Robin Mccullogh (MSc Thesis 2018)
Development of a point-of-care Loop-mediated Isothermal Amplification (LAMP) assay for Amoebic Gill Disease (AGD).
|Supervisor: Irene Cano Cejas (CEFAS, Weymouth).
|Atlantic salmon (Salmo salar) is one of the most popular species in finfish aquaculture, with an expensive multi-step husbandry process. However, within the last few decades, the viability of salmon aquaculture has been hindered by amoebic gill disease (AGD), caused by the parasitic infection of the amoeba Neoparamoeba perurans. The amoeba occupies the gill filaments proliferating and initiating hyperplastic lesions along the gill, causing the fusion of filaments and excessive production of mucus. Untreated infection leads to extreme cardiovascular distress and asphyxiation of the individual. Whilst there are several treatments, there are none which ensure complete eradication of the amoebae nor reinfection. Salmon farming industries invest millions of dollars annually in treatment and therefore, early diagnosis of the disease is essential in order to maximise profitability. Currently, laboratory diagnostics such as histopathological staining of the amoebae and PCR diagnostics are needed to confirm the presence of N. perurans, with positive confirmation taking weeks. In such time, the disease can progress and spread to other pens. Therefore, a reliable and rapid method is required to better address AGD infections and thus, it is proposed, the use of Loop-mediated isothermal amplification (LAMP), a PCR based technique that utilizes 6 loop-generating and displacement primers to bind to a specific region of a target gene as well as uses elevated amounts of DNA polymerase for high strand displacement. From the design of LAMP primers targeting the 18S gene of N. perurans, this study validates the LAMP reaction and optimizes a point-of-care protocol by use of a non-destructive sampling method via Isohelix swabs and field compatible DNA extraction protocol. Five different ‘quick and dirty’ DNA extraction methods were tested against a gold standard DNA extraction procedure, the EZ1 extraction robot. Through the utilization of the extraction buffer QuickExtract, a successful DNA extraction can occur in under 20 minutes. In testing this assay using in vivo sampling of AGD positive Atlantic salmon, the assay has a 73% success rate of positively identifying N. perurans, with the protocol taking an hour from sampling to assay. The success of this protocol brings the quality of molecular diagnostics into the field, allowing for rapid and dependable results. To address the reliability in results, a multiplex LAMP assay was designed, although, assay runs observed an unexpected cross reaction. Therefore, further modifications to the protocol require an altered Atlantic salmon elongation factor gene (EF1). Further, an in vivo, live imaging assay was developed using zymosan bioparticles in order to distinguish attachment behavior differences between virulent and non-virulent N. perurans clonal strains. Lastly, this study has initiated whole genome sequencing of N. perurans by DNA extraction from virulent and non-virulent clones, in prospect of incorporating greater specificity of the LAMP assay and successful multiplexing.