Development of multiplex polymerase chain reaction for the simultaneous detection of bovine mastitis-associated pathogens

Abstract

A multiplex polymerase chain reaction (mPCR) assay was developed for the simultaneous detection of five bovine mastitis-associated pathogens including Staphylococcus simulans, Staphylococcus hominis, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus genus-specific group. Results showed that the optimal annealing temperature for mPCR was 58 °C. The sensitivity of mPCR to detect the least DNA template concentration was 0.625 ng. The nucleotide sequences of PCR products were identified using the NCBI database. Results showed 99.15-100% homology to the gap gene of Staphylococcus simulans, 100% homology to the peptidase gene of Staphylococcus hominis, 98.76-99.69% homology to the clumping factor A gene of Staphylococcus aureus, 99.09-99.64% homology to the CAMP factor gene of Streptococcus agalactiae, and 99.13% to the elongation factor tu gene of the Streptococcus genus-specific group. This developed mPCR technique showed potential as a rapid, specific, highly sensitive, and low-cost diagnostic tool for the simultaneous detection of bovine mastitis-associated pathogens.