Detection of enterotoxin A and B genes with PCR-EIA and a hand-held electrochemical sensor

Two electrochemical assays for detecting Staphylococcus aureus enterotoxin A and B genes were developed. The assays are based on PCR amplification with biotinylated primers, hybridization to a fluorescein-labeled probe, and detection with horseradish peroxidase-conjugated anti-fluorescein antibody using a hand-held electrochemical detector. The limit of detection (LOD) for both assays was approximately 16 copies of the sea and seb genes. The assays were evaluated in blinded studies, each with 81 samples that included genomic and cloned S. aureus DNA, and genomic DNA from Alcaligens, Bacillus, Bacteroides, Bordetella, Borkholderia, Clostridium, Comanonas, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Streptococcus, Vibrio and Yersinia species. Both assays showed 100% sensitivity. The specificity was 96% for the SEA assay and 98% for the SEB assay. These results demonstrate the feasibility of performing probe-based detection of PCR products with a low-cost, hand-held, electrochemical detection device as a viable alternative to colorimetric enzyme-linked assays of PCR products.