Validation of a preformulated, field deployable, recombinase polymerase amplification assay for Phytophthora species

Authors: MCCOY, AUSTIN - Michigan State University; MILES, TIMOTHY - Michigan State University; BILODEAU, GUILLAUME - Canadian Food Inspection Agency; WOODS, PATRICK - California Department Of Food And Agriculture; BLOMQUIST, CHERYL - California Department Of Food And Agriculture; Martin, Frank; CHILVERS, MARTIN - Michigan State University

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2020
Publication Date: 4/7/2020
Publication URL: https://handle.nal.usda.gov/10113/6934608
Citation: McCoy, A.G., Miles, T.D., Bilodeau, G., Woods, P., Blomquist, C., Martin, F.N., Chilvers, M.I. 2020. Validation of a preformulated, field deployable, recombinase polymerase amplification assay for Phytophthora species. Plants. 9(4):466. https://doi.org/10.3390/plants9040466.
DOI: https://doi.org/10.3390/plants9040466

Interpretive Summary: Prior work in the Martin lab developed a Phytophthora genus and species specific diagnostic assay using the isothermal technology called recombinant polymerase amplification. The objective of this manuscript is to describe our collaboration with the company who provides the technology to formulate kits with all components of our Phytophthora assay present in the lyophilized pellet with the rest of the reaction components. These preformulated kits were evaluated in different labs using different instruments for data collection with similar results obtained. These results are the first phase of demonstrating the potential for commercialization of these preformulated kits.

Technical Abstract: Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time consuming DNA extractions. Historically RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but more recently have become available in liquid form; both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers, probes as well as additional RPA reagents contained within a single tube limits the risk of contamination and eliminates the need for refrigeration as the lyophilized reagents are stable at ambient temperatures. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers, probe and complete assay reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative-Polymerase Chain Reaction (qPCR) assay and commercially available RPA kits using three qPCR (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platforms (Axxin T16-ISO RPA), with experiments run in different labs. The assay was tested for sensitivity (ranging from 500 pg to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without plant extract added. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the utility and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instrumentation for measuring results.