Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates

Potato virus Y (PVY) has a worldwide distribution and infects several economically important crops from the Solanaceae family. The emergence and spread of the PVYNTN strain, which is the causative agent of potato tuber necrotic ringspot disease (PTNRD), has lead to large economic losses and highlighted the need for accurate discrimination of the different PVY strains. Detection and differentiation of PVY isolates is mainly based on a combination of ELISA, RT-PCR and bioassays; however, PVYNTN isolates are particularly difficult to differentiate from standard PVYN without the use of time-consuming bioassays. A strong correlation has been identified previously between the ability to induce PTNRD and the presence of a recombination point in the virus coat protein. An RT real-time PCR assay has been developed to enable detection of isolates with the recombination point, therefore, enabling rapid differentiation between potentially tuber necrotic PVYNTN isolates and standard PVYN isolates. The assay is also able to detect the presence of PVYO isolates. To aid with routine testing, immuno-capture and post-ELISA virus release were introduced; when coupled with RT real-time PCR the sensitivity of the assays were up to seven orders of magnitude higher than ELISA. The assay was shown to be a suitable method for rapid large-scale diagnostic testing of PVY in different types of plant material including tubers, and specific screening for potentially tuber necrotic recombinant isolates.