Digital amplification assay analysis method

A method of quantifying a plurality of different target nucleic acids amplified in a digital assay in the presence of a plurality of different probes, each different probe being specific for one of the plurality of different target nucleic acids and distinguishable from other different probes by having a unique Tm, wherein signals from different probes are collected from a plurality of partitions and comprise the same signal-generating reporter, the method comprising the steps of:

• a) performing a melt analysis to detect signals from the plurality of different probes in each partition at three or more different predetermined temperatures;
• b) calculating at least two relational values for the signals detected at the three or more different predetermined temperatures for each partition;
• c) plotting the at least two relational values against each other to identify subsets of partitions containing the same combinations of different target nucleic acids;
• and
• d) quantifying the plurality of different target nucleic acids.

A method for multiplexed detection in a digital PCR (dPCR) assay, the method comprising:

• a) amplifying by a dPCR procedure one or more of a plurality of different target nucleic acids in a sample distributed across a plurality of partitions, wherein the dPCR procedure utilizes a plurality of different signal generating probes, each different signal generating probe being specific for one of the plurality of different target nucleic acids that may be present in the sample and being distinguishable in the presence of its specific target nucleic acid by having a unique melting temperature (Tm) relative to other different signal generating probes in the dPCR procedure;
• b) detecting signals from the different probes in the plurality of partitions at three or more different predetermined temperatures;
• c) calculating at least two relational values for signals measured at the three or more different predetermined temperatures for each of the plurality of partitions;
• and
• d) plotting the at least two relational values against each other to determine which, if any, of the plurality of target nucleic acids are present in each of the plurality of partitions.