A highly selective enrichment broth combined with real-time PCR for detection of Staphylococcus aureus in food samples

Abstract

A new selective enrichment broth (OSA) was developed for detection of Staphylococcus aureus combined with real-time PCR. Acriflavine, nalidixic acid, potassium tellurite and sodium chloride were added as selective reagents and mannitol, sodium pyruvate, and yeast extract as promoters. The growth of S. aureus (10, 100, 1000 CFU/mL inoculation level) in OSA was superior to that in TSB with 7.5% NaCl (TNaCl). OSA showed a good inhibition ability against 8 strains including 4 Gram-positive and 4 Gram-negative. Further, OSA gave a good recovery of cold-, acid-, and heat-stressed cells comparable to TSBYE but superior to TNaCl. A real-time PCR targeting the nuc gene was developed and the standard curve was established from 9.1 × 107 to 9.1 × 102 cfu/mL with a high regression coefficient of determination (R2 = 0.999) and a high amplification efficiency of 96.362%. Using artificially contaminated food samples (kimbap, milk, and spam) with a level of ∼10 CFU/25 g or mL, OSA better supported the growth of S. aureus and all samples were readily detected after 12 h enrichment by real-time PCR. In summary, OSA was demonstrated as a promising selective enrichment broth for detection of S. aureus combined with real-time PCR with a total assay time of 15 h.