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OBJECTIVES
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.
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