Convergence amplification and fluoroswitch detection for the determination of hygiene-relevant ESKAPE pathogens

IGF 21186 N

Since cleaning and hygiene service providers are increasingly entrusted with surface disinfection in medical facilities they should be provided with an inexpensive rapid test device for checking the hygiene status of disinfected surfaces in-house.

On the one hand, the detection results in economic advantages for cleaning and hygiene service providers, and on the other hand it improves hygiene in medical facilities and reduces the risk of nosocomial infections in accordance with § 23 of the German Infection Protection Act (IfSG).

The goal was therefore to develop a cultivation-independent test for simultaneous detection of all six nosocomial ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.).

The simultaneous detection of individual cells is based on an isothermal convergence amplification of specific DNA target sequences, which produces a uniform product (the amplicon) as a sum parameter.

The amplicon itself is detected by an immobilised fluoroswitch in which it changes the conformation of the fluoroswitch in such a way that it switches from a quenched (switch off) to a fluorescent conformation (switch on).

During the project, suitable target sequences were identified in the 16S rDNA and the convergence amplification was successfully developed. By combining both amplification steps in a single reaction, the detection of target sequence concentrations of ≥2 nM was achieved.

The method enabled the amplification of single-stranded target sequences as well as short double-stranded PCR products. Differentiation between DNA of living and inactivated ESKAPE pathogens was successfully demonstrated by applying a propidium monoazide (PMA) treatment.

The fluoroswitch was developed as a model based on ssDNA with terminal fluorophores and quenchers. This model allowed the in-detail characterisation of its on/off switching kinetics and proved very good suitability as a fluoroswitch.

Its conformational change upon binding the amplicon, its immobilisation and its regeneration by RNase treatment could be all demonstrated experimentally. In addition, the coupling of gold nanoparticles (GNP) to ssDNA was successful and it demonstrated the quenching of fluorophores by the NSET effect.

The research results show that rapid detection of ESKAPE pathogens is possible by convergence amplification and downstream fluoroswitch detection, if the accessibility of genomic DNA for amplification is guaranteed, e.g. by a suitable sample preparation.

The research report is available on request from FRT.

The IGF-pro­ject 21186 N of the re­search as­so­ci­a­tion Europäische Forschungs­ge­mein­schaft Reini­gungs- und Hy­gien­etech­nolo­gie e.V., Cam­pus Ficht­en­hain 11, 47807 Krefeld, was sup­port­ed via the AiF with­in the fund­ing pro­gram „In­dus­trielle Gemein­schafts­forschung und -en­twick­lung (IGF)“ by the Fed­er­al Min­istry of Eco­nom­ic Af­fairs and Cli­mate Ac­tion due to a de­ci­sion of the Ger­man Par­lia­ment.