Real time quantitative PCR with intercalating dye for single and multiplex target DNA

摘要

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 (F_E at T_E), immediately below (F_MS at T_MS) and above (F_ME at T_ME) the melting of the target/amplicon. A change in slope from a baseline slope (S_B(F_MSF_E)/(T_MST_E)) to a melting phase slope (S_M(F_MEF_MS)/(T_MET_MS) indicates a specific amplification. The number of amplification cycles (C_T) it takes for the quantity (S_MS_B) 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 C_T for the sample with a standard curve. By selecting targets with distinguishable melting curve characteristics, multiple targets can be simultaneously detected.