Comparison of different approaches to quantify Staphylococcus aureus cellsby real-time quantitative PCR and application of this technique for examination of cheese

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting th e thermonuclease (nac) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantif ication proved to be less sensitive (60 nuc gene copies/mul) than using a f luorigenic TaqMan probe (6 nuc gene copies/mul). Comparison of the LightCyc ler system and the well-established ABI Prism 7700 SDS with TaqMan probes r evealed no statistically significant differences with respect to sensitivit y and reproducibility. Application of the RTQ-PCR assay to quantify S, aure as cells in artificially contaminated cheeses of different types achieved s ensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, de pending on the cheese matrix. The coefficients of correlation between log C FU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calc ulation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.