Application of microsphere based assay for multiple plant virus detection in globe artichoke

Background: Mixed viral infections are common in globe artichoke (Cynara cardunculus var. scolymus). Despite lack of evident symptoms, production losses are relevant. A rapid and accurate detection is advisable for selection and marketing of plant propagation material. Objectives: Our aim was the evaluation of a suspension microbeads array system for simultaneous detection of Artichoke latent potyvirus (ArLV), Artichoke Italian latent nepovirus (AILV), Artichoke mottled crinkle tombusvirus (AMCV), Tomato spotted wilt tospovirus (TSWV), Turnip mosaic potyvirus (TuMV), Cucumber mosaic cucumovirus (CMV), Potato virus X (PVX), Tobacco mosaic tobamovirus (TMV) and Pelargonium zonate spot anulavirus (PZSV). Methods: For PCR, oligonucleotides were designed from conserved regions in the viral genomes, using an Illumina´s Assay Design Tool. The actin gene of C. cardunculus was used as control. The microbeads-based procedure started with a first PCR target amplification. A further target-specific primer extension (TSPE), holding a unique 5' motif, was then performed. During this step biotinylated dCTPs were incorporated into the extension products. These were then linked to the holographic microbeads bearing an oligo complementary to the 5' motif. Probe labelling was performed with streptavidin-Alexa-647, for final scan using an Illumina BeadXpress Reader. Conclusions: Multiplex fluorescent microbeads assays allowed the contemporary detection of several viruses in a single sample. This highly flexible microarray approach used a suspension of barcoded microbeads bearing specific sequence motifs. It favoured the tailoring of user defined applications. The method is also suitable for identification of SNPs in viral strains, within a unique host phenotype.