Developing diagnostics for quarantine and quality- diseases in plants

These research methods must also be able to detect new diseases and pests so that swift and efficient action can be taken if incidents occur. It is only through knowledge and experience of the latest research methods that efficient action can be taken against notifications in the present and in the future. The comprehensive collaboration between knowledge institutes, testing laboratories and government in this project is essential to keep this sector future-proof.

Objective(s)

The development of cost-efficient and validated modern molecular methods for the identification of quarantine and quality diseases in plants for inspection services and the Netherlands Food and Consumer Product Safety Authority (NVWA). For this we employ the latest innovative technologies and strategies.

Methodology

Within the field of the detection and identification of quarantine and quality diseases in plants, four core areas were selected in which current diagnostics have proven inadequate and where improvements are urgently needed. They have been listed in order of priority:

1. Routine Use of Next Generation Sequencing
   The use of Next Generation Sequencing (NGS) and its concomitant analysis techniques can proactively support diagnostics. The availability of new sequences also facilitates the development and validation of specific tests for molecular multiplex detection.
2. The extraction of DNA/RNA from complex matrices (substrates, materials, soils, etc.)
   Extraction is of great significance for DNA and RNA based detection and diagnostics to allow for the robust detection of plant pathogens. Several matrices have remained problematic. In addition, the costs of DNA/RNA extraction (labour and reagents) are fairly high, which calls for the simplification of the methodology and for cost reduction.
3. Molecular multiplex (RNA and DNA) detection of different pathogens in the same sample
   This component is considered as a core expertise for inspection services and is therefore highly relevant. In the previous rounds this component was identified as being of great importance but the budget did not allow for the financing of this component. Multiplex detection is technically challenging but also of great importance for the efficient processing of samples for detection by the inspection services. For this particular component, we can collaborate with the Thermo Fisher company (previously known as Life Technologies), and acquire a great deal of new knowledge in specificity, amplification efficiency and pre-amplification in the real time PCR using the latest apparatus. Molecular detection techniques will play an increasingly important role due to their speed, sensitivity, objectivity and possibilities for automation. Therefore, these techniques should be employed effectively and cost-efficiently in the workflow of businesses and inspection services. Multiplex methods will result in reduction of costs as a result of the (i) use of a single sample, (ii) the limiting of reagents, apparatus, and time and through (iii) simplified data management. Moreover, reliability can be enhanced if increasing numbers of specific targets are detected per organism and if the same sample can provide multiple controls. The application of the new Open Array (12K) and Digital PCR techniques, are aspects that lend an increased relevance and highly innovate character to research in this core area.
4. Diagnostics for quarantine organisms and quality-compromising diseases
   Research towards the development of new diagnostics for these particular organisms is imperative. For all developed diagnostics, a high batch-to-batch consistency and standardised reference material is essential. Within this component, methods are being developed for the characterisation of reference materials by quality and quantity for Next Generation Sequencing, TaqMan PCR, immunoassays, biotests, etc.

Results

The project will be successful if usable and validated new tests and detection technologies and positive controls and reference materials are developed that can be applied by the NVWA and inspection services to their routine screening, thus effectively protecting and improving the quality of Dutch agricultural and horticultural products.

Next Generation Sequencing (NGS): validated pipeline for the analysis of NGS data; reference sequences for a large number of pathogens which are relevant for inspection services and NVWA; protocol for host depletion in NGS analysis; proof of principle for 48-hour service NGS.
DNA/RNA extraction: Extraction protocols for DNA/RNA from relevant matrices for inspection services and NVWA.

Multiplex methods: multiplex methods for the detection of relevant pathogens for inspection services and NVWA.

Diagnostics: protocols for the generating of control DNA for relevant quarantine pathogens for NVWA.

Further information

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