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PORTCHECK - Development of generic on site molecular diagnostics for EU pests and pathogens - SD0416

PORT CHECK is a combined RTD and demonstration activity aimed to deliver the tools and procedures to allow EU member state Plant Health competent laboratories and inspection services to perform molecular diagnostic assays “on-site” and at points of entry. The proposal will develop and evaluate real-time PCR (TaqMan) assays for a number of key harmful organisms, including Phytophthora ramorum (sudden oak death) and pinewood nematode; and transfer these assays to field portable real-time PCR platforms which were originally developed for bio-warfare and bio-terrorism applications. Particular attention will be paid to the problems associated with sampling and nucleic acid extraction in field conditions. The proposal mobilises an impressive consortium, including both academic and SME research partners, along with probably the most comprehensive and representative network of official Plant Health competent laboratory and inspection service organisations ever assembled for an EU RTD proposal. The consortium also includes partners with a proven track record of developing and successfully deploying on-site diagnostic kits to official Plant Health inspection services. Consultation with the principle stakeholders of the technology (inspection services, competent laboratories, DG SANCO, trade representatives etc.) at the outset and throughout the life of the project is given paramount importance. Adoption of the new technology should represent a step change in the way Plant Health services carry out inspections in support of Council Directive 2000/29/EC and will contribute to the reduced risk of the importation (and export) and establishment of harmful organisms. This in turn will help to prevent any consequential negative impacts on the sustainability of European agricultural systems, protect critically important natural forest ecosystems and other negative economic, social and environmental effects associated with the establishment of non-indigenous plant pests and diseases.
Over-all objective: To develop generic molecular diagnostic methods (including sampling, extraction and detection) for pests/pathogens of quarantine importance (including Pinewood nematode and Phytophthora ramorum (sudden oak death)), that could be used by authorities implementing Council Directive 2000/29/EC within the EU for detection on site and at ports of entry.
The principal objective of this project is to enhance the ability of member states to detect harmful organisms at the point of inspection, including points of entry, and thereby lessen the chances of their introduction. A secondary objective is to improve the efficiency of inspection services by significantly shortening the time taken to achieve a putative identification as well as increasing the accuracy of such inspections (classically based on visual symptoms or random sampling).

PORT CHECK as described in this proposal will also measurably improve and reinforce co-operation between the plant protection services of EU countries. The generic nature of the technology proposed should also, in the longer term, improve and reinforce co-operation between plant protection and custom services at the national level. Indirectly, improved diagnostics will yield valuable data on the incidence and critical pathways of harmful organism introductions and hence improve current EU risk assessments and ultimately lead to improved control measures.

Project Scientific and Technological objectives: The present project proposal aims to establish generic molecular methodologies for the direct detection of quarantine pathogens in suspect plant material using portable real time PCR at the point of inspection. In doing so, it will be necessary to achieve a number of scientific and technological objectives.

· Currently, the main rate-limiting step in the uptake of molecular techniques in the laboratory is the need for rapid and robust nucleic acid extraction techniques. To carry out these techniques at the point of inspection will require equally rapid and robust methods, but in addition methods developed within this project will need to be portable, and as such can not rely on the use of large laboratory based equipment such as centrifuges. Within the project a key objective will be to develop sampling strategies for each of the organisms, followed by techniques for grinding samples and extracting nucleic acid suitable for PCR amplification, using techniques that do not rely on the use of laboratory equipment or hazardous reagents
· Assays based on real time PCR will be developed for pathogens/pests where they are currently unavailable, whilst assays that are available will be evaluated for applicability to quarantine testing. The completed assays will then be evaluated using field portable PCR equipment, and the results compared to results generated in the laboratory, to investigate if the technology is suitable for use in a quarantine-testing situation. Investigations will be made into all relevant aspects of the assays, for example stabilisation of reagents.
· A key aspect will be the involvement of the principal stakeholders, i.e. the inspection services, from the outset and throughout the life of the project. A key objective will be to investigate the needs of the inspection services prior to the start of the project and to to define what requirements they have for testing of this kind. The final year of the project will then be spent evaluating the techniques developed, alongside inspection services in the EU and ring-testing final protocols in every member state.

Measurable objectives:

1. To define the needs and requirements of EU member state inspection services, in order to steer the work to achieve finished protocols that are of practical value to inspection services.
2. To develop nucleic acid extraction protocols (including sampling and grinding) that can deal with all the different quarantine material in the project, that are rapid, reliable and field portable.
3. To develop real time PCR assays for each of the organisms of interest in the project and to assess the performance of these assays when carried out on portable real time PCR equipment compared with the laboratory based equivalents.
4. To develop protocols for carrying out real time PCR testing, from taking samples through to interpreting results and to validate the protocol by submitting to a ring test by inspection services and competent laboratories from all the EU member states.
5. To disseminate results and achievements to the wider EU Plant Health community.

Project Documents
• Abstract : No report associated with this project   (19k)
Time-Scale and Cost
From: 2004

To: 2006

Cost: £113,000
Contractor / Funded Organisations
Central Science Laboratory
Alien Species              
Plant health              
Plant Pests and Diseases              
Plants and Animals