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Development of a Real-Time RT-PCR SYBR Green Assay for Tomato ring spot virus in Grape

September 2007 , Volume 91 , Number  9
Pages  1,083 - 1,088

Elwin L. Stewart , Xinshun Qu , Barrie E. Overton , Fred E. Gildow , and Nancy G. Wenner , Department of Plant Pathology, The Pennsylvania State University, University Park 16802 ; and Deborah S. Grove , Huck Institute, Wartik Laboratory, The Pennsylvania State University



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Accepted for publication 11 April 2007.
ABSTRACT

Grapevines infected with Tomato ring spot virus (ToRSV) pose an economic risk for growers in the northeastern United States. This study describes a one-step real-time reverse-transcription polymerase chain reaction (RT-PCR) SYBR Green assay for detecting ToRSV in grapevines. Two newly designed primer pairs based on the ToRSV coat protein gene sequence were evaluated for specificity and optimized for a SYBR Green assay. The primer pair ToRSV1f/1r yielded a 130-bp product with strong primer-dimer products, whereas the primer pair ToRSV2f/2r yielded a 330-bp product with weak primer dimer products. Real-time RT-PCR detected ToRSV in more naturally infected grapevines maintained in the greenhouse than did enzyme-linked immunosorbent assay. The nucleotide sequences of the fragments amplified from grapevine growing in Pennsylvania using real-time PCR were divergent from previously published sequences.


Additional keywords: Grape yellow vein virus, Vitis vinifera

© 2007 The American Phytopathological Society