Digital assays with a generic reporter
Digital assay system, including methods, apparatus, and compositions, for assay of one or more targets in a set of partitions containing a generic reporter of target amplification.
1. A method of performing a digital assay, the method comprising:
forming partitions each including a portion of a same mixture, the mixture containing a target and also containing a generic reporter that is sensitive to amplification of the target, wherein only a subset of the partitions each contain at least one copy of the target;
amplifying the target and at least one byproduct including a primer dimer to generate target and byproduct amplicons, respectively, in the partitions;
collecting amplification data from the generic reporter for a plurality of the partitions, wherein partitions exhibiting detectable amplification of the target, partitions exhibiting detectable amplification of the byproduct, and partitions exhibiting detectable amplification of neither the target nor the byproduct are distinguishable from one another in the data based on distinct amounts of the generic reporter being bound to the target and byproduct amplicons; and
determining a level of the target using the amplification data.
2. The method of claim 1 , wherein detectable amplification of the byproduct occurs stochastically in the plurality of partitions.
3. The method of claim 1 , wherein detectable amplification of the target in a partition inhibits detectable amplification of the byproduct.
4. The method of claim 1 , further comprising a step of assigning partitions of the plurality as positive or negative for the target based on a threshold that selectively includes partitions exhibiting detectable amplification of the target and selectively excludes partitions exhibiting detectable amplification of the byproduct.
5. The method of claim 4 , wherein the step of assigning includes a step of comparing a signal detected from each partition of the plurality with the threshold, and wherein the signal represents photoluminescence detected from the generic reporter.
6. The method of claim 4 , wherein the threshold corresponds to a strength of the signal that is intermediate respective signal strengths for partitions exhibiting detectable amplification of the target and partitions exhibiting detectable amplification of the byproduct.
7. The method of claim 4 , wherein partitions exhibiting detectable amplification of neither the target nor the byproduct have a signal strength that is less than the signal strength of partitions exhibiting detectable amplification of the byproduct.
8. The method of claim 4 , further comprising a step of choosing the threshold based on respective signal strengths of partitions exhibiting detectable amplification of the target and partitions exhibiting detectable amplification of the byproduct.
9. The method of claim 4 , wherein partitions exhibiting detectable amplification of the target have a stronger signal than partitions exhibiting detectable amplification of the byproduct.
10. The method of claim 4 , wherein the step of determining a level of the target includes a step of calculating a concentration based on a number of the plurality of partitions that are assigned as positive for the target or based on a number of the plurality of partitions that are assigned as negative for the target.
11. The method of claim 1 , wherein the target and byproduct amplicons have different lengths.
12. The method of claim 1 or claim 11 , wherein the generic reporter binds to an amplicon in a quantity proportional to a length of the amplicon.
13. The method of claim 1 or claim 11 , wherein the generic reporter binds to the amplicons nonspecifically.
14. The method of claim 1 or claim 11 , wherein the generic reporter comprises a dye that binds to the amplicons nonspecifically.
15. The method of claim 1 , wherein the determining step comprises classifying droplets positive only for the primer dimer as negative for the target.