Potato virus Y (PVY) detection in a single aphid by one-step RT-PCR with boiling technique

Potato virusY (PVY) (Potyviridae: potyvirus) is a serious emerging virus affecting seed potato worldwide. It affects the seed potato by transmitting non-persistently via aphids. Sometimes this virus induces symptomless infection and is hard to detect in potato. So, it requires a specific and quick diagnosis for efficient examination. Recently, a reverse-transcription polymerase chain reaction (RT-PCR)-based PVY detection method has been developed from plant as well as insect vectors. However, it is a complex and time consuming method. Here, we developed a simple PVY detection method that uses boiling for releasing the viral RNA from aphid stylets, and amplification by PVY-specific primers located in the viral coat protein gene. The method is suitable for various strains. This simplified method could save time compared to earlier detection methods because of the simplified RNA extraction step. Following this procedure, we tested this one-step RT-PCR-based PVY detection method by using three PVY vectoring aphid species (Myzus persicae, Aphis gossypii and Macrosiphum euphorbiae) as well as other sucking insects such as thrips, Frankliniella occidentalis. The reliability of a newly developed primer set was suitable for RT-PCR and procedures were successfully demonstrated for virus detection. This PVY detection method is rapid, easy to use and suitable for large-scale testing in laboratories of seed potato, and could potentially be applied to virus-free seed potato production.