Hyd5 gene based analysis of cereals and malt for gushing-inducing Fusarium spp. by real-time LAMP using fluorescence and turbidity measurements

The surface active class 2 hydrophobin Hyd5p (GenBank accession number DQ449530) has been identified as a causative agent for over foaming (gushing) of beer. In order to estimate the potential of brewing cereals and malt to induce gushing in beer we used a previously described set of primers to amplify a partial sequence of the hyd5 gene in Fusarium culmorum and closely related species in a real-time loop-mediated isothermal amplification assay. Real-time LAMP was optimized and performed on two different platforms using a turbidimeter and a fluorescence reader to monitor the reaction on line. Serial dilutions of purified target DNA were used to set up a calibration curve for quantitative estimation of DNA concentrations. Analysis of model barley samples prepared by mixing infected with non-infected material in different ratios demonstrated a positive correlation between the real-time LAMP results and respective infection levels. In order to investigate the performance of the newly established methods, samples of barley were analyzed for their gushing potential using the Hyd5 real-time LAMP assay and a reference in vitro test for gushing prediction (Modified Carlsberg Test, MCT) in parallel experiments. Results showed that the real-time LAMP assay was in accordance with the reference test in 50% of cases with both platforms used. It predicted a lower number of gushing-positive samples as compared to the current reference test. (C) 2013 Elsevier B.V. All rights reserved.