Visual and magnetic detection of antimicrobial resistance genes using nanoparticles and isothermal amplification

Sammanfattning: Antibiotic resistance is becoming an increasing concern, necessitating new ways to diagnose and treat it rapidly and accurately. One proposed solution is the use of nucleic acid amplification tests, capable of detecting resistance in just a few hours, as opposed to the several days that traditional testing methods may require. One concept for such a test involves the volume-amplified magnetic nanobead detection assay (VAM-NDA), which combines rolling circle amplification (RCA), an isothermal amplification method, with magnetic nanoparticles (MNPs) for the detection of the RCA products.In this thesis, improvements have been made to the VAM-NDA. Coupling of RCA products with microparticles allowed a greater hydrodynamic increase of the MNPs when bound to these products, facilitating the detection of shorter products. Moreover, circle-to-circle amplification (C2CA) was performed, a technique comprising two RCA reactions, increasing the sensitivity of the assay and leading to a 1 attomole limit of detection. Optimization of several reaction parameters has allowed for increased sensitivity. Of highest relevance are the changes made to the length of padlock probes, oligonucleotides used for target recognition. These changes resulted in faster amplification. Equally important is the work conducted on phi29 DNA polymerase. Different manufacturers and mutants of this enzyme were tested, resulting in an improved and faster amplification. However, a previously undiscovered feature of this enzyme was also investigated, where phi29 amplification is competitively inhibited by the presence of ssDNA oligonucleotides, regardless of whether digestion of those is possible or not.Finally, a novel detection method for RCA products was developed, where nanoparticles were made to aggregate with the DNA products, resulting in aggregates visible to the naked eye. This method was further optimized, and integrated with C2CA, as well as substituting MNPs for coloured polystyrene nanoparticles. The result is a fast assay that can detect down to 100 zeptomoles of target DNA visually, in less than two hours. The use of coloured nanoparticles also allowed multiplexing using two sets of nanoparticles with different colours, and two targets were detected in a single sample.In conclusion, this thesis brings a DNA detection method one step closer to its use in the identification of antimicrobial resistance. The improvements to VAM-NDA and magnetic detection, advances on padlock probe design, the discovery of a novel behaviour of phi29 DNA polymerase, and the potential for naked-eye detection and multiplexing are of high relevance for future basic and translational research.