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Multiplex PCR for the simultaneous detection of the SEA, SEB, SEC, SED and SEE genes of enterotoxigenic staphylococcus aureus

Abstract

The enterotoxins of Staphylococcus aureus (A, B, C, D and E) are known agents of food poisoning. Classical identification for these enterotoxigenic S. aurreus strains is laborious, time consuming and sometimes gives erronneous results. In this work, seven synthetic oligonucleotide primers were used in a multiplex PCR protocol to detect the genes encoding the Staphylococcal enterotoxins A to E simultaneously. The primers include two universal primers, which encode consensus sequences, and five primers, which encode unique sequences for toxin genes. None of the specific primer pairs (U2/A2, U1/B2, U1/C2, U2/D2 and U2/E2) cross-reacted with each other. Each primer was specific for the detection of its corresponding toxin gene, even though U2 and U1 are universal primers. The sizes of the amplified PCR products were 582 bp, 732 bp, 403 bp, 251 bp and 474 bp for enterotoxin genes, A, B,C,D and E, respectively. Unequivocal discrimination of the toxin genes was obtained by PCR using DNA extracted from strains of S. aureus whose toxigenicity had been previously established biologically and immunologically. When these primers were used to detect S. aureus in spiked foods containing 10 2 to 10 3 cell per ml of food homogenate, all five types of genes could be identified after six to eight hours of preincubation.

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