Back to results
US 12,542,195 B2Granted
Digital PCR detection apparatus, digital PCR quantitative detection method, multi-volume digital PCR quantitative analysis method, digital PCR detection method, nucleic acid detection microsphere, preparation method of nucleic acid detection microsphere, nucleic acid detection microsphere kit and high-throughput nucleic acid detection method
AssigneeSniper (Suzhou) Life Technology Co.
InventorGuang-Ji Sheng
Life of this patent
Estimating the term…
Abstract
The present application provides a digital PCR detection apparatus, a digital PCR quantitative detection method, a multi-volume digital PCR quantitative analysis method, a digital PCR detection method, a nucleic acid detection microsphere, a preparation method of the nucleic acid detection microsphere, a kit, and a high-throughput nucleic acid detection method.
Claims13
A digital PCR quantitative detection method comprising steps of:
- preparing a microdroplet generating device, a temperature controlling device, a fluorescence signal detecting device, and a quantitative analysis device, respectively controlled by a controller;
- preparing a nucleic acid amplification reaction liquid to be detected;
- performing, automatically by the controller, steps of:
- microdropletizing the nucleic acid amplification reaction liquid to be detected to form an array of microdroplets via the microdroplet generating device;
- carrying out, over a plurality of cycles, a nucleic acid amplification reaction for the array of microdroplets via the temperature controlling device;
- performing a bright field and dark field imaging for the array of microdroplets and acquiring a fluorescence image of all microdroplets after each cycle in the nucleic acid amplification reaction via the fluorescence signal detecting device;
- and
- providing the fluorescence images to the quantitative analysis device, wherein the quantitative analysis device performs the steps of:
- acquiring real-time fluorescence curves of microdroplets that have undergone a nucleic acid amplification according to the fluorescence images;
- acquiring Ct values of all of the microdroplets that have undergone the nucleic acid amplification from the real-time fluorescence curves;
- acquiring initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification according to a relationship between the Ct values and the initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification;
- acquiring a frequency distribution of initial nucleic acid copy numbers according to the initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification by:
- acquiring a maximum value and a minimum value of the initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification;
- determining a number of classes and a length of each class interval according to the maximum value and the minimum value;
- and acquiring the frequency distribution of the initial nucleic acid copy numbers;
- and
- calculating a parameter λ of a Poisson distribution according to the frequency distribution of the initial nucleic acid copy numbers and taking the parameter λ as a number of copies of a target DNA molecule contained in each microdroplet.
14 more elements
A digital PCR quantitative detection method comprising steps of:
- preparing a microdroplet generating device, a temperature controlling device, a fluorescence signal detecting device, and a quantitative analysis device, respectively controlled by a controller;
- preparing a nucleic acid amplification reaction liquid to be detected;
- performing, automatically by the controller, steps of:
- microdropletizing the nucleic acid amplification reaction liquid to be detected to form an array of microdroplets via the microdroplet generating device;
- carrying out, over a plurality of cycles, a nucleic acid amplification reaction for the array of microdroplets via the temperature controlling device;
- performing a bright field and dark field imaging for the array of microdroplets and acquiring a fluorescence image of all microdroplets after each cycle in the nucleic acid amplification reaction via the fluorescence signal detecting device;
- and
- providing the fluorescence images to the quantitative analysis device, wherein the quantitative analysis device performs the steps of:
- acquiring real-time fluorescence curves of microdroplets that have undergone a nucleic acid amplification according to the fluorescence images;
- acquiring Ct values of all of the microdroplets that have undergone the nucleic acid amplification from the real-time fluorescence curves;
- acquiring initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification according to a relationship between the Ct values and the initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification;
- selecting a portion of initial nucleic acid copy numbers from the initial nucleic acid copy numbers of all of the microdroplets that have undergone the nucleic acid amplification;
- acquiring a frequency distribution of the portion of the initial nucleic acid copy numbers;
- and
- performing a point estimation of a Poisson distribution according to the frequency distribution of the portion of the initial nucleic acid copy numbers to acquire a parameter λ of the Poisson distribution and taking the parameter λ as a number of copies of a target DNA molecule contained in each microdroplet.
12 more elements
Dependencies are inferred from the claim text.