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J Virol Methods. 2016 Jun;232:47-56. doi: 10.1016/j.jviromet.2016.01.013. Epub 2016 Feb 2.

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

Author information

1
National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Stillwater, OK, USA; Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA.
2
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA.
3
National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Stillwater, OK, USA; Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA; Departamento de Ciencias de la Vida y de la Agricultura, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador.
4
National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Stillwater, OK, USA; Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, USA. Electronic address: ochoaco@okstate.edu.

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.

KEYWORDS:

Biosecurity; Diagnostics; HRM; High Resolution Melting; SYBR-Green RT-qPCR; TaqMan RT-qPCR

PMID:
26850142
DOI:
10.1016/j.jviromet.2016.01.013
[Indexed for MEDLINE]

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