System and method for determining copies-per-unit-volume using PCR and flow control of droplets
Methods and systems for quantification of a target nucleic acid in a sample are provided. The method includes forming a plurality of discrete sample portions. Each of the plurality of discrete sample portions comprising a portion of the sample, and a reaction mixture. The method further includes amplifying the plurality of discrete sample portions to form a plurality of discrete processed sample portions. At least one discrete processed sample portion containing nucleic acid amplification reaction products. Fluorescence signals are detected from the at least one of the plurality of discrete processed sample portions to determine a presence of the at least one target nucleic acid. The method also includes determining the respective volumes of the plurality of the plurality of discrete processed sample portions, and estimating the number of copies-per-unit-volume of the at least one target nucleic acid in the sample. Estimating the number of copies-per-unit-volume is based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.
1. A system, comprising:
a droplet apparatus, in a first configuration, capable of forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample, a reference dye, and a reaction mixture, wherein the plurality of discrete sample portions comprises discrete sample portions of a plurality of sizes;
an amplification apparatus configured to amplify the plurality of discrete sample portions to form at least one discrete processed sample portion containing nucleic acid amplification reaction product of the target nucleic acid;
a detection apparatus configured to:
detect a fluorescence signal from the at least one of the plurality of discrete processed sample portions in response to a presence of the at least one target nucleic acid and a light scattering property, and
detect a real-time fluorescence signal from the at least one of the plurality of discrete sample portions to determine a Cq value;
an optical imager configured to produce an image of the plurality of discrete processed sample portions; and
a processor configured to:
determine the respective volumes of the plurality of discrete processed sample portions, from the image, by measuring an optical property of each discrete processed sample portion to determine a dimension of each discrete processed sample portion, and
determine an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein, and the Cq value.
2. The system of claim 1 , wherein the processor is further configured to determine the respective volumes of the plurality of discrete processed sample portions by analyzing a time-of-flight of the processed sample portions.
3. The system of claim 1 , further comprising a processor configured to estimate a portion size of the discrete sample portions to provide a predetermined average number of copies a target nucleic acid within the discrete sample portions.
4. The system of claim 1 , wherein the droplet apparatus is configured to form the plurality of discrete sample portions of at least one of a plurality of sizes, substantially two different sizes, or substantially a plurality of predetermined sizes.
5. The system of claim 1 , wherein the droplet apparatus, in the first configuration, is further capable of forming each of the plurality of discrete sample portions so that it is at least partially surrounded by a medium that is at least substantially immiscible with the plurality of discrete sample portions.
6. The system of claim 5 , wherein the medium that is substantially immiscible with the plurality of sample portions comprises at least one of a mineral oil, a silicone oil, a paraffin oil, a fluorinated fluid, or a perfluorinated polyether.
7. The system of claim 1 , wherein the droplet apparatus, in the first configuration, is further capable of forming the plurality of discrete sample portions to comprise at least one selected from the group consisting of: a porous bead and a magnetic bead.
8. The system of claim 1 , further comprising a processor configured to estimate the number of copies-per-unit-volume of the target nucleic acid in the sample based on the number of discrete processed sample portions determined to contain the at least one target nucleic acid therein.
9. The system of claim 1 , wherein the processor is further configured to determine the dimension of each discrete processed sample portion using at least one of the following group: a passive reference dye, optical refraction properties, optical imaging for measurement, optical reflection properties, optical absorbance properties, optical transmission properties, and a ladder of standard droplet sizes using different reference dyes.
10. The system of claim 1 , wherein the processor is further configured to determine the amount of the target nucleic acid based on the determined volumes of the plurality of discrete processed sample portions.
11. The system of claim 1 , wherein the processor is further configured to determine a size of discrete processed sample portion which provides a target percentage of discrete processed sample portions that test positive to determine the amount of the target nucleic acid.
12. A method, comprising:
forming a plurality of discrete sample portions of a sample comprising a target nucleic acid, each of the plurality of discrete sample portions comprising a portion of the sample, wherein the plurality of discrete sample portions comprises discrete sample portions of a plurality of sizes;
amplifying the plurality of discrete sample portions to form at least one discrete processed sample portion containing a nucleic acid amplification reaction product of the target nucleic acid;
detecting a fluorescence signal from the at least one of the plurality of discrete processed sample portions;
detecting a real-time fluorescence signal from the at least one of the plurality of discrete sample portions to determine a Cq value;
using an optical imager, determining the respective volumes of the plurality of the plurality of discrete processed sample portions by imaging the plurality of discrete processed sample portions;
determining a dimension of each of the discrete sample portions to determine a volume by measuring an optical property of each discrete sample portion; and
determining an amount of the target nucleic acid based on the number of discrete processed sample portions containing the target nucleic acid therein, and a Cq value.
13. The method of claim 12 , wherein the method further comprises determining the dimension of each discrete processed sample portion using at least one of the following group: a passive reference dye, optical refraction properties, optical imaging for measurement, optical reflection properties, optical absorbance properties, optical transmission properties, and a ladder of standard droplet sizes using different reference dyes.
14. The method of claim 12 , wherein determining the amount of the target nucleic acid is further based on the determined volumes of the plurality of discrete processed sample portions.
15. The method of claim 12 , further comprising determining a size of discrete processed sample portion which provides a target percentage of discrete processed sample portions that test positive to determine the amount of the target nucleic acid.