K. Walsh

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.

Detection of potato viruses using microarray technology: towards a generic method for plant viral disease diagnosis

Currently, most diagnostic methodology is geared towards detection of a very specific target species and often a number of assays need to be run in parallel to reach a result. The generic methods that are available for virus testing tends to give identification to the genus level only. The method described in this paper addresses this problem by exploiting a technology that has potential to test for a large number of targets in a single assay. Using the array constructed, the method was able to detect several common potato viruses (PVY, PVX, PVA, PVS) in single and mixed infections. The method was shown to be able to discriminate sequences with less than 80% sequence identity but was able to detect sequence variants with greater than 90% sequence identity. Thus the method should be useful for discriminating at the species level, but able to cope well with the intrinsic variability found within the genomes of RNA viruses. The sensitivity of the assay was found to be comparable with ELISA. The paper illustrates a significant step forward in the development of diagnostic methodologies by presenting for the first time a method that could theoretically be used not just for viruses, but for all the plant pathogens and pests that a modern diagnostic laboratory would want to test for, in a single completely generic and highly parallel format.

The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVYO, PVYN and PVYC strains using RT-PCR

Potato tuber necrotic ringspot disease (PTNRD) is a damaging disease of potatoes, causing unsightly necrotic rings on the surface of tubers. The causal agent is thought to be tuber necrotic isolates of Potato virus Y, known as PVY(NTN). The disease spoils tubers for processing and table use, and the lack of a diagnostic method makes control especially difficult. The development of an RT-PCR assay for the reliable detection of PVY(NTN) and discrimination of all the main strains of PVY (PVY(O), PVY(N) and PVY(C)) is described. An assay was developed, exploiting a recombination site in the coat protein of PVY(NTN), allowing more reliable diagnosis of these isolates. Although the conserved nucleotide differences observed between the strains was very small, competitive PCR and mutagenically separated PCR were both employed in the development of a robust assay. The assay was found to be more reliable than the most commonly used RT-PCR method, and should prove to be an important tool in the confirmation of symptoms and for the detection of PVY(NTN) in symptomless tissue, in disease surveys and seed health schemes.

Development of a sequence-specific real-time PCR to the melon thrips Thrips palmi (Thysan., Thripidae).

Abstract:  In recent years, the polyphagous pest Thrips palmi Karny has become a species of major quarantine concern, often intercepted on plant material in international trade. The ability to rapidly identify the species is a critical factor that will determine the success of any campaign to prevent its establishment in Europe or elsewhere. However, the immature stages cannot currently be identified to the species level with certainty by morphological methods. In this study, random amplified polymorphic DNA analysis was performed to identify putative markers, which were then screened by Southern blot analysis. One marker was sequenced and a real‐time polymerase chain reaction assay developed. The assay was screened against 21 thrips species including 10 other species of the genus Thrips and were found to be specific to T. palmi. The assay is rapid, could be completed in as little as 45 min and can be used to aid the identification of T. palmi.

Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.