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

A method for detecting in real time the amount of a target nucleic acid molecule in a sample, wherein the melting of the target nucleic acid molecule starts at a temperature TMS and completes at a temperature TME, the method comprising:

• A. Establishing a standard curve by:
• i) PCR-amplifying, in the presence of a suitable fluorescent dye, the target nucleic acid molecule, with a known starting concentration (C) through cycles of denaturing, annealing, and chain extension, wherein the fluorescence is increased when the dye is combined with a double-stranded nucleic acid molecule, wherein the chain extension occurs at a chain extension temperature TE;
• ii) measuring the fluorescence (F) during each amplification cycle at the temperature immediately before the temperature starts to increase from TE (FE at TE), at any temperature point (TB) in between TE and TMS (FB at TB), at TMS (FMS at TMS) and at TME (FME at TME);
• iii) calculating a baseline slope (SB), defined by negative (FB minus FE), divided by (TB minus TE), and an amplicon melting phase slope (SM), defined by negative (FME minus FMS) divided by (TME minus TMS);
• iv) recording the number of PCR cycles (N) required for the quantity (SM minus SB) to first become greater than zero;
• v) repeating steps i) through v) for a suitable range of concentrations of interest;
• and
• vi) plotting C against CT to obtain a standard curve for the target nucleic acid sequence;
• and
• B. Repeating steps (A)(i) through (A)(v) for a sample containing an unknown concentration of the target nucleic acid molecule, to obtain an CT value for the sample, and determining the target nucleic acid molecule concentration via the standard curve.