Standard addition method for rapid, cultivation-independent quantification of Legionella pneumophila cells by qPCR in biotrickling filters.

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.

A chemiluminescence-based heterogeneous asymmetric recombinase polymerase amplification assay for the molecular detection of mycotoxin producers.

The analysis of mold in indoor air is a prominent topic but it is hardly dealt with. The most affected fields of this issue are residential- and occupational safety since mold can have a number of impacts on human health. To date the most used methods for quantification of microorganism contamination in indoor air are culture- or microscopy-based and are not capable of translating the on-site situation to analytical data reliably. Here we present a chemiluminescence-based method to detect mycotoxin producers through isothermal amplification of mycotoxin biosynthesis genes using glass and polycarbonate carriers. In this proof-of-principle study, zearalenone producers were aimed to be detected by heterogeneous asymmetric recombinase polymerase amplification (haRPA). For this, an appropriate lysis method for fungal spores was developed allowing rapid access to DNA. A system calibration with spores of Fusarium culmorum as zearalenone-producing organism resulted in an LOD of 2.7 × 105 spores per ml. The system was shown to be specific for zearalenone producers. This work presents the first application of a heterogeneous isothermal amplification for rapid detection and quantification of mycotoxin producers. In the future, a multiplex detection can be possible by haRPA.