Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring.

LightCycler technology combines rapid in vitro amplification of DNA with real time detection and quantification of the amount of target molecules present in a sample. The system enables a 35-cycle PCR with 32 samples do be completed in 45 min, including quantification and identification of the product. It is therefore well suited for routine analysis of large numbers of samples in quality control and for defining HACCP concepts. Based on PCR primers specific to the tri5 gene, a quantitative group specific assay was established for Fusarium species producing trichothecenes. In the assay, SYBR Green I was used as fluorescent dye enabling real time detection of PCR products. Characterisation of the amplicons was achieved by melting point analysis (85 +/- 0.1 degrees C). Nonspecific products such as primer dimers could readily be distinguished from the product by their lower melting points. Composition of the amplification buffer was optimised and various hot start methods were tested in order to achieve the highest sensitivity of the assay. Uracil DNA glycosylase was added to prevent amplification of nonspecific products due to DNA carryover. The spectrum of species detected was generally in accordance with the results found in conventional PCR using the Tox5 primer pair. Reproducibility in six parallel experiments of the assay was determined to be 98% in the range between 0.05 and 6 ng of purified Fusarium graminearum DNA. The assay was used to analyse 30 wheat samples contaminated with toxigenic Fusarium spp. Contamination ranged from 0% to 78% as revealed by mycological analysis, and this is compared with results from the LightCycler. This is the first report on the use of the LightCycler system in combination with SYBR Green I for the quantification and identification of fungal DNA in pure cultures and sample material.