title: SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.
abstract: A SYBR(®) Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt-curve analysis (MCA) was optimized for the detection of nine grapevine viruses. The detection limits for simplex qRT-PCR for all nine grapevine viruses were estimated to be in the range of 214-1112 copies of the virus genome. Amplicons with melting temperatures (Tm) separated by at least 2°C in the MCA could differentiate two viruses in the same reaction. Therefore, eight of the nine viruses could be co-diagnosed in five different combinations of duplex assays. Of 305 grape leaf samples from the field or greenhouse, 162 were positive for at least one of the nine grapevine viruses using the duplex qRT-PCR assays. In contrast, only 127 samples were positive using endpoint RT-PCR and PCR assays, indicating the enhanced sensitivity of duplex real-time PCR. In addition, the duplex qRT-PCR assays were be used to detect Grapevine leafroll associated virus 3 (GLRaV-3) in its vector, the grape mealybug (Pseudococcus maritimus Ehrhorn), and Grapevine red blotch-associated virus (GRBaV) in Virginia creeper leafhopper (Erythroneura ziczac Walsh). The simplex and duplex real-time PCR assays developed in this study can be used to examine transmission of co-occruing viruses by insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines.
authors.affiliation: Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA, USA; Agriculture and Agri-Food Canada, Summerland Research and Development Centre, Summerland, British Columbia V0H 1Z0, Canada.
authors.forename: Sudarsana
authors.initials: S
authors.lastname: Poojari
authors.affiliation: Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA, USA; Department of Plant Pathology & Microbiology, Texas A&M AgriLife Research & Extension Center, 2401 East Highway 83, Weslaco, TX, USA.
authors.forename: Olufemi J
authors.initials: OJ
authors.lastname: Alabi
authors.affiliation: USDA-ARS, Root Disease and Biological Control Research Unit, Pullman, WA, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Okubara
authors.affiliation: Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA, USA. Electronic address: naidu.rayapati@wsu.edu.
authors.forename: Rayapati A
authors.initials: RA
authors.lastname: Naidu
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D029381:Closteroviridae / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D016679:Genome, Viral; D006430:Hemiptera / Q000821:virology; D057230:Limit of Detection; D009930:Organic Chemicals / Q000737:chemistry; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D027843:Vitis / Q000821:virology
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I
keywords: GLRaV; GRBaV; Grapevine viruses; Leafhoppers; Mealybugs; Melt-curve analysis