Real-time Fluorogenic PCR Assays for the Detection of entA, the Gene Encoding Staphylococcal Enterotoxin A

Abstract

Staphylococcal enterotoxin A (SEA) is among the most potent of the growing list of known enterotoxins produced by Staphylococcus aureus. SEA, a 27 kDa monomeric protein, is encoded by the entA gene. We have developed two real-time fluorogenic PCR assays for the detection of nucleic acid sequences in entA. The assays are useful in detecting and identifying strains of S. aureus that produce SEA and can serve a confirmatory role in determining the presence of SEA in food samples. The assays were tested in two real-time PCR formats, using either dye-labeled DNA probes corresponding to each primer set that are degraded by the 5' exonuclease activity of Taq polymerase, or a PCR master mix that contains the DNA-binding dye SYBR Green. In both formats the assays have a limit of detection of between 1 and 13 copies of a S. aureus genome that contains a copy of entA. Neither assay cross-reacted with genomic DNA isolated from other strains of S. aureus or other species.

Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A

“…These methods include polymerase chain reaction (PCR), mass spectrometry (MS), biosensor-based techniques, reversed passive latex agglutination (RPLA), immunoblotting, and ELISA [8,16,17,18,19,20]. …”

“…As little as only one to 13 copies of the S. aureus genome that contain a copy of the entA gene encoding the SEA protein can be detected by real-time PCR [17], but PCR cannot directly detect SEA and assess its levels in food. Nevertheless, it remains the preferred technique for confirming the presence of SEA-producing S. aureus strains in food samples.…”

The Inhibitory Effects of 2 Commercial Probiotic Strains on the Growth of Staphylococcus aureus and Gene Expression of Enterotoxin A

“…In the present study, the effect of Lactobacillus strains on the level of sea gene expression was investigated by RT-PCR given the latter's increased sensitivity and specificity compared to the conventional method. 8,26 RT-PCR has been used previously to study S. aureus SE gene expression. 27 Our data show that S. aureus co-culture with L. casei 01 resulted in the greatest level of down-regulation of sea gene expression at 25°C compared with L. acidophilus (LA5).…”

“…8 The inhibition of S. aureus growth and production of SE in foodstuffs is of importance in the public health, therefore, to detect the prevalence of enterotoxic strains in foods is required. From a food safety and human health point of view, lactic acid bacteria (LAB) may provide a promising strategy to combat against S. aureus.…”

An Update on Clinical Burden, Diagnostic Tools, and Therapeutic Options of Staphylococcus aureus

“…Molecular methods play an important role in detection and differentiation of pathogens. Numerous techniques have been reported for detection of S aureus virulence factors such as antibody,2–5 polymerase chain reaction (PCR),6,7 real-time PCRs (RT-PCRs),8 and aptamer-based9–11 methods. Many other sensitive methods such as immuno-PCRs,12 mass spectrometric analysis,13 and biosensor techniques14,15 are also reported.…”