Method of performing droplet-based assays

Patent metadata

Field	Value
Publication number	US-11612892-B2
Application number	US-202017110095-A
Country	US
Kind code	B2
Filing date	Dec 2, 2020
Priority date	Sep 23, 2008
Publication date	Mar 28, 2023
Grant date	Mar 28, 2023

Abstract

Method of analysis. In the method, a first emulsion and a second emulsion substantially separated from one another by a spacer fluid may be formed. The first emulsion, the spacer fluid, and the second emulsion may be flowed in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion. Amplification data may be collected from individual droplets of each emulsion downstream of the heating and cooling station. A level of the nucleic acid target present in each emulsion may be determined based on the amplification data collected from the individual droplets of the emulsion.

First claim

We claim:

1. A method of analysis, the method comprising:
forming a first emulsion and a second emulsion substantially separated from one another by a spacer fluid;
flowing the first emulsion, the spacer fluid, and the second emulsion in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, the channel forming a single-pass continuous fluid route from the fluid inlet to the fluid outlet, the fluid route traversing the temperature-controlled zones serially and repeatedly, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion;
collecting amplification data from individual droplets of each emulsion downstream of the heating and cooling station; and
determining a level of the nucleic acid target present in each emulsion based on the amplification data collected from the individual droplets of the emulsion.

2. The method of claim 1 , wherein the spacer fluid is miscible with a continuous phase of each emulsion.

3. The method of claim 1 , wherein the spacer fluid is gaseous.

4. The method of claim 1 , wherein the spacer fluid includes more than one spacer fluid that creates at least two spacer segments between the emulsions.

5. The method of claim 1 , wherein the spacer fluid is labeled.

6. The method of claim 5 , wherein the spacer fluid includes a dye configured to permit the spacer fluid to be distinguished from a carrier fluid of each emulsion.

7. The method of claim 1 , wherein flowing includes flowing the first emulsion and the second emulsion through at least three temperature-controlled zones of the heating and cooling station.

8. The method of claim 1 , wherein flowing drives a polymerase chain reaction in droplets of each emulsion.

9. The method of claim 1 , wherein the fluid route is helical.

10. The method of claim 1 , wherein a different nucleic acid target is amplified in each of the emulsions.

11. The method of claim 1 , wherein collecting amplification data includes detecting fluorescence from droplets of each emulsion.

12. The method of claim 1 , wherein determining a level of the nucleic acid target includes assigning individual droplets of each emulsion as amplification-positive or amplification-negative for the nucleic acid target based on the amplification data collected.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Ness Kevin D
• Masquelier Donald A
• Colston Jr Billy W
• Hindson Benjamin J

Classifications

• B29C45/006

  Joining parts moulded in separate cavities · CPC title

• B01L2400/0622

  distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves · CPC title

• B01L2200/0689

  Sealing · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• B01L2300/1822

  using Peltier elements · CPC title