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.

Transmission attributes and resistance to rose rosette virus

“…Indeed, the new test is more sensitive when compared to the Laney et al (2011) protocol and is able to detect the virus after 20 PCR cycles, conferring confidence to the results and also ensuring nucleic acid quality. Additional detection protocols for RRV were published after the completion of the research presented here (Babu et al, 2016;Dobhal et al, 2016). Notwithstanding, those protocols are based on sequences of 23 isolates whereas the one presented here was based on the sequences of 130 isolates and has been tested against more than 200 isolates collected from 19 states (Katsiani et al, 2017).…”

Resistance to Rose Rosette Virus and Transmission Attributes

“…Indeed, the new test is more sensitive when compared to the Laney et al [18] protocol, able to detect the virus after 20 PCR cycles conferring confidence to the results but also ensure nucleic acids quality (Figure 2). Additional detection protocols for RRV were published after the completion of the research presented here [3,11] and for this reason their sensitivity was not compared with the protocol presented here. Notwithstanding, those protocols are based on sequences of 23 isolates whereas the one presented here has been tested against more than 200 isolates collected from 19 States.…”

Facing Rose rosette virus: A risk to European rose cultivation

“…The first reported RRV detection method consists of an end‐point RT‐PCR with primers designed to amplify a fragment of RNA1 of the RRV genome (Table ; Laney et al ., 2011). Subsequent work showed the initial method to be inconsistent compared to other assays (Babu et al ., 2016), which has led to the development of additional methods.…”

“…Subsequent work showed the initial method to be inconsistent compared to other assays (Babu et al, 2016), which has led to the development of additional methods.…”

“…In the case of RRV, both techniques have a comparable limit of detection. Babu et al (2016) developed multiple primer/probe sets (Table S1) targeting three different regions of the RRV genome. Four primer/probe sets (RRV_2-1, RRV_2-2, RRV_3-2, RRV_3-5) and their corresponding product were tested in silico.…”