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Enrichment of Staphylococcus aureus via specific aptamers and detection by magnetic particle enhanced surface plasmon resonance spectroscopy

 
: Rüppel, Nadine
: Horn, Marcus

Bayreuth, 2016, VII, 88 pp.
Bayreuth, Univ., Master Thesis, 2016
English
Master Thesis
Fraunhofer IZI ()

Abstract
The facultative pathogen Staphylococcus aureus is a threat to general health-care due to the rising prevalence of strains that exhibit resistances towards substances of one or more classes of antibiotics. Rapid and precise diagnostic tests are pivotal to prevent further spread of these bacteria and ensure appropriate treatment of the patients. This work was performed to provide proof-of-concept for different methods that are planned to be implemented in a new diagnostic, PCR-based platform. The S. aureus specific gen of the thermostable nuclease (nuc) was used as example.
The first step in the hypothetical workflow is the enrichment and purification of S. aureus via aptamer coupled magnetic nanoparticles. This is possible due to the ability of the aptamers used to recognize specific epitopes on the pathogen’s surface. The experiments conducted revealed no considerable binding of the bacteria to the modified beads, even though functionality of several aptamers was confirmed before.
After enrichment, cell disruption of S. aureus is necessary. Thermal lysis with an adequate buffer proved to be most efficient when the lysate was analyzed by quantitative real-time PCR (qPCR) to detect the number of bacteria.
The next step is amplification of the released, genomic DNA by PCR-on-a-bead. In this method one primer is attached to the nanoparticles resulting in immobilization of the PCR-products. The functionality of this principle was verified by an enzymatic assay which was optimized for background reduction. In addition, determination of two methods for removing the complementary, uncoupled DNA strand was achieved by using this method.
Finally, the PCR-on-a-bead products are to be detected by a surface plasmon resonance (SPR) spectrometer developed by the Fraunhofer IWS via hybridization with a complementary oligonucleotide probe. Several probes were generated for this purpose and the specific association of an asymmetric PCR product was verified. Additionally, SH-PEG-OH was identified to be a suitable blocking agent for reducing nonspecific binding and the ability to reuse the SPR-chip at least three times by regeneration of the surface. Furthermore, the reproducibility of the experiments was investigated and functionalization by manual pipetting determined to give the most stable results. The proposed signal amplification by magnetic particles was confirmed. Nevertheless, no association of the PCR-on-a-bead products could be detected.

: http://publica.fraunhofer.de/documents/N-445775.html