Monitoring temperature with fluorescence

Patent metadata

Field	Value
Publication number	US-9850549-B2
Application number	US-201314433854-A
Country	US
Kind code	B2
Filing date	Oct 8, 2013
Priority date	Oct 9, 2012
Publication date	Dec 26, 2017
Grant date	Dec 26, 2017

Abstract

Systems, methods, and kits are provided wherein a temperature-sensitive reagent that emits a luminescent signal is used to adjust the identification of the temperature of a sample or to control thermocycling. In various illustrative embodiments, the sample is a PCR mixture.

First claim

We claim:

1. A method of measuring a temperature of a sample, comprising:
providing a sample that includes:
a nucleic acid;
a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
in the presence of the nucleic acid, is not directly proportional to an amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of the amount of the nucleic acid present in the sample; and
changes as a function of temperature in a known manner; and

a second fluorescent dye that produces a second fluorescent signal,

wherein the second fluorescent signal is indicative of the amount of the nucleic acid present in the sample;
exciting the temperature-sensitive fluorescent dye to induce emission of the temperature-sensitive fluorescent signal;
measuring the temperature-sensitive fluorescent signal emitted from the temperature-sensitive fluorescent dye; and
determining a fluorescence-determined temperature of the sample based on the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye.

2. The method as recited in claim 1 , wherein the temperature-sensitive fluorescent dye comprises sulforhodamine B.

3. The method as recited in claim 1 , wherein exciting the temperature-sensitive fluorescent dye comprises exposing the temperature-sensitive fluorescent dye to electromagnetic radiation light having a wavelength sufficient to induce emission of the temperature-sensitive fluorescent signal.

4. The method as recited in claim 1 , wherein the sample comprises an aqueous solution.

5. The method as recited in claim 1 , wherein the sample comprises a suspension.

6. The method as recited in claim 1 , wherein the sample comprises a PCR mixture.

7. The method as recited in claim 6 , further comprising measuring fluorescence emitted from the sample at one or more time points during PCR cycling.

8. The method as recited in claim 6 , further comprising controlling thermal cycling of the PCR mixture in accordance with a thermocycling profile by using feedback control, wherein a predetermined value of the temperature-sensitive fluorescent signal indicates an appropriate time to initiate a change to a next phase in the thermocycling profile,
wherein the predetermined value is selected from the group consisting of emission intensity of the temperature-sensitive fluorescent signal, a temperature calculated from the emission intensity of the temperature-sensitive fluorescent signal, and a value or parameter calculated from the emission intensity of the temperature-sensitive fluorescent signal, and
wherein the next phase is selected from the group consisting of: a start of a programmed temperature hold period for the PCR mixture; an end of the programmed temperature hold period for the PCR mixture; ramping to a predetermined temperature.

9. The method as recited in claim 1 , wherein variance between the temperature of the sample as determined based on the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye and a temperature measured through direct contact of the sample that includes the temperature-sensitive fluorescent dye is less than or equal to 1 degree Celsius.

10. The method as recited in claim 1 , wherein an intensity of the temperature-sensitive fluorescent signal emitted from the temperature-sensitive fluorescent dye is substantially independent of the amount of the nucleic acid present in the sample.

11. The method as recited in claim 1 , wherein the temperature-sensitive fluorescent dye is not a dsDNA binding dye, the temperature-sensitive fluorescent dye is not tethered to a nucleic acid, and the temperature-sensitive fluorescent signal of the temperature-sensitive fluorescent dye is not affected by dsDNA denaturation.

12. The method as recited in claim 1 , wherein
the second fluorescent signal is indicative of an amount of double-stranded nucleic acid present,
the determining step takes place while melting the nucleic acid and measuring the signal indicative of the amount of double-stranded nucleic acid present, and
further including the step of generating a melting curve based on the second fluorescent signal, the melting curve being adjusted based on the fluorescence-determined temperature.

13. The method as recited in claim 1 , wherein the determining step includes generating a ratio of the temperature-sensitive fluorescent signal to a signal from the sample that is generally temperature insensitive.

14. The method of claim 13 , wherein the temperature-sensitive fluorescent signal is at a first wavelength, and the signal that is generally temperature insensitive is a signal from the temperature-sensitive fluorescent dye at a second wavelength.

15. The method of claim 13 , wherein the signal that is generally temperature insensitive is the second fluorescent signal.

16. The method as recited in claim 1 , wherein an intensity of the temperature-sensitive fluorescent signal emitted from the temperature-sensitive fluorescent dye is not directly proportional to the amount of the nucleic acid present in the sample.

17. The method as recited in claim 1 , wherein an intensity of the temperature-sensitive fluorescent signal emitted from the temperature-sensitive fluorescent dye is not affected by dsDNA denaturation.

18. The method as recited in claim 1 , wherein a wavelength at which the temperature-sensitive fluorescent signal is emitted from the temperature-sensitive fluorescent dye is not directly proportional to the amount of the nucleic acid present in the sample.

19. The method as recited in claim 1 , wherein a wavelength at which the temperature-sensitive fluorescent signal is emitted from the temperature-sensitive fluorescent dye is not affected by dsDNA denaturation.

20. The method as recited in claim 1 , wherein the sample includes-complementary strands of the nucleic acid and the step of determining a fluorescence-determined temperature of the sample takes place while melting the double-stranded nucleic acid and measuring the signal indicative of the amount of double-stranded nucleic acid present in the sample.

21. The method as recited in claim 20 , wherein the controlling thermal cycling step comprises:
changing the temperature of the PCR mixture from a first temperature to a second temperature;
exciting the temperature-sensitive fluorescent dye to induce emission of the temperature-sensitive fluorescent signal therefrom;
detecting the temperature-sensitive fluorescent signal emitted by the temperature-sensitive fluorescent dye; and
changing to the next phase in the thermocycling profile upon detecting the predetermined value of the temperature-sensitive fluorescent signal.

22. The method as recited in claim 1 , wherein the second fluorescent dye is a dsDNA binding dye.

23. A method of measuring a temperature of a sample, comprising:
providing a sample that includes:
a nucleic acid;
a temperature-sensitive fluorescent dye that, in response to a stimulus, emits a temperature-sensitive fluorescent signal that:
in the presence of the nucleic acid, is not directly proportional to an amount of the nucleic acid present in the sample, is not affected by dsDNA denaturation, or is substantially independent of the amount of the nucleic acid present in the sample; and
changes as a function of temperature in a known manner; and

a second fluorescent dye that

Assignees

• Univ Utah Res Found

Inventors

• Sanford Lindsay N
• Wittwer Carl T

Classifications

• G01K11/20 Primary

  using thermoluminescent materials (G01K11/32 takes precedence) · CPC title

• G01N21/6428

  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) · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q3/00 Primary

  Condition responsive control processes (apparatus therefor C12M1/36; controlling or regulating in general G05) · 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