Open AccessDevelopment of a Real-Time RT-PCR SYBR Green Assay forTomato ring spot virusin Grape
Author(s) -
Elwin L. Stewart,
Xinshun Qu,
Barrie E. Overton,
F. E. Gildow,
N. G. Wenner,
David Grove
Publication year - 2007
Publication title -
plant disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.663
H-Index - 108
eISSN - 1943-7692
pISSN - 0191-2917
DOI - 10.1094/pdis-91-9-1083
Subject(s) - biology , primer (cosmetics) , virology , polymerase chain reaction , real time polymerase chain reaction , coat protein , sybr green i , microbiology and biotechnology , gene , genetics , rna , chemistry , organic chemistry
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.