SYBR(®) Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses.
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.
['Poojari|Sudarsana|S|', 'Alabi|Olufemi J|OJ|', 'Okubara|Patricia A|PA|', 'Naidu|Rayapati A|RA|']
D000818:Animals D052160:Benzothiazoles D029381:Closteroviridae D003959:Diamines D016679:Genome, Viral D006430:Hemiptera D057230:Limit of Detection D009930:Organic Chemicals D010935:Plant Diseases D018515:Plant Leaves D011804:Quinolines D012367:RNA, Viral D060888:Real-Time Polymerase Chain Reaction D020133:Reverse Transcriptase Polymerase Chain Reaction D012680:Sensitivity and Specificity D044366:Transition Temperature D027843:Vitis
Grapevine viruses Melt-curve analysis Mealybugs GRBaV Leafhoppers GLRaV