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

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.

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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

BioMed Research International ◽

10.1155/2015/417641 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 6

Author(s):

Ka Lok Hong ◽

Kailey Yancey ◽

Luisa Battistella ◽

Ryan M. Williams ◽

Katherine M. Hickey ◽

...

Keyword(s):

Molecular Recognition ◽

Human Serum ◽

Health Care Systems ◽

Sensitive Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Exotoxin A ◽

Single Stranded Dna ◽

Recognition Element ◽

Care Systems ◽

Molecular Recognition Element

Exotoxin A is one of the virulence factors ofPseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosingP. aeruginosainfections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant(Kd)of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.

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In VitroSelection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

Journal of Nucleic Acids ◽

10.1155/2014/102968 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 12

Author(s):

Ryan M. Williams ◽

Amanda R. Kulick ◽

Srilakshmi Yedlapalli ◽

Louisa Battistella ◽

Cyrus J. Hajiran ◽

...

Keyword(s):

Molecular Recognition ◽

Selection Method ◽

Detection Methods ◽

Sensing Element ◽

Single Stranded Dna ◽

Recognition Element ◽

High Affinity ◽

Recognition Elements ◽

Molecular Recognition Element

Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd=9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device.

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