Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products

• Published in: Journal of food protection 2001-05, Vol.64 (5), p.664-668
• Main Authors: TAMARAPU, Sudhir, MCKILLIP, John L, DRAKE, Maryanne
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cheese - microbiology; Colony Count, Microbial; Dairy Products - microbiology; DNA, Bacterial - analysis; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Milk - microbiology; Milk and cheese industries. Ice creams; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction - methods; Polymorphism, Restriction Fragment Length; Sensitivity and Specificity; Staphylococcus aureus - classification; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Time Factors
• ISSN: 0362-028X; 1944-9097
• DOI: 10.4315/0362-028X-64.5.664

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.