Method and device for digital PCR measurement

Patent metadata

Field	Value
Publication number	US-12018319-B2
Application number	US-201916973990-A
Country	US
Kind code	B2
Filing date	May 20, 2019
Priority date	Jun 14, 2018
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The present invention addresses the problem of providing a novel digital PCR analysis method. One embodiment of the novel digital PCR analysis method is a DNA detection method including the steps of: partitioning a DNA solution containing a fluorescent-labeled probe or a DNA intercalator and a target DNA to be detected into a plurality of compartments; carrying out a nucleic acid amplification reaction in the compartments; measuring a fluorescence intensity in association with a temperature change; calculating a melting temperature of a DNA double strand based on a change in the fluorescence intensity in association with the temperature change; and calculating a ratio of a fluorescence intensity at a second temperature that is lower than a first temperature in association with the temperature change to that at the first temperature.

First claim

The invention claimed is:

1. A DNA detection method comprising the steps of:
partitioning a DNA solution comprising a fluorescent-labeled probe or a DNA intercalator and a target DNA to be detected into a plurality of compartments;
carrying out a nucleic acid amplification reaction in the compartments;
for each of the compartments, measuring a fluorescence intensity in association with a temperature change;
for each of the compartments, calculating, in association with the temperature change, a melting temperature of a DNA double strand based on a change in the fluorescence intensity;
calculating, in association with the temperature change, for each of the compartments a ratio of a fluorescence intensity at a second temperature to a fluorescence intensity at a first temperature, the second temperature being lower than the first temperature.

2. The DNA detection method according to claim 1 , further comprising the step of specifying a compartment for which the calculated ratio of the fluorescence intensity is equal to or lower than a predetermined threshold as a compartment that does not contain the target DNA to be detected.

3. The DNA detection method according to claim 1 , further comprising the step of specifying a compartment for which the calculated ratio of the fluorescence intensity falls within a predetermined range as a compartment that contains the target DNA to be detected.

4. The DNA detection method according to claim 1 , wherein the DNA solution comprises a fluorescent-labeled probe and the melting temperature is a melting temperature of a double strand formed between the fluorescent-labeled probe and the target DNA to be detected.

5. The DNA detection method according to claim 4 , wherein the fluorescent-labeled probe has a fluorescent dye and a quencher for the fluorescent dye.

6. The DNA detection method according to claim 1 , wherein the DNA solution comprises a DNA intercalator and the melting temperature is a melting temperature of a double strand of the target DNA to be detected.

7. The DNA detection method according to claim 1 , wherein the plurality of compartments are arranged planarly.

8. The DNA detection method according to claim 1 , wherein the DNA solution is partitioned into the plurality of compartments in a form of droplets or by using wells.

9. A DNA detection device for detecting a target DNA to be detected in a DNA solution, comprising:
a heater for heating the DNA solution;
a fluorescence measurement unit for measuring an intensity of fluorescence emitted from the DNA solution; and
a calculator configured to calculate a melting temperature of a DNA double strand based on the change in the fluorescence intensity in association with a temperature change of the DNA solution, and then calculate a ratio of the fluorescence intensity at a second temperature to the fluorescence intensity at a first temperature, the second temperature being lower than the first temperature.

10. The DNA detection device according to claim 9 , further comprising an amplification unit for amplifying the target DNA to be detected.

11. The DNA detection device according to claim 9 , further comprising a monitor for displaying a detection result.

12. A non-transitory computer-readable storage medium containing instructions which when executed by a processor for allow a DNA detection device to execute a DNA detection method according to claim 1 .

13. The non-transitory computer-readable storage medium according to claim 12 , wherein the detection device is a DNA detection device for detecting a target DNA to be detected in a DNA solution, comprising:
a heater for heating the DNA solution;
a fluorescence measurement unit for measuring an intensity of fluorescence emitted from the DNA solution; and
a calculator for calculating a melting temperature of a DNA double strand based on the change in the fluorescence intensity in association with a temperature change of the DNA solution, and then calculating a ratio of the fluorescence intensity at a second temperature to the fluorescence intensity at a first temperature, the second temperature being lower than the first temperature.

14. The DNA detection method of claim 1 , further comprising plotting a graph of the ratio for each of the compartments on a graph.

15. The DNA detection method of claim 14 further comprising selecting compartments based on the graph.

Assignees

• Hitachi Ltd

Inventors

• Tanaka Junko
• Nakagawa Tatsuo
• Shimazaki Yuzuru
• Harada Kunio

Classifications

• C12Q2563/107

  fluorescence · CPC title

• C12Q2531/113

  PCR · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• B01L2400/0415

  electrical forces, e.g. electrokinetic · CPC title

• B01L2300/0883

  Serpentine channels · CPC title