Pathoproof Mastitis Pcr Assay and Sensitivity Validation of a New Test Kit for Rapid Identification of Staphylococcus Aureus, Streptococcus Agalactiae and Mycoplasma Bovis from Bovine Milk

PathoProof Mastitis PCR Assay is a rapid and accurate system for identifying mastitis pathogens from fresh or preserved bovine milk. The system includes: 1) reagent kits; 2) a real-time PCR instrument, i.e. the machine using the reagent kits for analysis of the milk samples; and 3) software for automated interpretation of the results. Rapid and efficient identification of the severe contagious mastitis pathogens Staphylococcus aureus, Streptococcus agalactiae and Mycoplasma bovis is important for the dairy industry and mastitis management. It is especially useful if these micro-organisms could be screened from DHI and/or bulk tank milk samples. We present an extension of the PathoProof product family to include a new kit (‘PathoProof Mastitis Major-3’ kit), which simultaneously identifies these contagious pathogens in fresh or preserved milk. The kit does not require any bacterial culturing efforts and it has a total laboratory throughput time of 4 hours. Sensitivity of the PathoProof system has been previously validated and reported for Staph. aureus and Strep. agalactae (1). We report experiments validating the sensitivity of the new kit for M. bovis based on two different approaches: i) detection limit in bacterial genome copy equivalents in PCR reaction; and ii) detection limit in bacterial colony forming units (CFU) in milk originating from cows with clinical mastitis.