Real‐time PCR assay for quantification of <i>Tilletia caries</i> contamination of UK wheat seed

Abstract

A quantitative real‐time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat‐seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log 10 (number of spores per seed + 1) on mean log 10 DNA ( µ g) produced a coefficient of determination ( R 2 ) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real‐time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed.