Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets

Abstract

The potential of DNA microarrays for detection of plant viruses is hampered by underutilization of sequence-independent amplification methods for target nucleic acid enrichment. A microarray system is described for an unbiased detection of plant viruses using both short (30 nt) and long (50 and 70 nt) oligonucleotide probes. The assay involves amplification of target nucleic acid using random primers followed by in vitro transcription whose cRNA product is labeled chemically, fragmented and used as target for hybridization. Initial optimization tests with Turnip vein clearing virus and Cauliflower mosaic virus showed increased hybridization efficiency with shorter cDNA targets (100 bp) and longer probes (50 and 70 nt). The system was validated in pure and mixed samples by detection of three Tymovirus species: Asclepias asymptomatic virus, Kennedya yellow mosaic virus and Turnip yellow mosaic virus. The method could detect sequence variants with 70–75% or higher sequence identity, indicating the possible utility of the approach for virus discovery. Array performance comparison of long probes demonstrated the competence of 50-mers to provide a satisfactory balance between detection sensitivity and specificity. The work described is a significant step towards a method to assess, in one assay, the presence of a large diversity of relatives of known viruses of plants.