In VitroSelection of a Single-Stranded DNA Molecular Recognition Element againstClostridium difficileToxin B and Sensitive Detection in Human Fecal Matter

Abstract

Toxin B is one of the major virulence factors ofClostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence ofC. difficileinduced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity(Kd=47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin,Staphylococcus aureusalpha toxin,Pseudomonas aeruginosaexotoxin A, and cholera toxin ofVibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations.