Applying CRISPR-Cas Technology to Bioaerosol Environmental DNA for the Monitoring of Pathogenic Species Such as P. Infestans
DAVID O'CONNOR, Weili Guo, Ciara Mcdermott, Steven Kildea, Anne Parle-McDermott, Dublin City University
Abstract Number: 671
Working Group: Bioaerosols in Agriculture: Sources, Risks and Mitigation
Abstract
Pathogenic plant microorganisms such as oomycetes, fungi, viruses and bacteria pose a serious threat to food security and negatively impact the agricultural ecosystem. Fungal infection along is accounted for up to 23% of global crop loss. Monitoring airborne plant pathogens is vital in terms of identifying pathogenic species, tracking their movement, therefore developing precision pesticides spraying program and preventing outbreaks.
Current molecular PCR-based detection methods are not suitable for ‘on-site’ early detection due to the requirement of sophisticated lab-based equipment such as Light Cycler. In recent year, environmental DNA (eDNA) has become a powerful tool for monitoring various species in surrounding environment (Williams et al., 2019). In this project, genetic material in eDNA extracted from ambient air samples will be evaluated using isothermal-based RPA method coupled CRISPR-Cas12a detection platform, the presence or absence of specific plant pathogen such as phytophthora infestans will be detected via both fluorescent and colorimetric techniques. Mycelia genomic DNA samples from phytophthora infestans and three close relative species were used for the assay development, mitochondrial sequences of phytophthora infestans and the three off-targets obtained from GenBank were used for gRNA and primer design, air samples collected by a multi vial cyclone sampler (Burkard, UK) were used for eDNA extraction.
Using the collateral cleavage activity of Cas12a, RPA coupled CRIPSR-Cas12a assay, as a proof-of-concept, revealed the specific detection of phytophthora infestans at constant temperature of 37 o C with single detection at 0.85fM. The presence of phytophthora infestans in one out of seventeen air samples were determined by both FAM-mediated fluorescent assay and colorimetric-based lateral flow assay.