title: Relative quantitation goes viral: An RT-qPCR assay for a grapevine virus.
abstract: Accurate detection and quantitation of viruses can be beneficial to plant-virus interaction studies. In this study, three SYBR green real-time RT-PCR assays were developed to quantitate grapevine leafroll-associated virus 3 (GLRaV-3) in infected vines. Three genomic regions (ORF1a, coat protein and 3'UTR) were targeted to quantitate GLRaV-3 relative to three stably expressed reference genes (actin, GAPDH and α-tubulin). These assays were able to detect all known variant groups of GLRaV-3, including the divergent group VI, with equal efficiency. No link could be established between the concentration ratios of the different genomic regions and subgenomic RNA (sgRNA) expression. However, a significant lower virus concentration ratio for plants infected with variant group VI compared to variant group II was observed for the ORF1a, coat protein and the 3'UTR. Significant higher accumulation of the virus in the growth tip was also detected for both variant groups. The quantitation of viral genomic regions under different conditions can contribute to elucidating disease aetiology and enhance knowledge about virus ecology.
authors.affiliation: Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
authors.forename: R
authors.initials: R
authors.lastname: Bester
authors.affiliation: Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
authors.forename: P T
authors.initials: PT
authors.lastname: Pepler
authors.affiliation: Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
authors.forename: J T
authors.initials: JT
authors.lastname: Burger
authors.affiliation: Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa; ARC Infruitec-Nietvoorbij (The Fruit, Vine and Wine Institute of the Agricultural Research Council), Private Bag X5026, Stellenbosch 7599, South Africa. Electronic address: hjmaree@sun.ac.za.
authors.forename: H J
authors.initials: HJ
authors.lastname: Maree
mesh_terms: D029381:Closteroviridae / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics*; D057230:Limit of Detection; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D027843:Vitis / Q000821:virology*
chemical_list: D017931:DNA Primers; D012367:RNA, Viral
keywords: GLRaV-3; Grapevine leafroll disease; RT-qPCR, Subgenomic RNA; Relative quantification; SYBR green