Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the Lightcycler, And the Smart Cycler Platforms (Technical Briefs‪)‬

Because disease-causing microorganisms can be used as aerobiological weapons, accurate and timely identification of these agents is necessary (1-4). Important agents include Bacillus anthracis (anthrax), Brucella species (brucellosis), Clostridium botulinum (botulism), Coxiella burnetii (Q fever), Francisella tularensis (tularemia), Staphylococcus aureus, and Yersinia pestis (plague) (5). Real-time PCR can rapidly detect the presence of nucleic acid markers with small reaction volumes and rapid cycling. Assay chemistry is increasingly important in this identification process because the choices and concentrations of enzymes, buffers, salts, primers, and probes affect assay detection limits (6-12). Real-time PCR assays for detecting biological warfare agents have been developed on the R.A.P.I.D.[R] (Idaho Technology, Inc.), LightCycler[R] (Roche), and Smart Cycler[R] (Cepheid) platforms and are compatible with various fluorescence-based methods such as TagMan[R] (13, 14), hybridization probes (15, 16), molecular beacons (17-19), Scorpion primers (20), LUXE primers (21-24), AEGIS primers (25-27), and SimpleProbes[R] (IT Biochem). We investigated whether these assays produce comparable sensitivity and specificity on these rapid cycling instruments.