Development of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus.

BACKGROUND Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus, causes significant loss in Cucurbitaceae plants. OBJECTIVES Development of a highly sensitive and reliable detection method for WSMoV. MATERIALS AND METHODS Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27 watermelon samples showing WSMoV infection symptoms. RESULTS The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and melting curves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Capsicum chlorosis virus (genus Tospovirus) and Cucumber mosaic virus (genus Cucumovirus). Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%. CONCLUSIONS A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detection of WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.