The requirements for prompt diagnosis of foot-and-mouth disease (FMD) during outbreaks, and the need to establish robust laboratory testing capacity within FMD-endemic countries, have motivated the development of point-of-care tests (POCTs) to support current diagnostic strategies. Despite numerous publications detailing the design of platforms and assays for this purpose, the majority have only been evaluated in laboratory settings, using protocols incompatible for use in challenging environments. To address this gap, this thesis describes the development of an end-to-end molecular toolbox for the detection and characterisation of FMD virus (FMDV) RNA in decentralised settings. A critical review and multiway comparison of seven assay formats and 11 sample preparation methods revealed that reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time reverse transcription PCR (rRT-PCR) POCT-formats exhibited comparable analytical and diagnostic sensitivity to their laboratory-based equivalents. Additionally, reagent lyophilisation provided a solution for cold chain and storage considerations, whilst not compromising assay performance. Both assays were compatible with simple sample preparation methods, removing the requirement for nucleic acid extraction. For example, dilution of samples in nuclease-free water enabled FMDV RNA to be detected in multiple sample types (epithelial tissue suspensions, serum, oesophageal-pharyngeal fluid and lesion swabs), from as early as one day post infection. Notably, when the robust field-ready protocols were deployed into challenging low-resource laboratory and field-settings within East Africa, POCT results (rRT-PCR = 144; RT-LAMP = 145) were consistent with clinical observations and a reference rRT-PCR, with FMDV detected from acutely infected as well as convalescent cattle. Furthermore, transitioning of East Africa-specific FMDV-typing rRT-PCR assays (for serotypes O, A, Southern African Territories [SAT] 1 and SAT 2) into a multiplex POCT-format enabled rapid identification of FMDV serotype in situ, confirming active outbreaks of both O and A. This thesis also describes the development of GoPrime, a novel real-time PCR (rPCR) primer/probe validation tool. By parameterising GoPrime with experimental data, collected to investigate the effects of primer/probe-template mismatches on cycle threshold and limit of detection, it was possible to quantitatively predict the performance of rPCR assays in silico. The work of this thesis supports the deployment of molecular POCTs into non-specialised, resource-limited and challenging settings for simple, highly sensitive and rapid detection and/or characterisation of FMDV.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:732721 |
| Date | January 2017 |
| Creators | Howson, Emma Lucy Anna |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/8607/ |
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