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Tobacco mosaic virus ( TMV), Hosta virus X ( HVX), Cucumber mosaic virus ( CMV), Tomato spotted wilt virus ( TSWV) and Impatiens necrotic spot virus ( INSV) are a few of the major viruses that infect ornamental and nursery plants. These viruses cause significant losses that impact growers and the ornamental industry. Often, a single ornamental plant is co-infected by more than one virus, which makes identification and discrimination of these viruses a difficult task, thus creating delays and limiting regulatory measures for effective quarantine. The aim of this study is to develop a sensitive, rapid, economic, and reliable multiplex Reverse Transcription PCR ( RT-PCR) for simultaneous detection and discrimination of these five viruses. Specific PCR primers were designed using the consensus sequences of corresponding capsid protein ( CP) genes of HVX and CMV, the nucleocapsid protein ( NP) genes of TSWV and INSV, and the movement protein ( MP) and CP genes of TMV. The primers were validated in vitro using single and multiplex RT-PCR assays. The detection limit of each primer set in multiplex RT-PCR was 100 fg ( TMV), 1 fg ( HVX), 10 fg ( CMV), 10 pg ( TSWV) and 10 pg ( INSV). Forty-six infected nursery samples collected from different locations in the USA were screened for virus infections using this multiplex RT-PCR. The multiplex RT-PCR has a potential for its application in routine diagnostics, quarantine, and epidemiological studies. The developed method is reliable, sensitive, and economic for testing a wide range of ornamental and nursery plants for detection of these viruses. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)