Poster: Diseases of Plants: Disease Detection & Diagnosis
535-P
Development of a field-based detection technique for Rose rosette virus using isothermal reverse transcription-recombinase polymerase amplification
B. BABU (1), B. Washburn (2), S. Miller (2), F. Ochoa-Corona (3), G. Knox (1) (1) North Florida Research and Education Center, University of Florida, U.S.A.; (2) Department of Biological Science, Florida State University, U.S.A.; (3) Department of Entomol
Rose rosette disease, caused by Rose rosette virus (genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and is inconsistent in detecting the virus even from symptomatic plants. Hence a novel probe-based isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, based on the nucleocapsid gene of the RRV, has been developed. The assay is highly specific as tested against other common rose infecting viruses (both inclusive and exclusive). Dilution assays using the in vitro transcript showed that the assay is highly sensitive, with a detection limit of 1 fg. Moreover a rapid technique for the extraction of viral RNA (< 5 min) has been standardized from RRV infected tissue sources, using a single buffer which facilitates virus adsorption, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants indicated that the virus could be detected from leaves, stems, petals, primary roots and secondary roots. The entire process including the extraction can be completed in less than 15 min, with less sophisticated equipments. The developed assay is currently evaluated for efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscape.