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Title: Exploring the application of nucleic acid aptamers for detection of food contaminants and viruses
Author: Derbyshire, Nicola
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Abstract:
Use of antibiotic, anti-fungal and anti-parasitic agents is required to protect from infections that can rapidly spread making entire crops unsuitable for human consumption or threaten the health of livestock. However, these agents can have toxic side effects on humans at high concentrations so their residual levels in food are regulated. Their levels are monitored through both random and routine surveillance checks that generate ~ 50 000 samples per year in the UK alone. This is not an easy task because many of these compounds are small molecules that require extensive sample extraction followed by analysis carried out by highly trained staff on sophisticated equipment, such as Liquid Chromatography–Tandem Mass Spectrometry. A simple, rapid and cheap method for screening these samples would therefore be beneficial in reducing the cost and time burdens of this monitoring. Nucleic acid aptamers are reagents that can be selected by a well-established in vitro protocol to bind to a wide range of target molecules. The flexibility of nucleic acids as primary capture ligands compared to antibodies means that they can be readily incorporated into rapid biosensors. Here I describe the selection and characterisation of aptamers against three classes of regulated residues, aminoglycoside (AMG) antibiotics, and the anti-fungal strobilurins and malachite green. The anti-AMG aptamers were toggle selected in an attempt to generate reagents able to recognise the entire class of drugs. Novel aptamers with such binding properties and Kd’s in the ~100 nM range were obtained. Some of these have been used in a simple and rapid biosensor based on aptamers physi-sorbed onto the surface of gold nanoparticles (GNPs). This proof of principle assay format showed promise in detecting aminoglycoside antibiotics at or below their permitted maximum residue levels. Next Generation DNA sequencing of the selected anti-AMG pools hints at the origin for the cross-reactivity. When further characterised, the previously selected anti-strobilurin aptamers appear to have specificity for their three selection targets. These aptamers were used to develop a high throughput screening method for aptamer target binding based on electrophoretic mobility shift assays with capillary electrophoresis. The assay has the potential to rapidly assess target binding of many sequences against many targets with limited user input. An anti-malachite green oxalate single stranded RNA aptamer that causes fluorescence on target binding has been previously described in the literature. I explored the potential of the single stranded DNA aptamers that I pseudo-toggle selected against malachite green oxalate and one of its metabolites to induce fluorescence on target binding. It appears that this phenomenon is unique to the single stranded RNA aptamer and malachite green oxalate In a separate attempt to illustrate the versatility of nucleic acid aptamers I have selected aptamers against the purified rabies virus glycoprotein (RVGP), which show both nano-molar affinity for the protein and also appear to have virus-neutralising properties. Rabies is endemic in many parts of the world and both pre- and post-exposure the major treatment relies on vaccination. Each year 100,000s of mice must be sacrificed following severe in vivo treatments in order to comply with the standard NIH-approved assay for vaccine potency. This level of in vivo testing is required because the anti-RVGP antibodies available are too variable in their response in ELISA assays to be a reliable indicator of potency. It is hoped that the selected aptamers will contribute to the development of such in vitro screens, thus reducing the need for animal testing.
Supervisor: Stockley, P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.577352  DOI: Not available
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