RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

Abstract

Plant diseases are often diagnosed by the method of DNA extraction followed by PCR. DNA extraction from plant tissue can be a recalcitrant and lengthy process, and sometimes ends up with inhibitors that reduce PCR amplification efficiency. Here we present a unique approach, RPA-PCR couple, to exclude the DNA extraction step from the standard plant diagnostic process. The process crudely macerates plant tissue in water for a few minutes, and then transfers the macerate supernatant to a Recombinase Polymerase Amplification (RPA) reaction. Following an incubation of 20 minutes at 39 {degrees}C, the RPA reaction can be directly utilized in PCR amplification. In RPA-PCR couple, the RPA reaction is run at slower reaction kinetics to promotes amplification of long amplicons and the slower reaction kinetics are achieved by lowering RPA components concentrations. In this proof of concept study, we targeted Phytophthora intergenic mitochondrial spacer between atp9 and nad9 genes and the two common Phytophthora pathogens of strawberry: P. fragariae and P. cactorum. We presented coupling of RPA with real time TaqMan and SYBR Green PCR assays, and conventional PCR amplification aimed at Sanger sequencing. We found the RPA-PCR couple specific and capable of detecting as low as 10 fg of Phytophthora genomic DNA. Moreover, comparing RPA-PCR couple with the routine method of DNA extraction followed by PCR generated comparable results for the field samples. The idea of RPA-PCR couple to exclude DNA extraction may have vast application in different fields such as clinical diagnostics, food inspection and soil sciences.