Validation of a real-time PCR method on pta gene for Clostridium tyrobutyricum quantification in milk

Abstract In the dairy industry, the late blowing defect in medium and long ripened cheeses causes important economic losses. This cheese defect, caused mainly by the anaerobic spore former Clostridium tyrobutyricum, is characterized by gas production resulting in holes formation in the paste, and in unpleasant aromas related to the butyrate production. Since longtime, the reference method used to detect the presence of total spore number in milk is the Most Probable Number (MPN), a semi-quantitative method which measures the bacterial growth and the gas production after heat treatment of the samples. This method often gives uncertain results, it requires long incubation periods and fails to differentiate between different species of spore-forming bacteria. A TaqMan real-time quantitative PCR targeting the phosphotransacetylase gene (pta) for C. tyrobutyricum was previously developed as an alternative technique to the traditional method, faster and more specific, and therefore of great interest to preventively determine the contamination of milk by C. tyrobutyricum. The aim of this work was the validation of this quantitative real-time PCR protocol for the C. tyrobutyricum detection in milk. Following the method validation, 120 samples of UHT and raw milk artificially contaminated with C. tyrobutyricum spores were analyzed in increasing concentrations, relating the results of the molecular method with those obtained by the MPN. Finally, 144 raw milk samples with a possible natural contamination were analyzed and demonstrated a positivity rate of 15.28% (CI95% 10.31–22.05%).