Validation of a Real-time PCR assay for fast and sensitive quantification of Brucella spp. in water buffalo milk

Water buffalo milk and derived dairy products, including mozzarella cheese, represent a possible source of Brucella contamination for consumers. Brucella is a severe pathogen for human health even at low concentrations. It is therefore fundamental to develop an assay that is faster and more sensitive than the traditional bacterial culturing method for the detection of the pathogen in the food matrix. We designed a Real-time PCR assay able to detect as low as 1 CFU/ml of Brucella spp. in water (80% probability) and 3 CFU/ml of Brucella spp. in buffalo milk (50% probability) in less than 3 h without any enrichment step. The assay was validated by calculating specificity, sensitivity, detection limit, precision, PCR efficiency, DNA extraction efficiency and food matrix inhibition. When this method was employed to detect and quantify Brucella spp. in 109 buffalo milk samples, the assay demonstrated a higher sensitivity in comparison to bacteriological analysis (27 positive samples and 2 positive samples, respectively).