Quantitating high titer samples by digital PCR

• US 9,222,886 B2
• Filed: 12/27/2011
• Issued: 12/29/2015
• Est. Priority Date: 12/27/2010
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Clemens, John M., Shain, Eric B.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/337,784
• Publication Number:
  US 20120164652A1
• Time in Patent Office: 1,463 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

Systems, devices, methods, kits, and compositions for nucleic acid analysis using digital PCR are provided. In particular, methods to analyze high titer samples that cannot be divided into a sufficient number of partitions containing zero nucleic acid molecules per partition to allow for Poisson analysis (digital PCR analysis) are described.

Claims

1. A method of quantitating target nucleic acid molecules in a sample comprising:

a) separating said sample into a plurality of partitions, wherein said sample comprises;

a mixture of nucleic acid molecules;

amplification reagent;

detection reagents; and

internal standard nucleic acid molecules having identical primer binding sequences as said target nucleic acid molecules, wherein said internal standard nucleic acid molecules are added to said mixture at a concentration to produce a number of partitions containing zero copies of said internal standard molecules per partition;

wherein said mixture of nucleic acid molecules is not diluted prior to addition of said amplification reagents, said detection reagents, and said internal standard nucleic acid molecules;

wherein a portion of said plurality of partitions contains zero copies of said internal standard nucleic acid molecules;

wherein a portion of said plurality of partitions contains zero copies of said target nucleic acid molecules;

wherein said portion of said plurality of partitions that contains zero copies of said target nucleic acid molecules is insufficient in number to allow application of Poisson statistics; and

wherein said portion of said plurality of said partitions that contains zero copies of said internal standard nucleic acid molecules is sufficient in number to allow application of Poisson statistics;

b) treating said plurality of partitions under amplification conditions such that said target nucleic acid molecules are amplified to produce detectable target amplicons in one or more of said partitions, and said internal standard nucleic acid molecules are amplified to produce detectable internal standard amplicons in one or more of said partitions, wherein said detectable target amplicons and said detectable internal standard amplicons are differentially detectable;

c) determining a change in amplification of said target nucleic acid molecules in said plurality of partitions in response to primer competition from said internal standard nucleic acid molecules; and

d) calculating an initial number of target nucleic acid molecules that are present in said sample before said sample has been separated into said plurality of partitions.

2. The method of claim 1, wherein said plurality of partitions comprises, on average, 2-100 nucleic acid molecules per partition.

3. The method of claim 1, wherein said detection reagents comprise a first labeled probe configured to bind to said target sequence, and a second labeled probe configured to bind to said internal standard sequence, wherein said first labeled probe and said second labeled probe are differentially detectable.

4. The method of claim 3, wherein said first labeled probe and said second labeled probe comprise different fluorescent labels.

5. The method of claim 1, wherein said sample is selected from an environmental sample, a biological sample, a clinical sample, and a forensic sample.

6. A method of extending the dynamic range of a non-symmetric nucleic acid amplification process comprising:

a) partitioning a sample into a plurality of partitions, wherein said plurality of partitions comprise, on average, 100 or more nucleic acid molecules per partition, wherein a portion of said plurality of partitions contains zero target nucleic acid molecules; and

wherein said portion of said plurality of said partitions that contains zero copies of said target nucleic acid molecules is insufficient in number to allow application of Poisson statistics; and

b) amplifying target nucleic acid molecules comprising a nucleic acid target sequence by said non-symmetric nucleic acid amplification process to produce target amplicons wherein said target nucleic acid molecules are not diluted prior to addition of amplification reagents for non-symmetric nucleic acid amplification.

7. The method of claim 6, wherein said non-symmetric amplification process is a linear-after-the-exponential PCR (LATE-PCR) amplification process.

8. The method of claim 6, further comprising:

c) detecting said target amplicons in said plurality of partitions using detection reagents.

9. The method of claim 8, wherein said detection reagents comprise fluorescent labels.

10. The method of claim 9, wherein said detection reagents comprise fluorescently labeled probes.

11. The method of claim 8, wherein said detecting is an end-point detection following completion of said non-symmetric amplification process.

12. A method of quantitating target nucleic acid molecules in a sample comprising:

a) separating said sample into a plurality of partitions wherein said target nucleic acid molecules differ in number per a plurality of partitions;

wherein said plurality of partitions comprise, on average, 100 or more nucleic acid molecules per partition;

wherein said sample comprises;

a mixture of nucleic acid molecules;

internal standard nucleic acid molecules having identical primer binding sequences as said target nucleic acid molecules;

amplification reagents for non-symmetric nucleic acid amplification; and

detection reagents;

wherein said mixture of nucleic acid molecules is not diluted prior to addition of said amplification reagents for non-symmetric nucleic acid amplification, said detection reagents, and said internal standard nucleic acid molecules;

wherein a portion of said plurality of partitions contains zero copies of said target nucleic acid molecules; and

wherein said portion of said plurality of partitions that contains zero copies of said target nucleic acid molecules is insufficient in number to allow application of Poisson statistics;

b) amplifying said target nucleic acid molecules by said non-symmetric nucleic acid amplification process to produce target amplicons;

c) detecting said target amplicons in said plurality of partitions using said detection reagents; and

d) correlating an intensity produced by said detection reagents following said non-symmetric nucleic acid amplification to an initial concentration of said target nucleic acid molecules in said sample.

13. The method of claim 12, wherein said amplification reagents comprise one excess primer and one limiting primer.

14. The method of claim 12, wherein said non-symmetric amplification process is a linear-after-the-exponential PCR (LATE-PCR) amplification process.

15. The method of claim 12, wherein said detection reagents comprise fluorescent labels.

16. The method of claim 15, wherein said detection reagents comprise fluorescently labeled probes.

17. The method of claim 12, wherein said detecting is an end-point detection following completion of said non-symmetric amplification process.

18. The method of claim 12, wherein said sample is selected from an environmental sample, a biological sample, a clinical sample, and a forensic sample.