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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Publications at this Location » Publication #418858
Research Project: Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries

Location: Crop Improvement and Protection Research

Title: Validation of a rapid, high throughput isothermal recombinase polymerase amplification screening assay for Phytophthora ramorum

Author
item NEUGEBAUER, KERRI - Michigan State University
item DAVENPORT, BRYANT - Agdia
item HARMON, CARRIE - University Of Florida
item BYRNE, JAN - Michigan State University
item MILES, LAURA - Michigan State University
item SNOVER-CLIFT, KAREN - Cornell University
item ROONEY-LATHAM, SUZANNE - California Department Of Food And Agriculture
item Martin, Frank
item Luster, Douglas
item MILES, TIMOTHY - Michigan State University

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2025
Publication Date: 5/20/2025
Citation: Neugebauer, K., Davenport, B., Harmon, C., Byrne, J., Miles, L., Snover-Clift, K., Rooney-Latham, S., Martin, F.N., Luster, D.G., Miles, T. 2025. Validation of a rapid, high throughput isothermal recombinase polymerase amplification screening assay for Phytophthora ramorum. PhytoFrontiers. 5(2):220-228. https://doi.org/10.1094/PHYTOFR-10-24-0114-FI.
DOI: https://doi.org/10.1094/PHYTOFR-10-24-0114-FI

Interpretive Summary: This manuscript discusses the commercialization by Agdia of a molecular diagnostic technique developed in an ARS lab using recombinant polymerase amplification (RPA) for detection of the quarantine pathogen Phytophthora ramorum. This technique is run at a constant temperature and is capable of pathogen detection in as little as 20 minutes without the need for DNA extraction. The specificity of the assay was validated against a wide array of Phytophthora species with culture and plant samples used for evaluating consistency when the assay was run in different labs.

Technical Abstract: Phytophthora ramorum is a water mold pathogen with at least 75 proven hosts and over 100 associated hosts. As a regulated pathogen the movement of ornamental plants infected with P. ramorum across the U.S. resulted in a surge of samples experienced by diagnostic laboratories and has emphasized the need for a specific and rapid screening assay. This work adapted and commercialized a P. ramorum RPA tool developed by Miles et al (2015). This assay was adapted using inclusivity testing to the four P. ramorum lineages and exclusivity testing on a panel of Phytophthora species and other commonly detected oomycetes. The developed assay showed sensitive and specific detection of P. ramorum down to 1fg/µl of each lineage while avoiding cross-reaction with over 100 different oomycetes in at least 30 hosts. The RPA assay was put through three tiers of validation. The first validation, a single lab multi-operator validation, produced diagnostic specificity and sensitivity scores of 100% and 92%, respectively, with 100% reproducibility and repeatability among operators. Tier 2 validation resulted in 100% diagnostic specificity and sensitivity on a blind sample set performed at two independent labs. The tier 3 validation was a blind performance study involving five independent labs across multiple fluorescent cycler platforms and operators. The developed RPA assay provides a rapid and simplistic approach for molecular detection and identification of P. ramorum.