Real-time PCR for quantification of toxigenic Fusarium species in barley and malt

Fusarium species are potential mycotoxin producers in cereals. In near future EU is going to set the maximum limit values for some Fusarium toxins in unprocessed cereals and cereal products. Mycotoxin analyses are expensive and time-consuming. Hence, a rapid and reliable quantification method for toxigenic Fusarium species is needed for evaluation of the mycotoxin risk in cereal-based industry. We have applied real-time PCR technique for the quantification of trichothecene-producing Fusarium species present in barley and malt samples (the TMTRI assay, S. Klemsdal unpubl. sequences). PCR results were compared to the amount of trichothecenes in the samples. Furthermore, highly toxigenic Fusarium gram inearum was quantified in cereals by real-time PCR (the TMFg12 assay, T. Yli-Mattila unpubl. sequences). DNA was extracted from ground kernels (0.1 g) using FastDNA Spin Kit for Soil and analysed in a LightCycler® system using fluorigenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium species and F. graminearum present in barley and malt samples, respectively. Both TaqMan assays were regarded as sensitive and reproducible. Linearity of the assays was at least 3-4 log units when determined using pure Fusarium DNA. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay correlated with the DON content in Finnish barley samples. The TMFg12 assay for F. graminearum gave a good estimation about the DON content in North American barley and malt samples. The amounts of DON and F. graminearum in Finnish barley were found to be naturally low.