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Title: Detection of Grapevine Leafroll-associated virus 7 using real-time qRT-PCR and conventional RT-PCR

Author
item Al Rwahnih, Maher - University Of California
item Osman, Fatima - University Of California
item Sudarshana, Mysore
item Uyemoto, Jerry
item Minafra, Angelantonio - Universita Di Bari
item Martelli, Giovanni - Universita Di Bari
item Rowhani, Adib - University Of California

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2011
Publication Date: 2/1/2012
Publication URL: http://www.sciencedirect.com/science/article/pii/S0166093411004630
Citation: Al Rwahnih, M., Osman, F., Sudarshana, M.R., Uyemoto, J.K., Minafra, A., Martelli, G., Rowhani, A. 2012. Detection of Grapevine Leafroll-associated virus 7 using real-time qRT-PCR and conventional RT-PCR. Journal of Virological Methods. 179:383-9.

Interpretive Summary: Nine isolates of Grapevine Leafroll-associated Virus 7 (GLRaV-7) from California have been sequenced to design more sensitive molecular diagnostic tools. We focused on the coat protein (CP) and the homologous heat shock protein (hHSP70) genes. Sequence identity among these isolates ranged between 90-100% at the nucleotide and amino acid levels. Primers for two qRT-PCR assays and one RT-PCR detection primers were designed from both the CP and hHSP70 conserved regions and evaluated using eighty six different grapevine plants, many with multiple infection, from the Davis Grapevine Virus Collection, Foundation Plant Services (FPS) collection and the USDA National Clonal Germplasm Repository (NCGR), Davis, CA. Results revealed that the qRT-PCR assay designed in the hHSP70 gene was more sensitive (29.07 % positives) than the one designed in the CP gene (22.09% positives). Both qRT-PCR assays were more sensitive than than RT-PCR. Phylogenetic analysis of GLRaV-7 isolates characterized in this study further supports the current classification of this virus as an unassigned member in the Closteroviridae family.

Technical Abstract: Nine isolates of Grapevine Leafroll-associated Virus 7 (GLRaV-7) from California have been sequenced to design more sensitive molecular diagnostic tools. These sequences were from the coat protein (CP) and the homologous heat shock protein (hHSP70) genes. Sequence identity among these isolates ranged between 90-100% on the nucleotide and amino acid levels. Two qRT-PCR assays and one RT-PCR detection primers were designed from both the CP and hHSP70 conserved regions and evaluated using eighty six different grapevine plants, many with multiple infection, from the Davis Grapevine Virus Collection, Foundation Plant Services (FPS) collection and the USDA National Clonal Germplasm Repository (NCGR), Davis, CA. Results revealed that the qRT-PCR assay designed in the hHSP70 gene was more sensitive (29.07 % positives) than the one designed in the CP gene (22.09% positives). Both qRT-PCR assays were more sensitive than than RT-PCR. Phylogenetic analysis of GLRaV-7 isolates characterized in this study further supports the current classification of this virus as an unassigned member in the Closteroviridae family.