This assay targets the tcdB gene, and employs real time PCR and molecular beacon technology.
After specimen preparation and lysis, the target (if present) is amplified through a real time PCR process. Amplified targets are detected with hybridization probes labeled with quenched fluorophores (molecular beacons). Amplification, detection, and interpretation of signals are performed automatically by the Cepheid SmartCycler® software.
The amplified targets are detected with a molecular beacon, a hairpin-forming, single-stranded oligonucleotide labeled at one end with a quencher and at the other end with a fluorescent reporter dye. In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridization, resulting in emission of fluorescence. The amount of fluorescence at any given cycle depends on the amount of amplicons present; the software continuously monitors the fluorescence emitted during amplification.