graminearum (isolate 212, University of Nottingham), F. culmorum (isolate 236, University of Nottingham), F. avenaceum (isolate 40, University of Nottingham), F. tricinctum (isolate 53, University of Nottingham), F. poae (isolate 246, University of Nottingham), F. langsethiae (isolate 227, University of Nottingham), M. nivale (isolate 226, University of Nottingham) and M. majus (isolate 224, University of Nottingham) were used to make DNA standard curves (100–10− 6 ng/μl). The amplification mix for each species consisted of 250 nM

of each primer (forward and reverse) and 2 × iQ SYBR Green Supermix (Bio-Rad, UK) reagent which was used according to manufacturer’s instructions. The volume of DNA sample in the reactions was 2.5 μl in a total reaction volume of 12.5 μl. In the negative control 2.5 μl of PCR-grade water replaced the DNA template. The detection limit for all eight assays is 10− 4 pg/ng check details total fungal DNA and all assays had an efficiency of E = 95–103%. Species specific primers www.selleckchem.com/products/Y-27632.html used for quantification of the species of interest, assay efficiency and references are presented in Table A.3. Mycotoxin analysis on all samples was performed by Campden BRI (Chipping Campden, UK) using UKAS accredited procedures. The trichothecenes

(DON, NIV,HT-2 and T-2) and zearalenone were extracted from flour oxyclozanide samples (25 g) into an acetonitrile/water mixture with further clean-up of the trichothecenes by solid phase extraction by passing the filtrate through Bond Elut Mycotoxin SPE columns (Agilent Technologies, Germany) (Klotzel et al., 2006). The LC/MS/MS analysis was performed on an Agilent 1200 Infinity LC system (Agilent Technologies, Germany) with a binary pump, coupled with Agilent 6490 MS/MS ESI (Agilent

Technologies, Germany) and equipped with an analytical column Agilent Poroshell 120 EC-C18 (2.1 × 100 mm, 2.7 μm, ID Agilent Technologies, Germany). The flow rate was set at 0.2 ml/min and the injection volume was 10 μl. Mobile phase A was water with 0.2% acetic acid and 5 mM ammonium acetate, mobile phase B was methanol with 0.2% acetic acid and 5 mM ammonium acetate. A linear binary gradient was applied from 20 to 70% phase B within 30 min. The content of phase B was then lowered to 20% within a minute followed by equilibration of the column for 10 min. Quantitative determination of all compounds was performed by operating the mass spectrometer in ESI positive and negative ionisation modes (Schuhmacher et al., 2005). The quantification of the samples was carried out using matrix-matched standards prepared in-house. Spiked samples were included in each batch to determine extraction recovery. The method had an acceptable recovery range for each trichothecene of 60–120%. The results were corrected for recovery.