Multiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins from dairy products
Multiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins from dairy products
Mohammad Javad Forouzani-Moghaddam,1,*
1. Msc of Food Microbiology, School of Paramedical, Iran University of Medical Sciences, Tehran, Iran
Introduction: Staphylococcal food poisoning remains one of the three most common types of food poisoning in the United States; approximately 25% of all foodborne illnesses reported in the United States are caused by staphylococcal intoxication. Staphylococcus aureus is a ubiquitous bacterium, being both a human and a zoonotic commensal. As a result, S. aureus may be found in milk, other dairy products, vegetables, and raw and fermented meats. The bacterium is also highly salt tolerant, resistant to nitrites, and capable of growth in foods with a low water activity. S. aureus is regarded as potentially hazardous in foods due to production of heat-stable enterotoxins.Due to these characteristics, S. aureus is an important foodborne pathogen. At least nine enterotoxins (A, B, C, D, E, G, H, I, and J) may be produced by S. aureus.
Methods: Cultures were incubated statically overnight in tryptic soy brothat 37 °C. Following confirmation of PCR primer specificity, S. aureus ATCC 25923 were used in food inoculations. Conditions were optimized for multiplex PCR targeting both entC and nuc genes simultaneously in a single tube. Magnesium chloride, primer, and dimethylsulfoxide concentration titrations were conducted. Final reaction conditions included 2.5 ml of extracted DNA, 1 ml of dimethylsulfoxide, 2 ml of 25 mM magnesium chloride (Life Technologies), 20 pmol of forward primer, 20 pmol of reverse primer (for each gene), and 44 ml of PCR Supermix. The cycling profile was the same as described above, except the annealing temperature was increased to 56 °C.
Results: DNA was successfully extracted from as few as 10 CFU/ml S. aureus from skim milk or 10 CFU/20 g cheese. S. aureus possesses a thick peptidoglycan cell wall and is resistant to many techniques that easily lyse other cells. The application of lysostaphin was necessary for successful extraction of S. aureus DNA. They also noted that DNA yields from cheese were higher than DNA yields from skim milk, which were similar to results reported here. The reason for the higher yields of DNA from cheese may be due to the presence of naturally occurring cheese microflora.
Conclusion: The developed multiplex PCR for S. aureus allows detection and differentiation of S. aureus and can be completed in 6 h. The sensitivity of the assay is far higher than the 106 number of S. aureus cells required for foodborne intoxication and the assay can be a useful tool for the food industry. This assay can be used by the dairy industry to monitor critical control points in a hazard analysis critical control point plan for detection of S. aureus either at milk receiving or to assess post processing product contamination.