Detection method for a target nucleic acid
Method of detecting a target nucleic acid. In an exemplary method, at least two thermal zones of different temperature may be created using a heating assembly. A first emulsion and a second emulsion may be formed. The first and second emulsions may be thermally cycled by passing them through tubing in a spaced relation to one another, with the tubing being wound around a central axis of the heating assembly and extending through each thermal zone multiple times. Thermally cycling may promote amplification of the target nucleic acid in droplets of each emulsion. Droplets of each emulsion may be passed through a detection channel located downstream of the tubing. Fluorescence may be detected from the droplets being passed through the detection channel.
1. A method of detecting a target nucleic acid, the method comprising:
creating at least two thermal zones of different temperature using a heating assembly;
forming a first emulsion and a second emulsion;
thermally cycling the first and second emulsions by passing them through tubing in a spaced relation to one another, the tubing being wound around a central axis of the heating assembly and extending through each thermal zone multiple times, wherein thermally cycling promotes amplification of the target nucleic acid in droplets of each emulsion;
passing droplets of each emulsion through a detection channel located downstream of the tubing; and
detecting fluorescence from the droplets being passed through the detection channel.
2. The method of claim 1 , wherein forming a first emulsion and a second emulsion includes generating droplets of each emulsion using at least one droplet generator that is connected to an inlet of the tubing.
3. The method of claim 2 , wherein generating droplets of each emulsion includes passing a sample-containing fluid and a carrier fluid through a channel junction of the at least one droplet generator to divide the sample-containing fluid into droplets of the emulsion encapsulated by the carrier fluid.
4. The method of claim 1 , wherein each emulsion includes a carrier fluid that encapsulates the droplets of the emulsion, and wherein the carrier fluid includes a fluorocarbon oil.
5. The method of claim 1 , wherein each droplet of the first emulsion includes a sample-containing fluid, the method further comprising aspirating a non-emulsified form of the sample-containing fluid from a well with an intake device connected to the tubing.
6. The method of claim 5 , wherein the well is provided by a microplate.
7. The method of claim 1 , wherein the heating assembly includes a thermoelectric cooler configured to transfer heat to and/or from at least one of the thermal zones.
8. The method of claim 1 , wherein the tubing forms a plurality of coils, and wherein thermally cycling includes moving droplets of each emulsion along a continuous portion of the tubing that remains in one of the thermal zones for at least one coil of the plurality of coils.
9. The method of claim 1 , wherein the emulsions are separated from one another in the tubing during thermally cycling by a volume of fluid that is immiscible with the droplets of each emulsion.
10. The method of claim 1 , further comprising illuminating a region of the detection channel with excitation light to induce the fluorescence.
11. The method of claim 10 , wherein the region of the detection channel has a diameter that is less than an average spherical diameter of the droplets of each emulsion.
12. The method of claim 10 , wherein illuminating includes illuminating the region of the detection channel with light of different wavelengths from a plurality of light sources.
13. The method of claim 12 , wherein detecting fluorescence includes detecting fluorescence induced by each light source of the plurality of light sources.
14. The method of claim 13 , wherein the fluorescence induced by each light source of the plurality of light sources is detected with the same optical sensor.
15. The method of claim 12 , wherein the plurality of light sources includes a plurality of LEDs.
16. The method of claim 12 , wherein illuminating includes illuminating the region of the detection channel with pulses of light from each light source of the plurality of light sources.
17. The method of claim 12 , wherein illuminating includes illuminating the region of the detection channel using only one light source at a time.
18. The method of claim 10 , wherein each emulsion travels along a flow path from an inlet of the tubing to an outlet of the detection channel, the method further comprising adding dilution fluid to each emulsion at a position along the flow path intermediate an outlet of the tubing and the region of the detection channel.
19. The method of claim 18 , wherein each emulsion includes a carrier fluid that encapsulates the droplets of the emulsion, and wherein the dilution fluid is miscible with the carrier fluid.
20. The method of claim 1 , wherein the fluorescence detected includes a fluorescence intensity for individual droplets, the method further comprising:
comparing the fluorescence intensity for each of the individual droplets with a threshold; and
assigning each individual droplet as positive or negative for a copy of the nucleic acid target.