Rapid and visual detection of grapevine fleck virus using recombinase polymerase amplification combined with lateral flow strips

Grapevine flack virus (GFkV) causes fleck disease and leads to severe symptoms when the infection occurs with multiple viruses. Restricting the propagation of GFkV-infected grapevines is the ideal management strategy to control the spread of this virus. Therefore, the development of rapid detection methods for GFkV is urgently required. In the present study, we developed a rapid and reliable assay based on the reverse transcription-recombinase polymerase amplification (RT-RPA) combined with lateral flow strips (LFS) using sequence-specific primers and a probe-based coat protein sequences for the GFkV detection in grapevines. The GFkV RT-RPA-LFS assay was optimized at 38 degrees C for 10 min and an extra 5 min LFS incubation time. The optimized RT-RPA-LFS assay had similar sensitivity to RT-PCR and showed no cross-reaction with major viruses infecting grapevines in Korea. The assay was successfully validated for GFkV detection in grapevine field samples. This study provides a reliable technique for rapidly detecting GFkV as an alternative diagnostic approach for producing virus-free grapevine nurseries.