Development of Aptamer Beacons for Rapid Presumptive Detection of <Emphasis Type="Italic">Bacillus</Emphasis> Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5′ ends and Iowa Black quencher on their 3′ ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.