Catalytic Aptazym reporter systems for fungal spore detection
IGF 19702 N
Mould infestation in buildings is an enormous economic problem. According to a study of the “Institut für Bauforschung e.V.” and the “Bauherren-Schutzbund e.V.” mould infestation damage represents more than 10 % of the overall building-related damage and annual costs of 4 billion € (only on residential buildings).
Furthermore, moulds inside buildings are not only a hygienic problem, but even a serious health hazard for immunocompromised persons. In 80 % of the cases, the fungal growth is not visible and detection as well as quantification of fungal spores in ambient air currently require complex and time-consuming microbiological methods.
With this in mind, a rapid and cost-effective detection of fungal spores in ambient air based on catalytic aptazyme reporter systems was developed: For this purpose, aptamers were identified that are directed against the target structures -glucan or chitin in fungal cell wall. By coupling the aptamers with a trans-acting DNAzyme (e.g. DET22-18) several aptazymes were generated.
The reporters were DNAzyme substrates with detection domains where the cleaving site is located between a fluorophore and a quencher. A fluorescence signal was generated when substrate is cleaved by the DNAzyme domain of trans-acting aptazymes bound to fungal spores via their aptamer domain.
After incubation with substrate, microscopic analysis of spores coated with aptazymes showed that substrate cleavage occurred at spore surface. As a result of the separation of fluorophore and quencher during substrate splitting, the spores began to fluoresce. A fluorescence microscopic detection as well as a quantification of fungal spores using catalytic aptazyme reporter systems proved to be possible.
In exemplary studies, no binding of the β-glucan aptamer and chitin aptamer or of aptamer domain of the developed aptazymes could be detected to bacterial germs collected from indoor air.
Such a detection system for fungal spores would enable cleaning service providers and facility management companies to recognize elevated spore concentrations in ambient air at an early stage to immediately take appropriate counter measures. Timely removal of mould infestations can reduce maintenance and restauration costs of the building and by application of the precautionary principle would minimize potential health risks.
The research report is available on request from FRT.