Rapid quantification of viable Legionella spp. by isothermal nucleic acid amplification test on chemiluminescence based DNA microarrays.

OBJECTIVE

To overcome the disadvantages of commonly used detection methods for Legionella, like the time-consuming culturing method with a high risk of underestimation or qPCR with a laborious experimental setup and a high risk of overestimation, an isothermal amplification method was chosen. The recombinase polymerase amplification (RPA) works at a constant temperature and can be coupled with a microarray for a simple readout via chemiluminescence. Together with this heterogeneous asymmetric RPA (haRPA), a distinction between life and dead Legionella is done by adding an DNA-intercalating dye, which can block free DNA so no amplification can occur. Because of the constant temperature and the simple readout system, on field measurements can be easily performed. Additionally to that, the microarray surface allows an immobilization of different primer pairs for multiplex analysis for a differentiation between L. pneumophila and other species. In this project, this assay should then be used to monitor air filters of pig farms. These bioactive filters are not suitable for any kind of disinfection, which could lead to a high risk for Legionella contamination. This would result in harmful aerosols for the health of animals and workers. Analyzing these air filter systems over 1 year will give an overview of the state of the art. Further (on field) measurements of a high number of bio filters in pig farms should then be performed to understand whether action need to be taken.

-     Isothermal detection method combined with a chemiluminescence based microarray for fast
      and easy detection of Legionella
-     Monitoring of different air filter systems over a period of one year
-     Evaluating the potential risk of legionella in air filter systems of pig farms
-     Field tests in pig farms

METHODS

Legionella spp. DNA from concentrated water samples is amplified by the haRPA using genomic sequences of the 16S rRNA on a DNA microarray. The amplification can be quantified by a visible chemiluminescence reaction detected with a CCD camera. To distinguish between viable and dead cells a DNA-intercalating dye propidium monazide (PMA) is added. It can pass the cell membrane of dead cells and inhibit the amplification of the haRPA.

INVOLVED PHD STUDENTS

M. Sc. Gerhard Schwaiger

PARTNERS

Thünen-Institut für Agrartechnologie

FINANCIAL SUPPORT

Bundesanstalt für Landwirtschaft und Ernährung (FKZ 28N-2-002-02)