DIGITAL ASSAYS WITH A GENERIC REPORTER

1 . A method of performing a multiplexed digital assay, the method comprising:

partitioning a mixture comprising a first target of a first length and a second target of a second length, a first pair of primers to amplify the first target, a second pair of primers to amplify the second target, and a nonspecific reporter that binds to an amplification product;

amplifying the first target and the second target in the partitions; and

detecting an intensity of light emitted by the nonspecific reporter from individual partitions.

2 . The method of claim 1 , wherein at least one primer of the first pair of primers is present at a lower concentration than each primer of the second pair of primers.

3 . The method of claim 1 , wherein at least one primer of the first pair of primers has a lower melting temperature than each primer of the second pair of primers.

4 . The method of claim 1 , wherein at least one primer of the first pair of primers is shorter than each primer of the second pair of primers.

5 . The method of claim 1 , further comprising:

selecting a concentration of at least one primer of the first pair of primers; and

preparing the mixture to contain the at least one primer at the selected concentration.

6 . The method of claim 5 , further comprising:

testing different concentrations of the at least one primer by detecting an intensity of light emitted by the nonspecific reporter after amplification of at least the first target using the different concentrations in different sets of partitions,

wherein the step of selecting a concentration is based on the step of testing.

7 . The method of claim 5 , wherein selecting the concentration comprises selecting a first concentration for at least one primer of the first pair of primers and a second concentration for at least one primer of the second pair of primers, and wherein the first concentration is different from the second concentration.

8 . The method of claim 1 , wherein the step of detecting is performed with the partitions at about a same temperature.

9 . The method of claim 1 , wherein the first length and the second length are different.

10 . The method of claim 1 , wherein the step of detecting is performed while the nonspecific reporter remains bound to the first target and the second target.

11 . The method of claim 1 , further comprising determining a level of each of the first and second targets in the mixture using a positive or negative count of partitions for the first or second target based on the intensity detected from the individual partition.

12 . The method of claim 11 , wherein determining the level comprises determining a concentration of each target.

13 . The method of claim 11 , wherein determining a level of each of the first and second targets comprises:

classifying each individual partition as positive or negative for each of the first and second targets based on the intensity detected from the individual partition;

obtaining a first number of the individual partitions that are classified as positive or negative for the first target and a second number of the individual partitions that are classified as positive or negative for the second target; and

determining a level of the first target using the first number and a level of the second target using the second number.

14 . The method of claim 1 , wherein the intensity detected is increased in individual partitions containing either one of the targets, at least predominantly by binding of the nonspecific reporter to nucleic acid amplicons.

15 . The method of claim 1 , wherein at least one of the first target and the second target is a spliced form of cDNA.