Systems and methods for performing digital PCR

Patent metadata

Field	Value
Publication number	US-11085074-B2
Application number	US-201615280160-A
Country	US
Kind code	B2
Filing date	Sep 29, 2016
Priority date	Sep 29, 2015
Publication date	Aug 10, 2021
Grant date	Aug 10, 2021

Abstract

Systems and methods are described for quantifying a target nucleic acid. A sample comprising a target nucleic acid is segregated into a first plurality of the reaction volumes containing at least one target nucleic acid molecule and a second plurality of the reaction volumes containing no target nucleic acid molecules. The reaction volumes are subjected to an amplification assay, wherein the amplification assay is configured to amplify the target nucleic acid. An indicator of amplification is detected or measured in at least some of the plurality of reaction volumes. The target nucleic acid is quantified based on the detection or measurement. After discontinuing the amplification assay, the plurality of reaction volumes may be heated and changes in the indicators of amplification of two or more of the at least some of the reaction volumes may be detected or measured.

First claim

What is claimed is:

1. A method of analysis for detecting and/or quantifying a nucleic acid in a sample, the method comprising:
within a plurality of sample reaction volumes, segregating a sample comprising a target nucleic acid associated with a first indicator of amplification, wherein the plurality of sample reaction volumes includes a first plurality of the sample reaction volumes each containing at least one molecule of the target nucleic acid, and a second plurality of the sample reaction volumes each containing no molecules of the target nucleic acid;
subjecting the first and second pluralities of sample reaction volumes to an amplification assay, wherein the amplification assay is configured to amplify the target nucleic acid to produce an amplified product of the target nucleic acid;
during the subjecting, taking a plurality of measurements of the first indicator of amplification for each of the first plurality of sample reaction volumes;
after taking the plurality of measurements, taking a post amplification assay measurement of the first indicator for each of the first plurality of sample reaction volumes;
determining that some of the first plurality of sample reaction volumes contain an amount of the amplified product based on the post amplification assay;
determining that the post amplification assay measurement of at least one of the first plurality of sample reaction volumes is not consistent with a sample reaction volume containing the amplified product;
determining an angle of launch of the at least one of the first plurality of sample reaction volumes based on at least two of the plurality of measurements of the first indicator of amplification;
determining that the at least one of the first plurality of sample reaction volumes contains an amount of the amplified product based angle of launch.

2. The method of claim 1 , further comprising:
taking at least a first and second post-amplification measurement of the first indicator of amplification at a first and second temperature, respectively, for at least one of the first plurality of sample reaction volumes;
identifying one or more changes in the first indicator of amplification based on the post-amplification measurements of the first indicator or amplification;
associating the at least one change to a post-amplification temperature;
comparing the post-amplification temperature associated with the at least one change to an expected melt temperature for the target nucleic acid; and
identifying an erroneous amplification product based on the comparing.

3. The method of claim 2 , wherein taking a plurality of measurements of the first indicator of amplification further comprises:
associating the measurements of the first indicator of amplification for the first plurality of sample reaction volumes to a quantification cycle value (C g );
comparing the quantification cycle values associated with the plurality of measurements to an expected quantification cycle value for the amplification assay; and
identifying an erroneous amplification product based on the comparing.

4. A system for detecting or quantifying a nucleic acid in a sample, the system comprising:
a reaction device;
an electronic processor;
a memory comprising instructions for performing the method of claim 1 ;
an input/output device comprising an input device and a display;
wherein the electronic processor performs the instructions and displays an amount of one or more target nucleic acids on the display or wherein the electronic processor performs the instructions and displays information regarding a detection of one or more target nucleic acids on the display.

5. The method of claim 1 , further comprising quantifying the amplification, wherein quantifying the amplification comprises determining a quantification cycle value (C g ) or a cycle threshold value based on the detecting or measuring the first indicator of amplification, wherein the cycle threshold value is a value at which the measured indicator of amplification reaches a predetermined threshold value.

Assignees

• Life Technologies Corp

Inventors

• Keys David N
• Majumdar Nivedita Sumi
• Straub Theodore E

Classifications

• B01L2300/024

  Storing results with means integrated into the container · CPC title

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• B01L7/52

  with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples · CPC title

• B01L2300/0829

  Multi-well plates; Microtitration plates · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title