What are the generally used detection methods in real time PCR (qPCR)?

Two detection strategies are generally applied to measure the amplification concentration in real-time PCR (qPCR), dye-based and probe-based chemistries.

In dye-based qPCR assays, DNA-intercalating fluorophores, such as Helixyte™ Green bind specifically to the minor groove of dsDNA to measure DNA amplification as it occurs throughout the PCR process. Alone such dyes display weak background fluorescence, but fluorescence intensity is enhanced significantly upon binding to dsDNA. As amplicon concentration increases with each successive cycle of amplification, so does the fluorescence intensity of the dye, to a degree proportional to the amount of dsDNA present in each PCR cycle.

In probe-based qPCR, sequence-specific fluorescent probes are used in combination with primers to detect the amplification product. Of the many probe-based qPCR chemistries available, including hybridization probes and molecular beacons, the most widely used employs the 5' nuclease assay associated with Taq DNA polymerase.