A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries

Abstract

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions.

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).…”

Development of a genome-informed loop-mediated isothermal amplification assay for rapid and specific detection of Xanthomonas euvesicatoria

“…oryzicola ; they interpreted that the variation in sensitivity was perhaps due to the differences in primer annealing efficiency. Similarly, reduced ability to detect target DNA in spiked assays possibly resulting from inhibitors present in host DNA 35 . Given that reproducibility is an essential and critical property of a diagnostic assay 36 , multi-operators performed the X .…”

Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay

“…; Dobhal et al . ). However, no testing protocol has been established for this multiplex probe‐based TaqMan real‐time RT‐PCR method to detect viruses in narcissus species.…”

“…Accordingly, the common multiplex real-time RT-PCR method was developed for plant material that was infected with multiple viruses, for example, the potato with the Potato mop top virus (PMTV) and the Tobacco rattle virus (TRV) (Mumford et al 2000), and citrus with the Citrus tristeza virus, the Citrus psorosis virus and the Citrus leaf blotch virus (Osman et al 2015). More recently, a branched, robust and high-throughput Taq-Man method has been described for viral detection in different ornamental crops (Boonham et al 2002;Wei et al 2012;Chen et al 2013a,b;Lin et al 2013;Tang et al 2014;Dobhal et al 2016). However, no testing protocol has been established for this multiplex probe-based TaqMan real-time RT-PCR method to detect viruses in narcissus species.…”