Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species.

Austin G Mccoy,Cheryl Blomquist,Patrick Woods,Guillaume J Bilodeau,Martin I Chilvers,Timothy D Miles,Frank N Martin

doi:10.3390/plants9040466

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 have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA 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 platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 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 added plant extract. 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 consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.

Highlights

Oomycetes within the genus Phytophthora constitute a large group of destructive plant pathogens.Phytophthora species cause root, crown, stem, foliar and fruit diseases on agriculturally and ecologically important species of plants [1,2,3,4]
Identification of ambiguous Phytophthora species has traditionally relied on techniques such as baiting, isolation onto a Phytophthora semi-selective medium, DNA extraction and polymerase chain reaction (PCR) or antibodies to identify the genus or species present [5,6,7,8]
Were Observed Between Preformulated and Commercially Available Recombinase polymerase amplification (RPA) Reactions

Summary

Introduction

Phytophthora species cause root, crown, stem, foliar and fruit diseases on agriculturally and ecologically important species of plants [1,2,3,4] These diseases can be difficult or impossible to distinguish by symptoms alone and in-lab diagnostic testing is required for accurate pathogen identification. Identification of ambiguous Phytophthora species has traditionally relied on techniques such as baiting, isolation onto a Phytophthora semi-selective medium, DNA extraction and polymerase chain reaction (PCR) or antibodies (i.e., immunostrips) to identify the genus or species present [5,6,7,8].

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Results

Conclusion