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Development of CRISPR-Cas and LAMP diagnostics to inform plant disease management

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  • Full or part time
    Dr J Tovar
    Dr L Bindschedler
  • Application Deadline
    No more applications being accepted
  • Competition Funded PhD Project (European/UK Students Only)
    Competition Funded PhD Project (European/UK Students Only)

Project Description

Fungal infections are a major threat to food security worldwide. In the UK alone, Septoria wheat leaf blotch – a fungal disease caused by Zymoseptoria tritici – and oilseed rape phoma leafspot and stem canker, diseases caused by the fungi Leptosphaeria maculans and L. biglobosa, cause combined agricultural loses of €180-300 million Euros per year. Other major crops such as barley and potato are also severely affected by fungal infection. The implementation of effective plant disease management strategies by farmers in the field requires fast, sensitive and specific diagnosis of infection, yet current diagnostic methods based on sample culture and microscopic identification are inadequate. Modern diagnostic tools that allow early intervention to minimise symptom development and agricultural loss are urgently needed.
This project seeks to develop novel nucleic acids diagnostic tests for fungal infections using Leptosphaeria and Zymoseptoria as case studies. We will use a set of genus-specific and species-specific DNA target biomarkers recently identified in Dr Tovar-Torres’s laboratory and CRISPR-Cas and loop-mediated amplification (LAMP) technologies to generate standardised Leptosphaeria and Zymoseptoria diagnostic kits for field and laboratory diagnosis. The project contains the following work packages:
1. Biomarker design and engineering of molecular reagents. Data mining and bioinformatic tools will be used to identify unique Leptosphaeria maculans and Zymoseptoria tritici target biomarkers for use in single and multiplex diagnostic assays. Expected outcomes: Guide CRISPR RNAs and LAMP primer sets that allow species-specific detection of disease-causing fungi.
2. Assay design and optimisation of reaction conditions. Isothermal and multi-thermal biochemical assays will be tested under a range experimental conditions to optimise reagent mixtures and reaction conditions. Expected outcomes: Standardised LAMP and in CRISPR-Cas assays for Leptosphaeria and Zymoseptoria diagnostics.
3. In-vitro assay evaluation: sensitivity and specificity testing. We will test the specificity and sensitivity of our standardised assays in vitro by establishing the minimum genomic equivalents or colony forming units that can be reliably detected using either serial dilution of DNA or spiked samples with quantified fungal conidia, followed by DNA isolation and detection. Expected outcomes: Monoplex and multiplex Leptosphaeria and Zymoseptoria assays with optimised sensitivity and specificity.
4. Validation of Leptosphaeria and Zymoseptoria diagnostic kits using field samples. We will validate our kits using field samples. Expected outcomes: A fully validated and standardised set of CRISPR-Cas and LAMP fungal diagnostic kits.
This proposal is expected to generate novel molecular diagnostic tools with substantial societal and economic impact. Farmers, biotech companies and diagnostic enterprises are all potential stakeholders. Early fungal diagnosis will support plant disease management leading to significant savings to the UK and global agriculture.

Interviews are provisionally planned for the period 25th February and 8th March 2019.
Please follow the application link below to our school web page for full instructions on eligibility and how to apply

Funding Notes

The studentship award will cover the cost of institutional tuition fees and provide an annual tax-free living stipend with at the standard RCUK rate with London weighting. The amount is currently at £16,777pa for the 18/19 Academic Year

References

Sashital, D. G. (2018). "Pathogen detection in the CRISPR-Cas era." Genome Medicine 10.
Zhou, L., et al. (2018). "The applications of CRISPR/Cas system in molecular detection." Journal of Cellular and Molecular Medicine 22(12): 5807-5815.
Knott, G. J. and J. A. Doudna (2018). "CRISPR-Cas guides the future of genetic engineering." Science 361(6405): 866-869.

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