Xueqin Rao1,2, Jie Sun1,2. 1. Department of Plant Pathology, College of Agriculture, South China Agricultural University, Guangzhou 510642, China. 2. Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.
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.
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.
Entities:
Keywords:
Real-Time RT-PCR; SYBR Green I; Watermelon silver mottle virus
Authors: D E Debreczeni; S Ruiz-Ruiz; J Aramburu; C López; B Belliure; L Galipienso; S Soler; L Rubio Journal: J Virol Methods Date: 2011-05-27 Impact factor: 2.014
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