Abstract
Defra currently spends approximately £1.6M p.a. on provision of diagnostic services in Plant Health. There is continuing pressure on the Plant Health service to increase the level of monitoring for statutory organisms in compliance with both National and EU Plant Health legislation. Thus either the total budget will need to be increased or the cost per sample reduced to achieve this aim. There exists considerable opportunity to both reduce the cost of testing, as well as improving the quality of response, through the development and adoption of appropriate novel diagnostic methodology in the centralised testing laboratory. In addition giving ‘first responders’ the ability to carry out diagnostic tests has already proven its worth in plant health with the successful adoption of lateral flow devices by inspectors. With development of new assay formats and improved extraction technology as well as speeding up and simplfying the process this should become increasingly possible with molecular methods. These methods will allow for future improvement of the provision and performance of diagnostic services carried out on behalf of Plant Health Division. The project will develop two themes using pests of current statutory concern as model systems: further development of molecular field-testing and development of novel methods for laboratory use. The research will be performed to meet 6 objectives and be applied in support of mycology, virology, bacteriology and invert ID. Theme I: Field testing (encompasing objective 1) The methods developed in the previous project and in the EU project PortCheck have developed a field test for P ramorum detection that can be completed in less than one hour, using minimal equipment. However, the equipment used is still expensive (i.e. smartcycler equipment) being capable of both careful temperature regulation (thermal cycling) and fluorescent detection (requiring the use of lasers within the instument). Feedback from the insepectors is also that the extraction method is still quite cumbersome and laboratory oriented. Single temperature (isothermal) amplification and non-fluorescent detection may resolve many of these problems; preliminary work on one such method (LAMP) looks promising, however several technological challenges need to be overcome. Firstly the rapid and simple extraction of DNA from the samples; secondly the assay chemistry (several are now available with different characteristics) and the use of internal controls (standard in all other lab and field tests); and finally simple end-point detection of target. The aim will be to reach molecular detection in less than 20 minutes from sample to result using a range of targets (Objective 1). Theme II: Laboratory testing (encompasing objectives 2-6) The two areas of laboratory testing of interest currently are the identification (including fine typing) of isolated pathogens or pests and the diagnosis of causal agents of disease. Work will continue with both elements. DNA microarray methods are based on hybridisation and currently have useful but limited sensitivity, the methods are being further explored in other projects (Biosecurity chip). Platforms are becoming available that combine parallel approaches with real-time PCR, offering the detection of multiple targets but with proven real-time PCR chemistry such as TaqMan, these methods will be explored in a diagnostic context. (Objective 2) For DNA barcoding the initial work on identifying genes for exploitation is well progressed, but the completion of web enabled analysis methods giving identification using the sequences needs work. Currently this is performed either using database homology search tools or clustering, both require considerable bio-informatics expertise and do not offer a routine solution. A dual approach based on homology and clustering with automated bio-informatics needs to be developed to bring the methods to a useable diagnostic tool in the laboratory. In addition fine typing for studying the epidemiology and transmission routes of plant pathogenic bacteria needs to be refined. (Objective 3) Methods for the rapid identification of virulence markers are of particular relevance to organisms such as bacteria, since identification at the species level does not always give an indication of virulence. Identification of these markers is currently challenging and limited numbers of markers are available. Thus work based on using rapid subtractive hybridisation approaches and fundctional genomic screens will be developed to rapidly identify and characterise virulence markers. This will be of great use for evaluating the pathogenesis of newly detected bacteria and also for epidemiological studies. (Objective 4)
United Kingdom