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.