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Title: Molecular detection of the Luteoviridae
Author: Chomic, Anastasija
Degree: Doctor of Philosophy
Institution: Lincoln University
Date: 2011
Item Type: Thesis
Abstract: The Luteoviridae is a family of single stranded positive sense RNA plant viruses which cause yield losses in many important food crops worldwide and are therefore of significant economic concern for some countries. Fast and accurate detection and identification is important for strategies designed to control the spread of Luteoviridae species and to reduce their economic impact. This study offers significant advances on current molecular protocols for their detection and differentiation by being taxonomically broad-ranging, time-efficient, sensitive to asymptomatic detection and with the potential to pick up unknown sequence isolates. Current molecular detection and identification tools for the Luteoviridae are mainly species specific, which limits their application for fast and accurate detection and identification. Firstly, the development of new generic primers for the family was investigated. Using the two-step reverse transcription polymerase chain reaction (RT-PCR), thirteen out of sixteen Luteoviridae species analysed were detected using three separate combinations of low-degeneracy generic primers, targeting the coat protein gene region. A synthetic positive control containing all primer sequence priming sites was designed as a generic tool for use with a variety of host plants and the Luteoviridae species. The Luteoviridae primers described in this study present a simple infection-detection tool which will be of benefit to biosecurity authorities in nursery-stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as-yet undiscovered species within the family. Secondly, the suitability of a two-step reverse transcription real time PCR (RT-qPCR) plus melting curve analysis (MCA) as a tool for the rapid detection and discrimination of Luteoviridae species was investigated. Melting temperature and shape of the melting peak were analysed for 13 Luteoviridae species using SYBR® GreenERTM fluorescent dye. Specific melting peaks were observed for all isolates investigated, however due to the high variability of sequences for some members of this family, different melting temperatures were also observed between different isolates of some species. Nevertheless, discrimination was achieved among 6 species. MCA, in this study, was demonstrated to be a faster and more discriminatory alternative to gel electrophoresis of end-point PCR products for the detection of Luteoviridae infection. Thirdly, this study evaluated two combinations of generic Luteoviridae primers for the detection of an early double-infection with BYDV-PAV and CYDV-RPV in barley and oats via RT-qPCR at the early stage of virus infection (3-15 days post inoculation (dpi)). The distribution of viruses in young and old leaves for optimising plant tissue collection strategies was also considered. Quantitative data from this study indicate that in some plants the titre of both luteoviruses is comparable to the expression of the plant mitochondrial gene nad5 (used as an internal RNA control) as early as 3 dpi and that titre differs greatly between individual plants. This study also suggests that virus distribution in different parts of the plant is probably host dependent; while young barley leaves at 9 dpi had a higher titre of both BYDV-PAV and CYDV-RPV viruses than old leaves, no such trend was observed in oats over the experimental period. Lastly, this study investigated the performance of the generic primers in several multiplex situations using both RT-PCR and RT-qPCR-MCA. Multiplex RT-PCR using seven generic primers resulted in some non-specific amplification, which although of non-viral origin, significantly impacted on the use of such an assay. Contrary to RT-PCR, multiplex RT-qPCR was shown to be a good solution for detection and discrimination of BYDV-PAV and CYDV-RPV infection in a range of samples and has the potential to be used in diagnostics. Using this work as a model, similar assays based on more versatile generic primers could be designed for other plant virus groups or other pathogens.
Supervisor: Armstrong, Karen
Pearson, Michael
Hampton, John
Persistent URL (URI):
Appears in Collections:Bio-Protection Research Centre
Doctoral (PhD) Theses

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