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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED STRATEGIES FOR ADVANCE MANAGEMENT OF FRUIT, NUT, AND OAK TREE DISEASES Title: Detection of Grapevine Leafroll-associated virus 7 using real-time qRT-PCR and conventional RT-PCR

Authors
item Al Rwahnih, Maher -
item Osman, Fatima -
item Sudarshana, Mysore
item Uyemoto, Jerry
item Minafra, Angelantonio -
item Martelli, Giovanni -
item Rowhani, Adib -

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 28, 2011
Publication Date: February 1, 2012
Repository 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.

Last Modified: 10/23/2014
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