A multiplex qPCR assay for detection and quantification of Plasmodiophora brassicae in soil

Abstract

Various physical and chemical factors in soil can inhibit the detection and quantification of soilborne plant pathogens using quantitative polymerase chain reaction (qPCR) assays. A multiplexed TaqMan qPCR assay, including a competitive internal positive control (CIPC), was developed to identify and (where necessary) compensate for inhibition in the quantification of resting spores of Plasmodiophora brassicae from soil. The CIPC amplicon was developed by modifying a sequence coding for green fluorescent protein so that it could be amplified with P. brassicae-specific primers. Addition of CIPC at 5 fg/μl to the singleplex qPCR assay designed to quantify P. brassicae genomic DNA did not reduce the sensitivity, specificity, or reproducibility of the assay. Each of the soil samples, either artificially inoculated or naturally infested with P. brassicae, exhibited no amplification of the CIPC. When the samples were diluted and reassessed, the quantification cycle of the CIPC relative to the control (water only) was delayed in each sample. The magnitude of the delay was used to adjust the estimate of resting spore concentration. The corrected concentration estimates were significantly higher than the unadjusted estimate, which indicated the presence of DNA inhibitors in samples even after dilution. The only exception was a mineral soil sample inoculated with a low concentration (10 3 spores/g) of resting spores. The assay was optimized for use on a range of soil types. A sample of 0.25 g for mineral soil and 0.10 g for high-organic-matter soil was optimum for recovery of DNA of P. brassicae. The assay represents an improvement over existing assays for estimating resting spore concentration in infested fields.