Development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses

The genus Potyvirus is the largest and one of the most economically important virus genera infecting plants. However, current diagnostic techniques are limited in their ability to identify multiple potyvirus infections. An assay that can identify multiple potyviruses simultaneously, with good specificity and sensitivity, is therefore highly desirable. To determine the feasibility of simultaneous detection of multiple potyviruses a 25-mer oligonucleotide microarray was developed targeting four distinct potyviruses: Dasheen mosaic virus (DsMV), Leek yellow stripe virus (LYSV), Potato virus Y (PVY) and Zucchini yellow mosaic virus (ZYMV). A total of 85 probes including 33 perfect-match and 52 mismatch probes were designed from conserved and variable sequence regions of the nuclear inclusion b (NIb) gene, RNA-dependent RNA polymerase (RdRp) gene, coat protein (CP) gene and the 3′ untranslated region (UTR), representing the four targeted potyviruses at both species and strain levels. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5′ terminus. The array showed high specificity when tested with nineteen different geographically diverse potyvirus isolates of the four target species, four distinct but closely related potyviruses, and four healthy plant species. The approaches and protocols developed in this study form a useful basis for developing other potyviruses arrays and the results also provide useful insights into generic issues for the development of arrays for detecting other pathogens.