Development of a visible label-free polymer-based diagnostic for detection of amplified DNA

Abstract

The current gold standard diagnostic for bacterial infections is the use of bacterial culture, which can be time consuming and can take up to five days for results to be reported. There is therefore an unmet clinical need for a rapid and label free alternative. This thesis demonstrates a method of detecting the presence of amplified DNA from bacterial samples to ~0.78 ng µl -1 using a sterically stabilised, cationic polymer latex and widely available equipment, providing an accessible alternative DNA detection technique within a 30 minute timescale at an overall volume of 50 µl. If DNA is present in a sample, successful amplification by polymerase chain-reaction (PCR) results in the amplified DNA inducing flocculation of the polymer latex followed by rapid sedimentation. This results in a visible and obvious change from a milky-white dispersion to a precipitated latex with a colourless and transparent supernatant, thus giving a clear visual indication of the presence or absence of amplified DNA. Specifically, the responses of polymer latexes with different morphologies were investigated. These latexes were primarily added to amplified DNA from Pseudomonas aeruginosa to observe flocculation. Evidence of sedimentation was analysed using disc centrifuge photosedimentometry (DCP), aqueous electrophoresis, dynamic light scattering, UV-Vis spectrophotometry and digital images. Using these techniques, it was found that the optimal candidate latex for detection of bacterial DNA using flocculation was ~700 nm sterically-stabilised PEGMA-P2VP prepared by emulsion polymerisation. The sensitivity and rate of sedimentation of the PEGMA-stabilised P2VP latex was demonstrated by varying the sequence length and concentration of amplified DNA. Further applications of this method of detecting amplified DNA were investigated using additional bacteria Escherichia coli and Staphylococcus aureus. Antibiotic resistance genes were amplified using PCR and it was also demonstrated that the PEGMA-P2VP latex flocculated in the presence of these bacteria and methicillin-resistance S. aureus (MRSA). Viral targets were also investigated and the specificity of this method was demonstrated by showing that this is specific to the species amplified, by using PCR product from a fungus (Candida albicans). Herein it has been demonstrated that this methodology could provide a user friendly, point-of-care initial test for bacterial infections.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Gavin Humphreys (Supervisor) & Lee Fielding (Supervisor)