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

ABSTRACT: 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.

SUBMITTER: Rao X 

PROVIDER: S-EPMC5435019 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature