Abstract: In order to establish a method for the rapid and simultaneous detection of three pathogenic bacteria of mastitis of Streptococcus agalactiae, Staphylococcus aureus and Pseudomonas aeruginosa, three pairs of specific primers were synthesized based on the Streptococcus agalactiae 16S rRNA gene, Staphylococcus aureus NUC heat-resistance gene, Pseudomonas aeruginosa ETA gene sequence and references. By adjusting and optimizing the parameters of the PCR reaction, a multiplex PCR detection system was established, and the sensitivity test and specificity test were simultaneously performed. The results showed that the detection method can simultaneously amplify specific fragments of 270 bp of Streptococcus agalactiae, 153 bp of Staphylococcus aureus and 375 bp of Pseudomonas aeruginosa and a generic 1 500 bp fragment of bacterial 16S rRNA. For other six kinds of pathogenic bacteria, only 1 500 bp universal fragments could be amplified, which had strong specificity. Three specific primer amplification bands were 99% homologous to the GenBank and bacterial gene sequences. After culture for 24 h, the minimum sensitivity of the system to Streptococcus agalactiae, Staphylococcus aureus and Pseudomonas aeruginosa was 8×10⁴, 4×10⁴ and 1.5×10⁵ CFU·mL^-1, respectively. The detection method established in this study can complete the rapid detection of Streptococcus agalactiae, Staphylococcus aureus and Pseudomonas aeruginosa, and is of great significance for the differential diagnosis and rapid detection of subclinical mastitis in dairy cows.

Key words: dairy subclinical mastitis, Streptococcus agalactiae, Staphylococcus aureus, Pseudomonas aeruginosa, multiplex PCR