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Q-AMP - Development of validated procedures for whole genomic amplification of DNA/RNA for quarantine plant pathogens and pests (EUPHRESCO ERA-Net - Real common pot pilot project) - PH0428

Description
Project Summary

Development of accurate identification tools for plant pathogens and pests is vital to support European Plant Health Policies. For this project Council Directive 2000/29/EC is important, listing some 275 organisms for which protective measures against introduction into and their spread
within the Community needs to be taken. Those threats are now greater than ever because of the increases in the volumes, commodity types and origins of trade, the introduction of new crops, the continued expansion of the EU and the impact of climate change.

Currently identifying pathogens (in particular new emerging diseases) requires a staff with specialised skills in all disciplines (mycology, bacteriology, etc.); which is only possible within big centralised laboratory facilities. Taxonomy, phytopathology and other fields which are vital for sustaining sound public policy on phytosanitary issues are threatened with extinction.

Modern molecular identification/detection techniques may tackle the decline in skills since they often require much less specialist skills to perform, are more amenable for routine purposes and can be used for a whole range of different target organisms. Most of those detection and identification methods rely lately on DNA/RNA.

In many fields of research, including agriculture, the scarcity of genomic DNA can be a severely limiting factor on the type and quantity of genetic tests that can be performed on a sample.

Diagnostic laboratories and National reference laboratories serving Plant Health Services often face those limited amounts of DNA/RNA quantities, due to the absence of in vitro cultivation methods of the target organism or the inability to culture obligate plant pathogens or simply because the methods available are time consuming and require expensive containment facilities. Moreover, quarantine specimens may not be available at the different reference laboratories within Europe due to the obstruction of transport of those organisms (EU regulation 95/44). One approach designed to overcome this problem is whole genome amplification (WGA). The objective
is to amplify a limited DNA sample in a non-specific way, in order to generate a new sample that is indistinguishable from the original but with a higher DNA concentration. The ideal WGA technique would amplify a sample up to a microgram level while respecting the original sequence representation or the marker to be used for positive dentification/detection. In this way rare samples can be preserved and a limitless supply of material can be made from the most limited of
resources. In the literature several WGA methods have been described which may be useful in this context.

In this project we will test several of these WGA methods for their utility in obtaining sufficient DNA to be stored and sent across Europe for use in diagnostic protocols as positive but also as negative controls in the area of Plant Health. Targets will be chosen from several groups of plant
pests/pathogens: insects, bacteria, viruses, nematodes, fungi for which TaqMan PCR’s are available. Validated protocols for WGA will be written and made available for Plant Protection Organizations across Europe.

In addition we will develop protocols for optimal storage and transport of DNA from the different plant pests/pathogens and the WGA amplified samples to be send across Europe to be used as positive and negative controls in their diagnostic methods.

Objective
Aims

To develop procedures for optimal whole genome amplification (WGA), storage and transfer of
DNA/RNA material from quarantine plant pathogens.

Objectives

• To develop protocols for whole genome amplification (WGA) of extracted DNA and RNA
samples from quarantine and regulated plant pests/pathogens
• To validate those protocols
• To develop protocols for the correct storage of extracted DNA/RNA and/or WGA amplified
material from quarantine and regulated plant pests/pathogens
• To develop protocols for the correct transportation of extracted DNA/RNA and/or WGA
amplified material from quarantine and regulated plant pests/pathogens
Time-Scale and Cost
From: 2009

To: 2010

Cost: £204,048
Contractor / Funded Organisations
Plant Research International, Central Science Laboratory
Keywords
Plant health              
Plant Pests and Diseases              
Plants and Animals              
Fields of Study
Plant Health