An Immunomagnetic PCR Signal Amplification Assay for Sensitive Detection of Staphylococcus aureus Enterotoxins in Foods

摘要

Enterotoxigenic strains of Staphylococcus aureus produce a variety of heat-stable staphylococcal enterotoxins (SEs) that are a prevalent cause of food poisoning in the United States and other countries. Many immunological and biochemical assays often work well in buffer systems but are hindered when tested in the complex chemical environment of foods. To overcome these biases and improve the limits of detection, we implemented an immunomagnetic PCR signal amplification assay (iPCR-SA) for recovery and detection of SEA and SEB in foods. Anti-SEA or anti-SEB primary antibodies were coated onto COOH-modified magnetic beads using l-ethyl-3-(3-dimethyl aminopropyl) carbodiimide reagent. Secondary antibodies were covalently linked to amino-modified reporter DNA oligonucleotides (563 bp) via the linker molecule succinimidyl-4[N-maleimidomethyl]-cyclohexane-1-carboxylate. An internal 159-bp portion of the reporter DNA retained by the captured toxin molecule was then amplified by real-time PCR. A semiautomated Bead Retriever proved extremely helpful in both the application of the conjugation chemistries and required washes and the recovery and washing of bead-conjugated toxin from tested food samples. The procedure was simple, and analyses were completed in 5 to 6 h. The assay was sufficiently robust that we were able to detect SEA and SEB in tryptic soy broth, milk, lemon cream pie, tuna salad, deli turkey, and ground turkey at levels as low as 7.5 fg/ml. SE was still detected at high sensitivity after heating in food samples for typical pasteurization or cooking regimens. Sensitivity was diminished only when samples were subjected to extreme heating.