A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase
Binoy Babu1, Brian K Washburn2, Tülin Sarigül Ertek3
1North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Quincy, FL 32351, USA.
Journal of Virological Methods
|June 7, 2017
Summary
A new rapid, field-deployable assay accurately detects Rose rosette virus (RRV) in plants. This isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) method offers a faster, more accessible alternative to current diagnostics for managing this rose industry threat.
Area of Science:
- Plant Pathology
- Molecular Virology
- Agricultural Diagnostics
Background:
- Rose rosette disease, caused by Rose rosette virus (RRV), poses a significant threat to the U.S. rose industry.
- Current diagnostic methods like RT-PCR and RT-qPCR are time-consuming, lab-dependent, and not suitable for field use.
- Effective management relies on early detection and eradication of infected plants to limit disease spread.
Purpose of the Study:
- To develop a novel, rapid, and field-deployable diagnostic assay for Rose rosette virus (RRV).
- To create a sensitive and specific method for detecting RRV in various rose plant tissues.
- To provide an accessible alternative to existing laboratory-based diagnostic techniques.
Main Methods:
- Development of a probe-based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay targeting the RRV nucleocapsid gene.
- Standardization of a rapid viral RNA extraction technique (<5 minutes) using PBS-T buffer.
- Validation of the RT-exoRPA assay on various rose plant tissues (leaves, stems, petals, pollen, roots) and cultivars across different U.S. states.
Main Results:
- The developed RT-exoRPA assay demonstrated high specificity, showing no cross-reactivity with other rose-infecting viruses.
- Sensitivity was high, with a detection limit of 1 fg/μl for in vitro transcripts.
- The complete diagnostic process, including RNA extraction, was completed in under 25 minutes using minimal equipment.
- RRV was successfully detected in all tested plant parts and across diverse rose varieties.
Conclusions:
- The novel RT-exoRPA assay provides a rapid, sensitive, and specific tool for field-based detection of Rose rosette virus (RRV).
- This assay significantly improves upon existing diagnostic methods, enabling faster and more widespread application in nurseries and landscapes.
- The developed method facilitates efficient large-scale field testing, crucial for managing RRV and protecting the rose industry.
