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 of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection 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 (K d = 47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio 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.

In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against the Pesticide Fipronil and Sensitive Detection in River Water

“…The equilibrium dissociation constant obtained for R12.51 is comparable to other small molecule binding ssDNA MREs identified with other variants of SELEX [ 33 , 34 , 35 ]. This high affinity further validates the stringency of this SELEX scheme [ 36 , 37 ].…”

“…This assay was modified from a previously described bead-based assay [ 24 , 25 , 26 ]. The data is presented relative to binding between R12.51 and fipronil as has been previously described [ 24 , 25 , 36 , 37 ]. The ssDNA MRE preferentially binds to fipronil, compared to atrazine ( p = 0.030), propanil ( p = 0.005), and malathion ( p = 0.039) in buffer conditions ( Table 2 ).…”

Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum

“…Initially, 30–50 randomly selected sequences were analyzed for the presence of consensus sequence families after rounds 3, 6, 9, and 12. Toward the end of the selection, in order to monitor the convergence of families more frequently, 30–50 random sequences from both rounds 13 and 14 were analyzed as previously described [ 34 , 35 , 38 ].…”

“…This difference is likely due to different methods of immobilizing the ssDNA MRE as noted above and thus leads to a lower level of immobilization and detection responses. The current study utilizes direct covalent conjugation of 5′ amino modified ssDNA to the SAM on gold surface [ 34 , 35 , 37 ]. However, covalently attached DNA provides a more stable immobilization as compared to streptavidin/biotin and thiolated DNA attachment under a wide range of storage conditions.…”

“…The M13R primer, complementary to a region upstream of the PCR insert, was sequenced (Eurofins MWG Operon, Huntsville, AL, USA) along with purified plasmid. A total of 30–50 randomly selected sequences for each respective round were subjected to analysis for consensus sequence families [ 34 , 35 ].…”