A real-time immunocapture PCR (RT-IPCR) without interference of protein A for convenient detection of staphylococcal enterotoxin B from food and environmental samples

Abstract

Purpose: A real-time immunocapture PCR (RT-IPCR) has been fabricated for the detection of Staphylococcus aureus enterotoxin B (SEB) from food and environmental samples. Methods: Considering the fact, anti-SEB immunoglobulin G (IgG) has affinity towards protein A, produced by nearly all S. aureus, and generates false-positive read out in all immuno-based assay. We have employed avian anti-SEB antibody (SEB-IgY) as capture probe, since IgY interact less efficiently to protein A and biotinylated SEB-specific monoclonal antibody (SEB -MAb) conjugated with reporter DNA as revealing probe for real-time PCR amplification and signal generation. Sensitivity and selectivity of the assay were evaluated employing closely related enterotoxins and other toxins. Results: The RT-IPCR is highly specific and sensitive (100 fg/mL). The practical applicability of the assay was tested using spiked food sample as well as naturally contaminated food samples. The sensitivity and specificity of RT-IPCR were not compromised by the foods tested and was able to detect SEB conveniently. Further, the assay was validated comparing with the in-house developed PCR, and plausible result was obtained. Conclusion: The developed assay can be utilized as a low-cost detection system of SEB in routine food testing laboratories.