Simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus using real-time PCR and high resolution melting analysis

In this study, the real-time PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed in a real-time PCR using a primer set for both of them or duplex real-time PCR that included one specific primer set for each virus. These two strategies allowed us to confirmed virus infection in all tested samples. In 17 field samples the technique revealed samples positive for CNRMV or CGRMV as well as positive for both viruses. In addition, the HRM analysis made it possible to differentiate clearly between CNRMV and CGRMV. Sequence variations among CNRMV and CGRMV isolates observed from the HRM peaks were confirmed by sequencing. To test the capability to use this method in field, forty one sweet cherry samples were examined by HRM analysis. The HRM data showed that seven samples were positive for CNRMV and three were infected with CGRMV. The results presented in this study indicated that real-time PCR followed by HRM analysis provides sensitive, automated and rapid tool to detect and differentiate between CNRMV and CGRMV isolates.