. The PCR-chip device combines a temperature controlling unit and a fluorescence intensity measurement. It was designed to achieve rapid real-time DNA amplification, using an approach of real-time fluorescence read out with the intercalating dye EvaGreen® and melting curve analysis, without requiring multiple probes. To show applicability, sensitivity and specificity of this detection system, the identification of different Legionella species is demonstrated. The presented results exhibit reproducibility and good sensitivity, showing that the setup is suitable for robust and cost-efficient detection and monitoring of a variety of Legionella spp.in urban water samples.