A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets

• Published in: Journal of virological methods 2017-02, Vol.240, p.78-84
• Main Authors: Babu, Binoy, Washburn, Brian K., Miller, Steven H., Poduch, Kristina, Sarigul, Tulin, Knox, Gary W., Ochoa-Corona, Francisco M., Paret, Mathews L.
• Format: Article
• Language: English
• Subjects: chemical control; corolla; detection limit; diagnostic techniques; disease control; DNA Primers; Emaravirus; Flowers - virology; genes; Isothermal; leaves; Nucleic Acid Amplification Techniques - methods; Plant Leaves - virology; plant propagation; Plant Stems - virology; Plant Viruses - genetics; Plant Viruses - isolation & purification; quantitative polymerase chain reaction; Recombinase polymerase amplification; Recombinases - genetics; reverse transcriptase polymerase chain reaction; Reverse Transcription; RNA Viruses - classification; RNA Viruses - genetics; RNA Viruses - isolation & purification; Rosa; Rosa - virology; rose rosette disease; Rose rosette virus; Sensitivity and Specificity; stems; Temperature; Viral Proteins - genetics; viruses
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2016.11.014

•Designed multiple RT-RPA primer sets for Rose rosette virus (RRV).•RT-RPA assay for detection of RRV was developed.•The RPA primer sets were highly specific to RRV.•Primer sets were highly sensitive, detecting up to 1fg of virus.•Developed assays are rapid, sensitive and reliable.•Developed assays successfully detected RRV from leaves, stems and flower petals. Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/μL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3–4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment.