Detection by multiplex PCR of Staphylococcus aureus , S. intermedius and S. hyicus in artificially contaminated milk

Abstract

ABSTRACT: This research aimed to detect coagulase-positive Staphylococcus (CPS) directly in samples of artificially contaminated milk, using multiplex PCR (mPCR). Standard and isolated bacterial strains of S. aureus, S. epidermidis, S. hyicus, S. intermedius, Listeria monocytogenes and Escherichia coli species were used, evaluating the specificity and detection limit of mPCR, for artificially contaminated UHT milk. Primers specific for the nuc gene (NUC1-NUC2 were used for S. aureus, NUC3-NUC4 for S. hyicus and NUC5-NUC6 for S. intermedius). It was possible to detect the three target species by mPCR, directly from bovine whole milk, with adequate specificity and acceptable detention limit for identification of coagulase-positive Staphylococcus (CPS) in foods. The specificity was determined by the amplification of species-specific fragments, and the detection limit was assessed by the detection thresholds obtained for the three species (103 CFU mL-1). From these results, the mPCR described, with the proposed set of primers, has the potential for use in precise identification and differentiation between CPSs in milk samples.

Keywords

• Staphylococcus aureus
• Staphylococcus hyicus
• Staphylococcus intermedius
• Multiplex PCR
• nuc gene.