Topic A: Whole genomic amplification methods

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Topic title

Validation and harmonisation of Whole Genome Amplification techniques in support of the diagnosis of quarantine pests/pathogens of plants relevant to the Netherlands and UK, and the wider EU.

Background

Within the European Commission, quarantine plant pest species are the policy responsibility of The European Commission's DG SANCO. National diagnostic or National Reference Laboratories (NRL) play a key role in diagnosis of quarantine/regulated plant pests and diseases. Highly qualified reference DNA is required to develop, standardize, validate and harmonize molecular diagnostics. Furthermore, reference DNA is needed as positive/negative controls for monitoring reliability of the results of molecular assays (recommended in EPPO standard 7/84 Basic requirements for quality management in plant pest diagnosis laboratories; obligatory for ISO 17025 accreditation).

Diagnostic laboratories serving national Plant Health Services often face limited DNA quantities, due to the absence or time-consuming in vitro cultivation methods of the target organisms. The use of cloned target DNA fragments can be a solution to create an unlimited source of nucleic acid. However, these clones do not represent the complete genome and can only be used for assays amplifying specific target DNA. Whole Genome Amplification (WGA) is a way to increase minute quantities of DNA from small and/or precious samples into microgram yields and, contrary to cloning, representing the complete genome for a diversity of down-stream processes, e.g. molecular identification and detection. WGA is particularly useful for non-cultivable organisms in their natural matrices (plant tissue) or for archived material in collections

Research need

The scientific community needs reliable, validated and harmonised protocols for WGA methods applicable to plant pathogens and pests and for the durable storage of DNA produced by WGA. The WGA methods need to be evaluated both in relation to the quantity and quality of the WGA-DNA. The work should produce:

Validated WGA protocols suitable for relevant plant pathogen and pest groups, where validation might comprise:
- Testing commercial WGA kits (PCR-based or Multi Multiple Displacement Amplification) for their yield, in particular for organisms of low availability.
- Coverage of genome by amplification of randomly chosen fragments of the genome, tested on reference species from a range of pest groups with complete genome sequence.
- Comparison of the original genome with amplified genomes of the organism of interest using typing techniques, e.g. AFLP.
Protocols for the durable storage of WGA-DNA samples from representative quarantine plant pests and pathogens.

Specific project outputs should therefore be: (i) published/publicly available generic, harmonised protocols for WGA on reference species and species of concern to the funding countries UK, NL and to the wider EU; (ii) published/publicly available protocols (accepted and ring tested) for durable storage of WGA-amplified DNA samples; (iii) validation reports.

Expected benefits

The project outputs will help Government bodies in the funding countries (The Netherlands and the UK) and wider EU (especially those that are tasked with implementing the EC Plant Health Directive) to meet the quality standards on WGA and DNA storage imposed by authorized reference laboratories in the domain of Plant Health.
The project will facilitate sharing of expertise and the development of increased European science capacity/capability in the area of molecular diagnosis of quarantine plant pests and pathogens.
The scientific community working in this area, through collaborative research on shared needs and through exchange of expertise.

Organisations interested in participating in a research consortium

Name: David Kenyon Organisation: SASA

Expertise: Expertise in DNA sequencing and the utilisation of a range of marker systems including microsatilites and AFLP's. Experiance of developing noval diagnostic probes for the detection of quarantine organisms using real-time PCR.

Name: dhdykksw Organisation: None

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