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Title:   Author  item
                                            AL RWAHNIH, MAHER - University Of Californiaitem
                                            OSMAN, FATIMA - University Of Californiaitem
                                            Sudarshana, Mysoreitem
                                            Uyemoto, Jerryitem
                                            MINAFRA, ANGELANTONIO - Universita Di Bariitem
                                            MARTELLI, GIOVANNI - Universita Di Bariitem
                                            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
                                    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.


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