|POOJARI, SUDARSANA - Washington State University|
|OLFEMI, ALABI - Washington State University|
|NAIDU, RAYAPATI - Washington State University|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2016
Publication Date: 5/28/2016
Citation: Poojari, S., Olfemi, A., Okubara, P.A., Naidu, R.A. 2016. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses. Journal of Virological Methods. 235(5):112:118.
Interpretive Summary: The 70 viruses known to infect grape plants complicate diagnosis and disease management. Sensitive and specific molecular diagnostic assays were developed for nine major grapevine viruses found in the state of Washington. Each virus could be diagnosed individually using single assays or, in six cases, in combination with a second virus. The six double (duplex) assays were used to show that our molecular diagnostics were more sensitive in detecting viruses in field- and greenhouse-grown grape samples, and in two insect vectors than were existing assays. Our assays have the potential for improving disease management and for determining virus transmission rates of insect vectors.
Technical Abstract: A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range of 214 to 1112 copies/µl of target DNA. Amplicons with melting temperatures (Tm) separated by least 2oC 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 petiole 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 detected 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) vector. The singleplex and duplex real-time PCR assays developed in this study can be used to determine transmission efficiencies of insect vectors as well as for rapid and sensitive detection of viruses in infected grapevines.