In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.
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.
Bruno
J G
JG
Department of Radiology, University of Texas Health Science Center at San Antonio 78284-7800, USA. john.bruno@aloer.brooks.af.mil
Kiel
J L
JL
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
D004269:DNA, Bacterial