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Cultivation-independent molecular biological methods are essential to rapidly quantify pathogens like Legionella pneumophila (L. pneumophila) which is important to control aerosol-generating engineered water systems. A standard addition method was established to quantify L. pneumophila in the very complex matrix of process water and air of exhaust air purification systems in animal husbandry. Therefore, cryopreserved standards of viable L. pneumophila were spiked in air and water samples to calibrate the total bioanalytical process which includes cell lysis, DNA extraction, and qPCR. A standard addition algorithm was employed for qPCR to determine the initial concentration of L. pneumophila. In mineral water, the recovery rate of this approach (73%–134% within the concentration range of 100–5000 Legionella per mL) was in good agreement with numbers obtained from conventional genomic unit (GU) calibration with DNA standards. In air samples of biotrickling filters, in contrast, the conventional DNA standard approach resulted in a significant overestimation of up to 729%, whereas our standard addition gave a more realistic recovery of 131%. With this proof-of-principle study, we were able to show that the molecular biology-based standard addition approach is a suitable method to determine realistic concentrations of L. pneumophila in air and process water samples of biotrickling filter systems. Moreover, this quantification strategy is generally a promising method to quantify pathogens in challenging samples containing a complex microbiota and the classical GU approach used for qPCR leads to unreliable results. [ABSTRACT FROM AUTHOR]