Improving and developing diagnostics for high throughput identification of viruses, with a focus on begomoviurses

Abstract

Begomoviruses are a large group of over 400 plant viruses that infect many dicot vegetables and crops throughout the subtropical and temperate regions of the world. Australia currently has only five known begomovirus species. Thus, the biosecurity threat posed by potential incursions is significant. To date, detection of the different species and strains relies on several different generic PCR’s. To simplify begomovirus detection, we set out to develop assays with the capacity for in-field diagnostics. We developed a novel tissue blot hybridization chain reaction (TB-HCR). In this assay, plant stems are blotted onto nitrocellulose membrane tissue blots and then probed with specific probes designed to bind to either a family or group of viruses or specific viruses. During the assay one probe or several different probes can be used for screening at the same time. After the probe binds to the virus, labelled RNA hairpins (HP) are added and bind to the probe initiating a hybridization chain reaction. We have also developed a generic begomovirus Recombinase Polymerase Amplification (RPA) lateral-flow assay which to date, has successfully detected eleven different begomoviruses from Australia, Timor-Leste and PNG. We have also tested specific LAMP assays for agriculturally important begomoviruses in Australia. Our results with begomoviruses and other virus species, and the advantages and disadvantages of these novel assays will be discussed.