Simultaneous detection of mastitis pathogens in milk by multiplex real-time polymerase chain reaction

The objective was to develop a multiplex real-time polymerase chain reaction (PCR) for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. Milk samples (n=192) were analyzed by multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. The multiplex real-time PCR technique correctly identified 97.7% of all quarter milk samples, 91% of Staph. aureus, 98% of Strep. agalactiae, and 100% of Strep. uberis. The overall sensitivity of the multiplex real-time PCR assay to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5% and the specificity was 99.6%. Results of this study indicate that multiplex real-time PCR procedure is a rapid and accurate method for simultaneous identification of Staph. aureus, Strep. agalactiae and Strep. uberis directly from quarter milk samples.