title: Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.
abstract: Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.
authors.affiliation: North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Quincy, FL 32351, United States. Electronic address: binoybabu@ufl.edu.
authors.forename: Binoy
authors.initials: B
authors.lastname: Babu
authors.affiliation: Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, United States.
authors.forename: Ayyamperumal
authors.initials: A
authors.lastname: Jeyaprakash
authors.affiliation: Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, United States.
authors.forename: Debra
authors.initials: D
authors.lastname: Jones
authors.affiliation: Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, United States.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Schubert
authors.affiliation: Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, United States.
authors.forename: Carlye
authors.initials: C
authors.lastname: Baker
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306, United States.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Washburn
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306, United States.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Miller
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306, United States.
authors.forename: Kristina
authors.initials: K
authors.lastname: Poduch
authors.affiliation: North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Quincy, FL 32351, United States.
authors.forename: Gary W
authors.initials: GW
authors.lastname: Knox
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Ochoa-Corona
authors.affiliation: North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Quincy, FL 32351, United States. Electronic address: paret@ufl.edu.
authors.forename: Mathews L
authors.initials: ML
authors.lastname: Paret
mesh_terms: D017931:DNA Primers; D018076:DNA, Complementary; D019251:Nucleocapsid / Q000235:genetics; D010935:Plant Diseases / Q000517:prevention & control / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D027826:Rosa / Q000821:virology*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Diagnostics; Emaravirus; Rose rosette virus; TaqMan RT-qPCR