Digital PCR measuring device

Patent metadata

Field	Value
Publication number	US-12203852-B2
Application number	US-202017435310-A
Country	US
Kind code	B2
Filing date	Feb 26, 2020
Priority date	Mar 19, 2019
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

A digital PCR measuring apparatus capable of measuring a melting curve with high accuracy is provided. The digital PCR measuring apparatus includes a temperature adjuster that controls a temperature of a sample container including a plurality of minute regions, a fluorescence measurement part that measures fluorescence intensity of a plurality of minute regions, and a controller that controls the temperature adjuster and the fluorescence measurement part. The controller controls the temperature adjuster to raise a temperature of the sample container, and, after removing air bubbles generated in the sample container, measures fluorescence intensity of a plurality of minute regions while controlling the temperature adjuster to lower the temperature of the sample container, and measures a melting curve of a plurality of the minute regions.

First claim

The invention claimed is:

1. A digital PCR measuring apparatus that detects DNA contained in a sample, comprising:
a temperature adjuster that controls a temperature of a sample container including a plurality of minute regions to which a fluorescently labeled probe and a sample containing DNA to be detected are supplied, the plurality of minute regions are a plurality of wells arranged in a planar array, and each of the plurality of wells having a diameter of 10 μm to 100 μm;
a fluorescence measurement part that measures fluorescence intensity of the plurality of minute regions; and
a controller that controls the temperature adjuster and the fluorescence measurement part, wherein
the controller controls the temperature adjuster to increase a temperature of the sample container, and, after removing an air bubble generated in the sample container, measures fluorescence intensity of the plurality of minute regions while controlling the temperature adjuster to lower a temperature of the sample container, and measures a melting curve of the plurality of minute regions.

2. The digital PCR measuring apparatus according to claim 1 , further comprising an air bubble removing part that removes an air bubble by adjusting an inclination angle of the sample container with respect to a horizontal plane.

3. The digital PCR measuring apparatus according to claim 1 , further comprising an inclination table configured to be able to hold the sample container in a manner inclined with respect to a horizontal plane.

4. The digital PCR measuring apparatus according to claim 3 , wherein
the fluorescence measurement part has a fluorescence imaging system that guides fluorescence emitted from the sample container to an imaging element, and
the fluorescence imaging system has an optical axis inclined with respect to the horizontal plane in accordance with an inclination of the sample container with respect to the horizontal plane.

5. The digital PCR measuring apparatus according to claim 3 , wherein the inclination table is configured to be able to incline the sample container at an angle in a range of 10° to 20° with respect to the horizontal plane.

6. The digital PCR measuring apparatus according to claim 1 , wherein the temperature adjuster increases a temperature of the sample container to a temperature higher by 5° C. or more than a melting temperature of the DNA to be detected and the fluorescently labeled probe in a case of increasing the temperature.

7. The digital PCR measuring apparatus according to claim 1 , wherein the temperature adjuster makes a temperature change when increasing a temperature of the sample container faster than a temperature change when lowering a temperature of the sample container.

8. The digital PCR measuring apparatus according to claim 1 , wherein the controller is configured to correct positional deviation between a plurality of images captured by the fluorescence measurement part while lowering a temperature of the sample container.

9. The digital PCR measuring apparatus according to claim 1 , wherein
the fluorescence measurement part includes a plurality of filters, the plurality of filters having different transmission wavelengths, and
the controller is configured to capture a fluorescence image by the fluorescence measurement part while switching the plurality of filters when measuring fluorescence intensity of the plurality of minute regions while lowering a temperature of the sample container.

10. The digital PCR measuring apparatus according to claim 9 , wherein the controller performs fluorescence measurement on each of the plurality of filters to acquire a plurality of melting curves, acquires differential curves of the plurality of melting curves, and determines whether or not DNA to be analyzed is contained in the minute region according to a property of the melting curves or the differential curves.

11. The digital PCR measuring apparatus according to claim 10 , wherein the controller determines an influence of leakage light by comparing a plurality of the melting curves and the differential curves.

12. The digital PCR measuring apparatus according to claim 10 , wherein the controller determines whether or not DNA to be analyzed is contained in a minute region based on a ratio or a difference between fluorescence intensity at a first temperature lower than a melting temperature and fluorescence intensity at a second temperature higher than the melting temperature.

Assignees

• Hitachi Ltd

Inventors

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

Classifications

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N21/6456

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G01N21/15

  Preventing contamination of the components of the optical system or obstruction of the light path · CPC title

• G01N21/03

  Cuvette constructions · CPC title

• G01N2021/054

  Bubble trap; Debubbling · CPC title