Undergraduate Thesis Or Project

Development of a diagnostic assay for Phytophthora ramorum lineage detection

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  • Phytophthora ramorum is the causal agent of Sudden Oak Death (SOD), which is an economically and environmentally important disease causing up to 80% mortality of tanoaks in Pacific Northwest coastal forests (LeBoldus et al., 2022). There are currently four clonal lineages in the United States and Europe, named North America 1 (NA1), North America 2 (NA2), European 1 (EU1), and European 2 (EU2); only the first three are found in the Pacific Northwest. There is a critical need for fast, accurate, and inexpensive diagnostic probes to identify lineages. Specific High Enzymatic Reporter unLOCKing (SHERLOCK) (a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based assay) can be used to identify lineage from a sample based on polymorphisms unique to each clonal lineages’ genome. With a detection limit of 100 copies of target DNA region template per µL, the SHERLOCK assay is a specific test that can be used in <1 hour, allowing for in field diagnostics. In the current work, we developed an assay specific for the NA1 clonal lineage. DNA from each of the lineages was extracted using a Qiagen DNeasy kit, with concentrations verified using a Nanodrop device. Genomes were scanned to identify diagnostic primers and guide RNA loci for each lineage, using the krisp-vcf algorithm. Krisp-vcf identified a region located on Phytophthora ramorum Pr102 scaffold 0001, position 225003 to 225626 that contained a unique single nucleotide polymorphism (SNP) within the diagnostic sequence for NA1 as compared to the other three lineages. Results of the NA1 SHERLOCK assay, including NA1 strains as positive controls as well as other lineges as negative controls, indicated the assay is lineage specific. This work provides a rapid assay for specific identification of samples for lineage NA1, the oldest discovered lineage of P. ramorum in the continental United States.
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