Multiplexing and quantification in PCR with reduced hardware and requirements

A method for performing a Polymerase Chain Reaction (PCR) comprising:

• performing a multiplexed PCR in a single channel using a thermal cycler, using a limited set of primers to control plateau levels of a plurality of different amplicons, thereby obtaining different plateau levels for different amplicons, and without performing an end-point melting curve analysis;
• detecting, using a detector, an amplicon of the plurality of different amplicons, the plurality of different amplicons generated during the multiplexed PCR;
• and
• associating, using a processor controlled analyzer, a cycle threshold (CT) value to an amplicon of the plurality of different amplicons generated during said multiplexed PCR, wherein the associating step comprises:
• (i) receiving a digital representation of a sum amplitude signal, wherein said sum amplitude signal comprises a sum amplitude curve corresponding to a total detected intensity generated by the plurality of different amplicons of said multiplexed PCR, wherein said total detected intensity comprises the signal intensity over a plurality of different wavelengths detected in the single detection channel;
• (ii) measuring an amount of asymmetry in a derivative of said sum amplitude curve;
• (iii) determining a relative position of any two CT values corresponding to any two amplicons from the plurality of different amplicons by using the measured asymmetry;
• and
• (iv) associating a CT value to an amplicon of said plurality of amplicons, wherein associating the CT value is performed using a characteristic of said sum amplitude signal without determining melting temperatures (Tm) for each of said plurality of different amplicons;
• and
• wherein the processor controlled analyzer is connected to the thermal cycler for temperature measuring and modulation.