Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identi␣cation and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% speci␣city against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc␣ S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc+ S. aureus log-phase broth culture showed a good linearity of response (R2 0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV à 3.02). The overall limit of quanti␣cation (LOQ) for both rt-PCR assays (about 100% PCR ef␣ciency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were con␣rmed in raw milk arti␣cially contaminated with log-phase broth cultures of either a single egc+ S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc+ mixture in the presence of 106 cfu/mL of egc␣ S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ=100cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ = 10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic speci␣city and 96–107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high speci␣city, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc+ S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.