Partner und Internationale Organisationen
(Englisch)
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AT, BE, BG, CH, CY, CZ, DE, DK, ES, FI, FR, GR, HU, IT, LT, NL, NO, PL, PT, SI, UK
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Abstract
(Englisch)
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Plant Inspection is the first line of defense in crop biosecurity. Rapid and sensitive detection is key to guarding against the accidental or intentional introduction and spread of potentially devastating agricultural pests in Switzerland and Europe. Inspection also ensures that export plant material is pest-free, thereby protecting the international trade value of Swiss products. The European and Mediterranean Plant Protection Organization (EPPO) designated the most dangerous pests in lists recommended for quarantine regulation including 26 phytopathogenic bacteria, 11 phytoplasmas, 15 phytoparasitic nematodes [see Annex A]. Detecting all 52 of these pests essentially requires 52 different protocols specific to each agent. This is time consuming (3-7 days for culturing when possible, 20+ days for plant infection tests) and often depends on the availability of specialized expertise (particularly for nematode identification). Molecular methods (PCR and ELISA) have speeded up the detection process, but species-specific reactions and antibodies are still needed, and for new quarantine organisms no such diagnostic methods are available. Fast, simple and sensitive methods to detect a broad-spectrum of target pests are essential for a water-tight plant inspection. Microarrays are a relatively new innovation that has greatly aided investigations of pathogen-host gene interactions. Recently, this technology has been extended for clinical/veterinary diagnostics. Basically, a gene common to all target organisms is chosen (eg., 16S rRNA) and oligonucleotide probes are designed based on regions of that gene sequence unique for each species. Probes are mechanically spotted on a slide in a precise microarray arrangement and chemically fixed. A chip can be used when needed, by hybridizing the product of a single PCR reaction performed on mixtures of unknown organisms (or clinical sample) to the microarray. The identity of the organism is deduced based on the unique pattern of labelled probes that react with the sample. Hierarchical taxonomic discrimination of organisms can be easily increased through the use of probes designed from several common genes and PCR for each gene combined in a single multiplex reaction. Each microarray can carry literally hundreds of probes allowing the detection of a large number of organisms with one tool. Simply said, microarrays are 'laboratories-on-a-chip'. Because only one PCR procedure is needed for all organisms, and all organisms can be detected with a single chip only minimal training is needed. The equipment needed to run the PCR, hybridize and read the chip is affordable and increasingly standard in diagnostic laboratories. Results can be obtained as quickly as ½ day from the time a plant sample is received. Microarrays offer a uniform tool to simply, reliably and rapidly inspect plant material for dangerous pests improving interception of contaminated shipments but allowing clean material to be traded without delay. Using the microarray design methods that we have successfully applied to distinguish rodent species in ecological studies and plant viruses in crop samples, we have developed PLANT-INSPECTION-on-a-CHIP using single microarrays to detect a wide-range of bacteria and phytoplasmas listed by EPPO with a single preparatory PCR step, thus simplifying the presumptive diagnostic process. We have laid the foundation for a third chip that will include quarantine nematodes.
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