Development of TaqMan assays for 3ADON, 15ADON and NIV Fusarium genotypes based on Tri12 gene

Fungi of the genus Fusarium are important pathogens of numerous crops including cereals. Infection of the grain with Fusarium spp. often results in the accumulation of various mycotoxins which poses a significant risk to food safety. The Fusarium graminearum species complex, together with F. culmorum, are the main source of trichothecenes in cereal grains. They are comprised of fungal strains producing one of three specific trichothecenes: either 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) (not present in F. culmorum) or NIV (nivalenol). Recently, qualitative PCR approaches have been developed for rapid determination of 3ADON, 15ADON and NIV genotypes within F. graminearum/F. culmorum. This paper presents the development of three TaqMan genotyping assays for quantitative diagnostics of 3ADON, 15ADON and NIV genotypes within F. graminearum/F. culmorum. Primers and probes were designed on the basis of 95 nucleotide site within the Tri12 gene which allowed for specific determination of trichothecene producers. Additionally, to address the problem of detecting inhibitors, an internal positive control (IPC) was developed employing primers and a probe derived from conserved fungal sequence of 5.8S rDNA. The assays developed were successfully applied for direct determination of trichothecene genotypes from Fusarium-damaged wheat kernels (FDK)