Validation of a Rapid, High-Throughput Isothermal Recombinase Polymerase Amplification Screening Assay for <i>Phytophthora ramorum</i>

Phytophthora ramorum is an invasive, federally regulated pathogen in the United States, with at least 75 confirmed hosts and over 100 associated hosts. The movement of ornamental plants infected with P. ramorum, as a regulated pathogen, across the United States resulted in a surge of samples experienced by diagnostic laboratories, underscoring the need for a specific and rapid screening assay. This work adapted and validated a previously published P. ramorum recombinase polymerase amplification (RPA) diagnostic tool for commercial application. This assay was validated through inclusivity testing for the four P. ramorum lineages and exclusivity testing on a panel of Phytophthora species and other commonly detected oomycetes. The reformulated assay demonstrated sensitive and specific detection of P. ramorum down to 1 fg for each lineage, without cross-reacting with over 100 different oomycetes across at least 30 hosts. The RPA assay underwent three tiers of validation. In Tier 1, a single-lab, multi-operator validation, it achieved diagnostic specificity and sensitivity of 100 and 92%, respectively, with 100% reproducibility and repeatability among operators. Tier 2 validation, performed on a blind sample set at two independent labs, resulted in 100% diagnostic specificity and sensitivity. Tier 3 involved a blind performance study across five independent labs using multiple fluorescent cycler platforms and operators. The reformulated RPA assay offers a rapid and simple approach for the molecular detection and identification of P. ramorum. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .