Staphylococcus aureus is a spherical,Gram-positive pathogenic bacterium commonly associated with bovine mastitis and clinical infections.It is also recognized as a pathogen causing the outbreak of food poisoning.The objective of this study was to develop and evaluate a rapid and reliable technique that combines propidium monoazide(PMA)staining with real-time PCR to detect and quantify viable cells of S.aureus in milk powder and meat products.The inclusivity and exclusivity of the assay were evaluated using 58 strains belonging to 14 species.Serial dilutions of S.aureus cells were used to establish a standard curve and to confirm the effect of PMA treatment.Milk powder and meat products were used as the spiked food and the ability PMA-qPCR to eliminate the dead cells was performed on milk powder.Furthermore,meat products were inoculated with different density of S.aureus and 105 CFU/g of Bacillus cereus and Salmonella enterica to test the interference of non-target microorganism.PMA treatment prior to DNA extracted and the results showed PMA is able to eliminate the false-positive results with little effect on viable cells.The PMA-qPCR assay was specific and more sensitive than conventional PCR that the LOD was 3.0 × 102 CFU/g in spiked milk powder.Additionally,there was no significant interference for the detection of viable S.aureus from other non-target bacteria.At last,the PMA-qPCR protocol can be an effective and rapid method to quantify the viable cells of S.aureus in food samples.These studies demonstrated that the PMA-qPCR assay is specific and reliable,offering a valuable diagnostic tool for routine analysis in food and clinical diagnostic research at a reasonable cost.It can also be used on liquid milk e.g.bovine,goat and donkey milk etc.