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.

中文翻译：

---

在生物滴滤器中通过 qPCR 对嗜肺军团菌细胞进行快速、独立于培养的定量的标准添加方法

独立于培养的分子生物学方法对于快速定量嗜肺军团菌（L. pneumophila ）等病原体至关重要，这对于控制产生气溶胶的工程水系统非常重要。建立了标准添加方法来定量畜牧业废气净化系统的工艺水和空气的非常复杂的基质中的嗜肺军团菌。因此，将活嗜肺军团菌的冷冻保存标准品添加到空气和水样中，以校准整个生物分析过程，包括细胞裂解、DNA 提取和 qPCR。 qPCR 采用标准添加算法来确定嗜肺军团菌的初始浓度。在矿泉水中，该方法的回收率（在每毫升 100-5000 个军团菌的浓度范围内为 73%-134% ）与使用 DNA 标准品进行传统基因组单位 (GU) 校准获得的数据非常一致。相比之下，在生物滴滤器的空气样品中，传统的 DNA 标准方法导致了高达 729% 的显着高估，而我们的标准添加则给出了 131% 的更实际的回收率。通过这项原理验证研究，我们能够证明基于分子生物学的标准添加方法是确定生物滴滤系统的空气和工艺水样品中嗜肺军团菌实际浓度的合适方法。此外，这种定量策略通常是一种很有前景的方法，可以对含有复杂微生物群的挑战性样品中的病原体进行定量，而用于 qPCR 的经典 GU 方法会导致结果不可靠。