CONTROLS AND CALIBRATORS FOR TESTS OF NUCLEIC ACID AMPLIFICATION PERFORMED IN DROPLETS

1 . A method of performing a droplet-based assay, comprising:

detecting a signal from each of a plurality of droplets;

comparing a width of the signal from each droplet to a permitted range;

excluding droplets for which the signal has a width that is not in the permitted range, to identify a set of included droplets; and

determining a concentration of a target provided by a sample disposed in the plurality of droplets using data collected from the included droplets and without any contribution of data collected from the excluded droplets.

2 . The method of claim 1 , wherein the step of excluding droplets includes a step of comparing a width of a peak formed by the signal from each droplet to a width maximum and a step of excluding each droplet for which the corresponding peak has a width that is greater than the width maximum.

3 . The method of claim 2 , wherein the step of excluding droplets includes a step of comparing a width of a peak formed by the signal from each droplet to a width minimum and a step of excluding each droplet for which the corresponding peak has a width that is less than the width minimum.

4 . The method of claim 1 , wherein the width corresponds to a time interval during which the signal is detected from a droplet.

5 . The method of claim 1 , wherein the step of determining a concentration is based on an intensity of the signal from included droplets.

6 . The method of claim 1 , further comprising a step of thermally cycling the plurality of droplets to promote amplification of the target.

7 . The method of claim 1 , wherein the step of detecting a signal includes a step of detecting a first signal and a second signal from each droplet of the plurality of droplets, and wherein the data used for determining a concentration is obtained from the first signal.

8 . The method of claim 1 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal.

9 . The method of claim 1 , wherein the step of detecting a signal includes a step of detecting a signal from each droplet traveling through a detection region.

10 . The method of claim 1 , wherein the signal has an intensity that varies according to whether or not the target is present in a droplet.

11 . A method of performing a droplet-based assay, comprising:

detecting a signal from at least two types of calibration droplets, the signal for each type of calibration droplet being of different intensity;

detecting sample data from sample droplets; and

determining if amplification of a target occurred in each of the sample droplets based on the sample data and the signal of each different intensity detected from the calibration droplets.

12 . The method of claim 11 , wherein the at least two types of calibration droplets include a first type and a second type configured to provide respective signal intensities corresponding at least generally to sample droplets that are negative or positive for amplification of the target.

13 . The method of claim 12 , wherein each sample droplet contains a PCR mixture for amplification of the target.

14 . The method of claim 11 , wherein the step of determining includes a step of determining a threshold using the signal detected from the calibration droplets and a step of comparing data for individual sample droplets to the threshold, to distinguish sample droplets that are negative from those that are positive for amplification of the target.

15 . The method of claim 11 , wherein the step of detecting a signal includes a step of detecting a signal of different intensity from at least three distinct types of calibration droplets.

16 . The method of claim 11 , wherein the step of detecting a signal and the step of detecting sample data are both performed at a same wavelength or wavelength range.

17 . The method of claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed with a same detector.

18 . The method of claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed with the calibration droplets and the sample droplets arranged in separate groups.

19 . The method of claim 18 , further comprising a step of detecting a signal from the at least two types of calibration droplets with the at least two types intermixed.

20 . The method of claim 11 , wherein each type of calibration droplet contains a different amount of a same dye.

21 . The method of claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed on droplets flowing through a same detection region.

22 . The method of claim 21 , further comprising a step of loading the calibration droplets and the sample droplets into a flow channel that intersects the detection region, wherein the calibration droplets are loaded before the sample droplets.

23 . The method of claim 11 , further comprising a step of thermally cycling the sample droplets.

24 . The method of claim 23 , further comprising a step of thermally cycling the calibration droplets, wherein each different intensity of the signal detected from the calibration droplets is not affected substantially by the step of thermally cycling.

25 . The method of claim 23 , wherein the calibration droplets are not thermally cycled.

26 . The method of claim 11 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal from each type of calibration droplet, and wherein the step of detecting sample data includes a step of detecting sample data as fluorescence intensity.

27 . A method of performing a droplet-based assay, comprising:

generating droplets from an aqueous phase including a first dye and a second dye, the second dye being an internal reference;

detecting sample data from the first dye included in the droplets, the sample data being related to a reaction performed in the droplets;

detecting reference data from the second dye included in the droplets;

transforming the sample data with the reference data to reduce variability in the sample data that is independent of the reaction; and

determining if the reaction occurred in each of the sample droplets based on sample data that has been transformed with the reference data.

28 . The method of claim 27 , further comprising a step of amplifying a nucleic acid target in the droplets, wherein the step of detecting sample data includes a step of detecting amplification data from the first dye.

29 . The method of claim 27 , wherein the second dye is not conjugated to a nucleic acid.

30 . The method of claim 27 , wherein the step of generating droplets includes a step of generating monodisperse droplets.

31 . The method of claim 27 , wherein the step of transforming the sample data includes a step of dividing a sample data value by a reference data value for each droplet.

32 . A method of performing a droplet-based assay, comprising:

detecting a signal from each of a plurality of droplets flowing through a detection region;

transforming an intensity of the signal for each of the plurality of droplets according to a duration of such signal to obtain transformed signals; and

determining whether amplification of a target occurred in individual droplets based on the transformed signals.

33 . The method of claim 32 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal.

34 . The method of claim 32 , wherein each signal forms a peak, and wherein the duration corresponds to a width of the peak.

35 . The method of claim 34 , wherein the step of transforming an intensity of each signal includes a step of transforming a value corresponding to a height or an area of the peak formed by such signal.

36 . The method of claim 32 , wherein the step of transforming includes a step of dividing the intensity of each signal by the duration of such signal.

37 . The method of claim 32 , further comprising:

comparing a duration of each signal to a permitted range; and

excluding each signal having a duration that is not in the permitted range.

38 . The method of claim 37 , wherein the step of comparing includes a step of comparing a duration of each signal to a duration maximum and a step of excluding each signal having a duration that is greater than the duration maximum.

39 . The method of claim 32 , further comprising a step of thermally cycling the plurality of droplets to promote amplification of the target.

40 . A method of performing a droplet-based assay, comprising:

generating droplets from an aqueous phase including a sample and first and second dyes;

detecting sample data from the first dye in the droplets, the sample data being related to amplification of a test target from the sample;

detecting control data from the second dye in the droplets, the control data being related to amplification of a control target in individual droplets;

analyzing the sample data and the control data to determine respective concentrations of the test target and the control target; and

correlating the concentration of the test target with the concentration of the control target.

41 . The method of claim 40 , wherein the test target and the control target are both endogenous to the sample.

42 . The method of claim 40 , wherein the test target is endogenous to the sample and the control target is not endogenous to the sample.

43 . The method of claim 40 , wherein the step of correlating includes a step of determining a validity of the test target concentration based on the control target concentration.

44 . A method of performing a droplet-based assay, comprising:

obtaining a first set of droplets configured to amplify a test target from a sample disposed in the first set of droplets, and a second set of droplets configured to amplify a control target in the second set;

detecting test amplification data from the first set of droplets and control amplification data from the second set of droplets; and

analyzing the test amplification data and the control amplification data to determine a concentration of the test target and the control target; and

correlating the concentration of the test target with the concentration of the control target.

45 . The method of claim 44 , wherein the step of detecting includes a step of detecting data from the first set of droplets as a group and from the second set of droplets as a separate group.

46 . The method of claim 44 , wherein the first set and the second set of droplets each includes a same sample that provides the test target and the control target.

47 . The method of claim 44 , wherein test target is provided by a sample that does not provide the control target.

48 . The method of claim 44 , wherein the step of correlating includes a step of determining a validity of the test target concentration based on the control target concentration.