On the sensitivity of qPCR diagnostics for the canola clubroot pathogen Plasmodiophora brassicae

Abstract

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other Brassica crops. Polymerase chain reaction (PCR), particularly probe-based quantitative PCR (qPCR), is widely used for the detection of P. brassicae in soil samples. To improve consistency in clubroot detection while maintaining efficiency, diagnostic laboratories would benefit from adopting a single, highly efficient qPCR system for routine testing. In this study, we analyzed the primer and probe sequences of all published PCR and qPCR systems for P. brassicae detection. Based on these analyses, three independently developed probe-based qPCR systems were selected and their performance was evaluated using synthesized target DNA (gBlock). One probe-based qPCR system exhibiting superior sensitivity on gBlock was subsequently evaluated on P. brassicae genomic DNA. This system consistently detected DNA equivalent to four resting spores per reaction, corresponding to a soil sample containing 1,000 spores per g soil when the DNA extraction protocol was considered as a component of the qPCR system. The sensitivity of the system was further validated using DNA extracted from soil samples collected from multiple locations across Alberta, where P. brassicae was detected at levels below those associated with visible clubroot symptoms. Based on these results, we recommend this qPCR system for routine clubroot diagnostics in laboratories across Canada.