The detection of Tomato spotted wilt virus (TSWV) in individual thrips using real time fluorescent RT-PCR (TaqMan).
Tomato spotted wilt virus (TSWV) is an important virus, economically in the UK, causing damaging disease in ornamental and vegetable crops. The virus is vectored by several species of thrips, most importantly the western flower thrips (Frankliniella occidentalis Pergande [Thysanoptera: Thripidae]). The vector thrips themselves constitute a damaging pest and are difficult to control completely. Monitoring thrips numbers is an important part of the control of virus, but does not give information on how many of the thrips are viruliferous. Monitoring the presence of viruliferous thrips at an early stage of an epidemic may lead to improved disease control, since virus can be spread effectively whilst vector pressure is low and symptoms may take several weeks to appear on some hosts. This paper describes the development of a sensitive and robust, high-throughput method for the detection of TSWV in individual insects based on TaqMan chemistry. The method incorporates a novel RNA specific internal control to increase the reliability of the results. Results are also presented on comparisons of different extraction methods, including insects taken from sticky traps, for high-throughout testing. Implementation of a method such as this for the reliable detection of TSWV in individual thrips would aid the understanding of the progress of TSWV epidemics, and offer an early disease warning system for growers.
Boonham
N
N
Central Science Laboratory, Sand Hutton, YO41 1LZ, York, UK. n.boonham@csl.gov.uk
Smith
P
P
Walsh
K
K
Tame
J
J
Morris
J
J
Spence
N
N
Bennison
J
J
Barker
I
I
D000818:Animals; D007303:Insect Vectors / Q000821:virology*; D007313:Insecta / Q000821:virology*; D018551:Solanum lycopersicum / Q000469:parasitology; D010935:Plant Diseases / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism; D013997:Time Factors; D014026:Nicotiana / Q000821:virology; D017865:Tospovirus / Q000235:genetics / Q000302:isolation & purification*
D019914:Taq Polymerase