Final full-text version of the paper available at: http://dx.doi.org/10.1016/S0740-0020(02)00180-6.A two-step PCR procedure for detection of genes encoding Staphylococcus aureus thermonuclease (nuc) and enterotoxins (se_s) was developed. For this, the reaction conditions for multiplex-PCR assays were optimized and generic and specific se-primers designed. In a step detection of enterotoxigenic staphylococci using a set of primers targeting nuc, and group-generic se[a, d, e, j] and se[b, c] sequences, is accomplished. The generated amplicons are 468-, 670- and 540-bp long, respectively. In the second step aliquots of the 670- and/or 540-bp amplicons are analysed by nested-PCR using the following sets of primers: the forward se[adej]—together with the reverse sea, sed, see and sej, and/or the forward se[bc]—plus reverse seb and sec. The six se-genes can be distinguished since each of them yield an amplicon of different size. The procedure was validated by using 163 strains of S. aureus, 15 strains of other species, and 31 cake samples; and also by comparison of the obtained results with conventional-PCR-assays and immunoassays (SET-RPLA). The first PCR-step showed that all S. aureus strains, but none of the strains representing other species, were nuc-positive and 52 of them, and only them, were se-positive. Twelve out of the 31 cake samples were probed to be nuc-positive, but only eight were se-positive, and enterotoxigenic S. aureus were collected from them. The second step confirmed that all se-positive strains carried one or more se-genes. A positive correlation between the presence of se-genes by multiplex-PCR, conventional-PCR and SET-RPLA was found. This procedure allows it to be extended to include other se-genes.Peer reviewed