Improving and developing diagnostics for high throughput identification of viruses

Abstract

Assays for high throughput screening of crops and weeds for virus monitoring and management need to be quick, easy, and ideally, low cost. Current methods include the use of tissue blot immuno assays (TBIA), where plant stems are blotted onto a nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIA are fast and cheap, but are limited by antibody availability and specificity. One approach we recently used to increase TBIA specificity, was to generate an antibody against the polerovirus soybean dwarf virus (SbDV) via protein expression. The coat protein of SbDV was cloned into an expression vector, expressed, purified, and used to generate a specific SbDV antibody. The generated antibody shows good specificity and sensitivity of SbDV positive isolates in TBIA. This method can used to generate antibodies for specific viruses where there are currently no antibodies available or they are hard to purify, such as poleroviruses. Another and more novel high throughput approach we are developing is the tissue blot hybridization chain reaction (TB-HCR). Similar to TBIA, plant stems are blotted onto a membrane. However, TB-HCR involves using specific probes that can be designed to bind to either a family or group of viruses or specific viruses. During the assay, one probe or a number of different probes can be used for screening at the same time. Following probe binding, labelled small DNA hairpins are added and bind to initiator sequences on the probe causing a cascading unfolding and hybridisation of the hairpins (hydridisation chain reaction). Current advances in the development of this technique in the identification of viruses in pulse crops will be discussed.