Oral: A Multidisciplinary Approach to Combating Rose Rosette Disease: Science to Practice
Development of efficient diagnostic tools to enable rapid, easy-to-use, accurate and affordable detection of Rose rosette virus.
F. OCHOA-CORONA (1), A. A.M. Salazar Aguirre (2), S. Molina Cárdenas (2), A. Olmedo-Velarde (2), S. Dobhal (3), J. Olson (1), B. Babu (4), M. Paret (4) (1) Oklahoma State University, U.S.A.; (2) Universidad de las Fuerzas Armadas ESPE, Ecuador; (3) Kansas
Rose rosette virus (RRV, Emaravirus) is a major threat to the rose industry and early detection and selective eradication of plants are the only alternative available for disease management. Molecular-based assays with potential for technology transfer and/or on site implementation should be easy to use, offering visual detection, reliability and sensitivity to the end user. Loop mediated isothermal amplification (LAMP) and thermophilic helicase dependent amplification (tHDA) are isothermal DNA amplifications that combines some of these criteria and not requiring thermocycler. Primer design criteria for both LAMP and tHDA with self-quenched primers (SqP) were investigated. A rapid 15 min probe based isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay was developed. Broad detection of Emaravirus and species discrimination was targeted with Reverse Transcription polymerase chain reaction (RT-PCR) coupled to High Resolution Melting (HRM) analysis. Also a single primer-set suitable for use with three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting was developed including a clonable synthetic, non-infectious multi-target artificial positive control containing a specific RRV probing site and other five sites for viruses commonly infecting ornamentals. The contribution of these methods within a holistic perspective of RRV diagnostics is presented.