Development of a multiplex PCR for detection of Staphylococcus aureus in skim milk and cheddar cheese

Staphylococcus aureus is an important foodborne pathogen that can enter into dairy products through raw ingredients or food handling. The organism is salt tolerant and can survive in hard cheese. Rapid detection of this organism in dairy products is important for product quality and safety. The objectives of this study were to develop a method for extraction of S. aureus DNA from milk and hard cheese and to identify a multiplex PCR method to detect pathogenic S. aureus. A modified solvent extraction procedure that included addition of lysostaphin was developed for extraction of S. aureus DNA from foods. Primers were identified from the thermostable nuclease (nuc) gene and the enterotoxin C (ent C) gene. PCR parameters for the simultaneous amplification of both targets were optimized for annealing temperature, annealing time, magnesium chloride, and DMSO concentration. Specificity was tested with pathogenic Staphylococci, nonpathogenic Staphylococci and other genera. Sensitivity of the developed assay was determined by the extraction and amplification of S. aureus DNA from serial dilutions (100 to 108 CFU/mL) in skim milk and hard cheese (10 mL volumes). The developed assay will be a useful tool for detection and differentiation of S. aureus contamination in dairy foods.