Development of automated nucleic acid technologies for marine point of sample diagnostics
Wilson, M. (2021). Development of automated nucleic acid technologies for marine point of sample diagnostics. PhD Thesis. University of Southampton; Faculty of Engineering, Science and Mathematics; School of Ocean and Earth Sciences: Southampton. 150 pp.
Unsafe water, poor hygiene and inadequate sanitation are responsible for about 90% of diarrheal associated deaths worldwide, which are the second leading cause of death in children under five, globally (1.2 million deaths in 2005). The economic cost associated with unsafe water is also significant, with the World Bank estimating that a lack of access to safe water and sanitation amount to equivalent global economic losses of US$260 billion, annually. Coastal communities also face challenges from harmful algal blooms, which deplete oxygen and essential nutrients, and produce toxins that threaten human health; this impacts on locally important industries such as aquaculture, mariculture, leisure and tourism. Current, widely used methods for the detection of biological hazards in water sources rely on culturing or visual inspection of water samples via light microscopy. These techniques are time consuming, require well stocked laboratories and expensive equipment, are dependent on highly trained technical staff, and are often unable to differentiate morphologically similar organisms. Molecular techniques, on the other hand can provide better discrimination between related taxonomical groups, and are suited for miniaturisation and automation, enabling the analysis to be undertaken outside of the laboratory, and by minimally trained personnel.Recent advances in microfluidic technologies have demonstrated that molecular-biology techniques (e.g. genetic sequence amplification and detection) can be applied using lab-on-a-chip (LOC) systems for testing samples for the target organisms at the ‘point of sample’ or ‘point of care’. Nucleic acid sequence-based amplification (NASBA) and recombinase polymerase amplification (RPA) are particularly attractive for LOC applications as, unlike the current gold standard Polymerase Chain Reaction (PCR), they require simple, isothermal heating and a generally lower reaction temperature. NASBA is also attractive for LOC-based methods targeting RNA (e.g. RNA viruses) as it directly amplifies RNA removing the need for an additional reverse transcription step. This study indicates several, novel advances towards the provision of point of sample genetic testing for a range of harmful microorganisms and viruses. For the first time, the isothermal amplification of a fragment of the Hepatitis E virus using NASBA is reported with comparable sensitivity to a clinically relevant PCR-based assay. Further, amplification of a fragment of the Alexandrium minutum ITS1-5.8s-ITS2 rRNA gene region via RPA was achieved with a limit of detection of 10 cells exceeding the required sensitivity of 40 cells L-1 for statutory A. minutum surveillance in seawater. In addition to this, two methods for the long-term, dry storage of amplification reagents are presented. Trehalose and sucrose sugars combined with lyophilisation were sufficient to retain reagent activity after storage for four weeks at room temperature. Air drying in the presence of pullulan maintained reagent functionality for six weeks. Alongside these advances, a portable, handheld prototype concept for nucleic acid amplification was developed and tested, with the A. minutum ITS1-5.8s-ITS2 RPA assay being successfully performed on the handheld device achieving the same sensitivity as when performed on laboratory equipment. These advances demonstrate that nucleic acid amplification on a miniaturised device is possible, and that in future, rapid, specific and sensitive testing for harmful species may be able to be performed at the point of sample by non-technically trained operators.
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