Abstract: The PCR-based, dsDNA quantification method monitors the fluorescence of a target, whose melting characteristics is predetermined, during each amplification cycle at selected time-points. Fluorescence is measured immediately after the annealing phase (FE at TE), immediately below (FMS at TMS) and above (FME at TME) the melting of the target/amplicon. A change in slope from a baseline slope (SB=−(FMS−FE)/(TMS−TE)) to a melting phase slope (SM=−(FME−FMS)/(TME−TMS) indicates a specific amplification. The number of amplification cycles (CT) it takes for the quantity (SM−SB) to become greater than zero correlates with the starting concentration of the target (C). The concentration of the target in a sample is determined by comparing the value of CT for the sample with a standard curve. By selecting targets with distinguishable melting curve characteristics, multiple targets can be simultaneously detected.

Abstract: The PCR-based, dsDNA quantification method monitors the fluorescence of a target, whose melting characteristics is predetermined, during each amplification cycle at selected time-points. Fluorescence is measured immediately after the annealing phase (FE at TE), immediately below (FMS at TMS) and above (FME at TME) the melting of the target/amplicon. A change in slope from a baseline slope (SB=−(FMS−FE)/(TMS−TE)) to a melting phase slope (SM=−(FME−FMS)/(TME−TMS) indicates a specific amplification. The number of amplification cycles (CT) it takes for the quantity (SM−SB) to become greater than zero correlates with the starting concentration of the target (C). The concentration of the target in a sample is determined by comparing the value of CT for the sample with a standard curve. By selecting targets with distinguishable melting curve characteristics, multiple targets can be simultaneously detected.