Real-time PCR is useful in pathogen detection, gene expression and regulation, and allelic discrimination. This PCR technique utilizes the 5' nuclease activity of "Taq" DNA polymerase to cleave a nonextendible, fluorescence-labeled hybridization probe during the extension phase of PCR. A second fluorescent dye, e.g., 6-carboxy-tetramethyl-rhodamine, quenches the fluorescence of the intact probe. The nuclease cleavage of the hybridization probe during the PCR releases the effect of quenching resulting in an increase of fluorescence proportional to the amount of PCR product, which can be monitored by a sequence detector.