title: Amplification-free detection of grapevine viruses using an oligonucleotide microarray.
abstract: A single-colour microarray hybridization system was designed and evaluated for the detection of viruses infecting grapevine. Total RNA (≥0.5μg) from infected plants was converted to cDNA and labelled with Cy3 using two different strategies. While amine-modified and labelled cDNA was adequate for the detection of nepoviruses, the 3DNA technique, a post-hybridization detection method that uses intensely fluorescent dendrimer reagents, was required for the detection of closteroviruses in infected plants. Threshold detection levels were based on the ratio between viral specific and 18S rRNA positive control signal intensities. Oligonucleotides between 27 and 75 nucleotides in length were evaluated and compared. Viruses detected include eight nepoviruses, two vitiviruses, and one each of closterovirus, foveavirus, ampelovirus, maculavirus and sadwavirus. Results of this work demonstrate the potential of microarray technique to detect viral pathogens without sequence bias amplification of template RNA.
authors.affiliation: Sidney Laboratory, Centre for Plant Health, Canadian Food Inspection Agency, 8801 East Saanich Road, Sidney, British Columbia, Canada V8L 1H3.
authors.forename: Ismail
authors.initials: I
authors.lastname: Abdullahi
authors.forename: Yana
authors.initials: Y
authors.lastname: Gryshan
authors.forename: Michael
authors.initials: M
authors.lastname: Rott
mesh_terms: D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D050091:Dendrimers; D005453:Fluorescence; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D010942:Plant Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D014773:Virology / Q000379:methods*; D027843:Vitis / Q000821:virology*
chemical_list: D018076:DNA, Complementary; D050091:Dendrimers