title: In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.
abstract: Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.
authors.affiliation: Department of Radiology, University of Texas Health Science Center at San Antonio 78284-7800, USA. john.bruno@aloer.brooks.af.mil
authors.forename: J G
authors.initials: JG
authors.lastname: Bruno
authors.forename: J L
authors.initials: JL
authors.lastname: Kiel
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015374:Biosensing Techniques; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D019020:Directed Molecular Evolution; D004563:Electrochemistry; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction; D013171:Spores, Bacterial / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial