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Content archived on 2024-04-16

THE DEVELOPMENT AND APPLICATION OF NUCLEIC ACID PROBE TECHNOLOGY FOR RAPID AND RELIABLE DETECTION AND IDENTIFICATION OF PLANT QUARANTINE BACTERIA

Objective

The successful implementation of the common plant health policy in the European Community is largely dependent upon the availability of reliable test procedures for high risk statutory organisms. Essentially standardized methods for laboratory tests which are harmonized in all Member States are a prerequisite.

Recent advances in molecular biology have generated powerful fingerprint methods for rapid, specific and sensitive detection and identification of plant pathogens with obvious advantages over the common conventional methods, in particular serological tests.

The project aims to introduce these technologies in methods for reliable test-monitoring of the bacteria under the EC Plant Health Directives.
The successful implementation of the common plant health policy is the European Community is largely dependent on the availability of reliable test procedures for high risk statutory organisms. It is necessary to have standardized methods which are harmonized in all Member States. Recent advances in molecular biology have generated powerful fingerprint methods for rapid, specific and sensitive detection and identification of plant pathogens with obvious advantages over the common conventional methods, in particular serological tests. Work is being done to improve the detection, diagnosis and identification of bacteria which are quarantine or high risk statutory organisms by the development and exploitation of nucleic acid probe techniques.

Probes are now available for 5 quarantine bacteria. Protocols were prepared for deoxyribonucleic acid (DNA) hybridization tests for 2 organisms, ring tests made with bacterial cell suspensions and hybridization conditions, labelling and detection systems evaluated. Protocols have been evaluated for colony hybridization tests and direct blotting and ring tests made with bacterial cell suspensions and contaminated plant material. Primers were prepared for polymerase chain reaction (PCR) amplification tests, reagents distributed and the first round of tests made for sensitivity.
The programme consists of (1) testing of probes in dot blot hybridization, (2) development and quantification of target DNA amplification by the Polymerase Chain Reaction (PCR), (3) identification and genetic characterization in Restriciton Fragment Length Polymorphism (RFLP) analysis and (4) rapid detection of single cells with fluorescent RNA oligonucleotide probes.

The work consists of the preparation of the test protocols and experiments to evaluate the methods, which are performed in three consecutive stages :

1) testing of cell suspensions of the target bacteria, related and non-related pathogenic and saprophytic bacteria, in particular those naturally present in association with the plant material,

2) reconstruction tests with artificially contaminated plant extracts,

3) ringtests using samples plant material with different levels of natural contamination.

Topic(s)

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Call for proposal

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Funding Scheme

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Coordinator

RIJKSSTATION VOOR PLANTENZIEKTEN
EU contribution
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Address
BURGEMEESTERS VAN GANSBERGHELAAN 96
9820 MERELBEKE
Belgium

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Participants (10)