Macroarray detection of grapevine leafroll-associated viruses

Grapevine leafroll-associated viruses (GLRaVs) are an emerging group of viruses that represent a significant threat to the global productivity and sustainability of the grapevine industry. Their control is achieved through the identification and elimination of infected vines, and the use of planting material derived from virus-tested, certified stocks. As such, much effort has been invested in developing reliable molecular diagnostic techniques. In this work, we report the development of a macroarray assay for the detection of the principal GLRaVs. In total 314 70-mer oligonucleotides specific to GLRaV-1, -2, -3, -4, -7, and GLRaV-4 strains 5, 6, 9 and Pr were spotted onto a 11 × 7 cm nylon membrane. Thirty-four grapevine samples from various origins were tested by the macroarray, RT-PCR and ELISA. Thirty were positive for virus infection using RT-PCR, 28 by ELISA and 25 by the macroarray. Mixed infections were identified by macroarray in two samples and confirmed by RT-PCR or ELISA. There were a few discrepancies between methods that were most likely due to differences in the sensitivity of detection, and in the case of the macroarray, limitations in the sequence data available for certain virus species in the design of the oligonucleotides. This work demonstrates the successful application of macroarray methodology using randomly primed and sequence-nonspecific amplified cDNAs derived from grapevine total RNA extracts, and provides a proof-of-principal for unbiased multiplex detection using a single robust platform.