Calibrations and controls for droplet-based assays

Patent metadata

Field	Value
Publication number	US-9417190-B2
Application number	US-201313945661-A
Country	US
Kind code	B2
Filing date	Jul 18, 2013
Priority date	Sep 23, 2008
Publication date	Aug 16, 2016
Grant date	Aug 16, 2016

Abstract

System, including methods and apparatus, for performing droplet-based assays that are controlled and/or calibrated using signals detected from droplets.

First claim

We claim:

1. A method of performing a droplet-based assay, comprising:
forming droplets from a same mixture, wherein only a subset of the droplets contain a target;
irradiating a region of a channel;
detecting a forward scattering signal and a fluorescence signal from a plurality of the droplets as each droplet passes through the region of the channel, wherein the fluorescence signal is detected from a label present in each droplet and varies according to whether the target is present in the droplet;
identifying accepted droplets of the plurality for which the forward scattering signal meets a condition and rejected droplets of the plurality for which the forward scattering signal does not meet the condition, wherein the condition corresponds to a permitted size of a droplet; and
determining a concentration of the target based on the fluorescence signal from the accepted droplets, and without any contribution of the fluorescence signal from the rejected droplets.

2. The method of claim 1 , wherein the forward scattering signal has an intensity that is at least substantially independent of whether or not the target is present in the droplet.

3. The method of claim 1 , wherein the forward scattering signal detected from each droplet forms a peak, and wherein the condition corresponds to a permitted width of the peak.

4. The method of claim 1 , wherein the step of determining includes a step of determining a fraction of the accepted droplets that are positive or a fraction that are negative for the target.

5. The method of claim 1 , wherein the forward scattering signal is produced by refraction of incident radiation.

Assignees

• Bio Rad Laboratories Inc

Inventors

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

Classifications

• G01N2201/1242

  Validating, e.g. range invalidation, suspending operation · CPC title

• G01N21/6456

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G01N21/6486 Primary

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• G01N2021/6441

  with two or more labels · CPC title