TaqMan real time-quantitative PCR targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese

Late blowing defect affects hard cheese production and Clostridium tyrobutyricum appears as the primary cause of this spoilage. Our aim was to develop a quantitative TaqMan real-time PCR method to detect this agent in different matrices along the whole agro-dairy chain, using specific primers targeting the phosphotransacetylase gene (pta). The optimised assay appeared to be sensitive and specific to quantify C tyrobutyricum. Results demonstrated a 100% efficiency with low numbers of cells and spores (approx. 14 mL(-1) or g(-1)) also detected in broth and in milk matrix artificially contaminated with C tyrobutyricum. Combined with a DNA extraction method optimised to lyse both cells and spores, our method is suitable for silage and hay, faeces, milk and hard cheese samples without any enrichment. The molecular quantification by qPCR was found to be precise and could be used for the rapid determination of the presence of C tyrobutyricum. (C) 2013 Elsevier Ltd. All rights reserved.