DIGITAL ASSAYS WITH MULTIPLEXED DETECTION OF TWO OR MORE TARGETS IN THE SAME OPTICAL CHANNEL

• US 20130040841A1
• Filed: 07/12/2012
• Published: 02/14/2013
• Est. Priority Date: 07/12/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Saxonov, Serge, Dube, Simant, Regan, John Frederick
• Granted Patent: US 8,951,939 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Abstract

System, including methods and apparatus, for performing a digital assay with multiplexed detection of two or more distinct targets in the same optical channel.

Claims

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

amplifying two or more targets in droplets containing a reporter for each target;

detecting a signal from the droplets representing combined emission of light from the reporters for the targets, wherein each reporter provides a portion of the signal having an intensity that varies according to whether or not the corresponding target is present in a droplet, and wherein a plurality of the droplets contain more than one target and have a distinguishable intensity of the signal relative to droplets containing only one target;

determining for each target a number of droplets that are positive for only one of the targets;

determining at least one number of droplets that are positive for more than one target; and

determining a quantity representative of a level of at least one target.

2. The method of claim 1, wherein the quantity representative of a level is a concentration of the at least one target.

3. The method of claim 2, wherein the step of determining a quantity representative of a level further comprises a step of ascertaining a total number of droplets based on the signal, and obtaining a concentration of the at least one target based on the total number of droplets.

4. The method of claim 1, wherein the quantity representative of a level is a copy number of the at least one target in a sample disposed in the droplets.

5. The method of claim 1, wherein the step of determining a quantity representative of a level further comprises a step of obtaining a copy number of a first target by comparing the number of droplets positive for the first target with the number of droplets positive for a second target, the first and second targets being among the two or more targets.

6. The method of claim 5, wherein the copy number of the second target is known, and wherein the copy number of the first target is determined by calculating the ratio of the number of droplets positive for the first target to the number of droplets positive for the second target and multiplying the ratio by the copy number of the second target.

7. The method of claim 5, wherein the copy number of the second target is unknown, and wherein the copy number of the first target is obtained relative to the copy number of the second target as the ratio of the number of droplets positive for the first target to the number of droplets positive for the second target.

8. The method of claim 1, wherein the reporters each are labeled with the same luminophore.

9. The method of claim 8, wherein the luminophore is selected from the group consisting of FAM, VIC, ROX, TAMRA, and JOE.

10. The method of claim 1, wherein the two or more targets include a first target and a second target, wherein the step of determining for each target includes a step of counting a number of droplets positive for the first target and not the second target and a number of the droplets positive for the second target and not the first target, and wherein the step of determining at least one number of droplets includes a step of counting a number of droplets positive for both the first target and the second target.

11. The method of claim 1, the signal being a first signal, further comprising:

detecting from the droplets a second signal representing emission of light from an additional reporter for an additional target, the additional reporter providing at least a portion of a second signal having an intensity that varies according to whether or not the corresponding additional target is present in a droplet, wherein the first signal and second signal correspond to emission of light in different wavelength regimes;

counting a number of the droplets that are positive for the additional target based on the second signal; and

determining a quantity representative of a level of the additional target in the droplets.

12. The method of claim 11, wherein the first signal is collected from a first luminophore and the second signal is collected from a second luminophore.

13. The method of claim 12, wherein the first luminophore is one of FAM, VIC, ROX, TAMRA, and JOE, and wherein the second luminophore is another of FAM, VIC, ROX, TAMRA, and JOE.

14. The method of claim 1, wherein the droplets include a first probe reporting a presence or absence of a first target in individual droplets, a second probe reporting a presence or absence of a second target in individual droplets, and a third probe reporting a presence or absence of a third target in individual droplets, and wherein a signal from the first and second probes represents light emitted in a first wavelength regime and a signal from the third probe represents light emitted in a second wavelength regime distinct from the first wavelength regime.

15. The method of claim 1, wherein each reporter is a probe including an oligonucleotide, and wherein each target is a nucleic acid.

16. A method of performing a digital assay, comprising:

amplifying two or more targets in droplets containing a reporter for each target, wherein amplifying is performed with a plurality of different annealing temperatures on respective sets of the droplets;

detecting a signal from each set of droplets representing combined emission of light from the reporters for the targets, wherein each reporter provides a portion of the signal having an intensity that varies according to whether or not the corresponding target is present in a droplet;

comparing the signals from the sets of droplets;

selecting an annealing temperature for which amplification of each target is distinguishable based on the signal detected from the corresponding set of droplets;

determining for each target a number of droplets that are positive for amplification at the selected annealing temperature; and

determining a quantity representative of a level of at least one target.

17. The method of claim 16, wherein the step of detecting is performed on a fraction of each set of droplets, further comprising a step of selectively repeating the step of detecting a signal for the set of droplets corresponding to the selected annealing temperature after the step of selecting an annealing temperature.

18. The method of claim 16, wherein a plurality of the droplets contain more than one target and have a distinguishable intensity of the signal relative to droplets containing each target alone after amplification at the selected annealing temperature.

19. A method of performing a digital assay, comprising:

amplifying at least three targets in droplets containing a respective reporter for each target, wherein each reporter includes a different luminophore;

detecting at least two signals from the droplets, each signal representing combined emission of light from at least two of the reporters detected in a different optical channel;

determining for each target a number of droplets that are positive based on the at least two signals; and

determining a quantity representative of a level of at least one target.

20. The method of claim 19, further comprising a step of determining at least one number of droplets that are positive for more than one of the targets.