Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring <i>Staphylococcus aureus</i> from Food Samples

In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring <i>Staphylococcus aureus</i> from food and clinical samples. The method employs immunocapture of <i>S. aureus</i> and amplification of <i>seb</i> gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3′,5,5′-tetramethylbenzidine) in the presence of H₂O₂ produce colorimetric signal. A linear relationship of optical signal with the initial template of <i>seb</i> was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 10² CFU/mL of <i>seb</i> gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of <i>seb</i> from food and clinical sources

Selection and Characterization of Aptamers Using a Modified Whole Cell Bacterium SELEX for the Detection of <i>Salmonella enterica</i> Serovar Typhimurium

This study describes the selection of single-stranded DNA (ssDNA) aptamers against <i>Salmonella enterica</i> serovar Typhimurium using a modified whole cell systematic evolution of ligands by exponential enrichment (whole cell SELEX). For evolving specific aptamers, ten rounds of selection to live <i>Salmonella</i> cells, alternating with negative selection against a cocktail of related pathogens, were performed. The resulting highly enriched oligonucleotide pools were sequenced and clustered into eight groups based on primary sequence homology and predicted secondary structure similarity. Fifteen sequences from different groups were selected for further characterization. The binding affinity and specificity of aptamers were determined by fluorescence binding assays. Aptamers (SAL 28, SAL 11, and SAL 26) with dissociation constants of 195 ± 46, 184 ± 43, and 123 ± 23 nM were used to develop a nanogold-based colorimetric detection method and a sedimentation assay. The former showed a better sensitivity limit of 10² CFU/mL using aptamer SAL 26. This approach should enable further refinement of diagnostic methods for the detection of <i>Salmonella enterica</i> serovar Typhimurium and of other microbial pathogens