Duplex amplification method for in situ rapid detection of hygiene-relevant germs on flexible endoscopes
IGF 20900 N
Aim of the research project was an in-situ rapid detection of hygiene-relevant germs on internal and external endoscope surfaces in accordance with Annex 10 of the guideline for the validation of endoscope reprocessing procedures.
The detection of individual hygiene-relevant germs was based on selective labelling with aptamers from DNA, which can be quantitatively detached from the germs again after labelling by thermal denaturation and eluted from the endoscope.
Using flow cytometry, it could be determined that up to 40 % of the cells could be labelled using an aptamer against Enterococcus faecium.
Thermal detachment of bound aptamers could be demonstrated at 60 °C. Recovered aptamers could be bound by hybridisation to complementary DNA segments (oligonucleotides) on specific evaluation fields for the detection of individual microbial species (e.g. S. aureus, Enterobacteriaceae, etc.).
Spherical DNA hybrid aggregates (SDHA) were developed for the detection of the bound aptamers; these could be prepared using gold nanoparticles with a diameter of 20 nm or using carboxylated latex beads with a diameter of 1 µm by binding oligonucleotides with a partially complementary sequence to the aptamers to be detected.
Other oligonucleotides immobilised on the surface of the SDHA served as initiators to trigger a high number (1st amplification) of hybridisation chain reactions (HCR).
In order to be able to form fluorescent hybridisation products on the surface of the SDHA by means of HCR, special fluorophore-functionalised metastable hairpin oligonucleotides were developed, which enabled the formation of high-molecular and strongly fluorescent hybridisation products within 10 min, composed of more than 500 individual hairpin oligonucleotides (2nd amplification), thus enabling sensitive visual detection.
The duplex amplification method provides cleaning and hygiene service providers, who are increasingly entrusted with the reprocessing of medical devices, and the reprocessing units for medical devices operated by the medical facilities themselves, with a rapid detection method that can be used in-house and cost-effectively for independent, in-house testing of the reprocessing quality of flexible endoscopes.
The research report is available on request from FRT.