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CN-121992088-A - Single-cell digital PCR (polymerase chain reaction) based on digital microfluidic and application thereof

CN121992088ACN 121992088 ACN121992088 ACN 121992088ACN-121992088-A

Abstract

The invention relates to a single-cell digital PCR method based on digital micro-fluidic and application thereof. According to the invention, single-cell DNA droplets are obtained, after being cracked, the single-cell DNA droplets are mixed with a digital PCR pre-reaction liquid, the single-cell droplets are separated again to enable the copy number of target genes contained in the single-cell droplets to be 0 or 1, then the single-cell DNA droplets are subjected to digital PCR amplification, and finally absolute quantification of target genes of single cells is carried out through fluorescent detection, so that specific digital PCR detection of single cells is realized, and the copy number of target genes is obtained. The method can realize the digital PCR quantitative detection of the target gene in the single cell, has higher detection sensitivity and detection efficiency, and can realize the special detection of the specific single cell.

Inventors

  • MA HANBIN
  • SHI MUDE
  • LIANG JIANCONG

Assignees

  • 佛山奥素博新科技有限公司
  • 广东奥素液芯微纳科技有限公司

Dates

Publication Date
20260508
Application Date
20241108

Claims (10)

  1. 1. The single-cell digital PCR method based on digital micro-fluidic is carried out in a digital micro-fluidic system and is characterized by comprising the following steps: s1, obtaining single-cell liquid drops to be detected on a digital microfluidic chip; S2, single-cell lysis and single-cell DNA extraction, namely injecting a DNA extraction reagent on the microfluidic chip, tearing the DNA extraction reagent liquid drop into small liquid drops with the same volume as the single-cell liquid drop, fusing the DNA extraction reagent liquid drops and the single-cell liquid drops in the volume ratio of 1:1 to obtain mixed liquid drops, and setting lysis conditions for cell lysis to obtain single-cell DNA liquid drops; S3, configuring a digital PCR reaction system Mixing the single-cell DNA liquid drop with a digital PCR pre-reaction liquid to obtain a digital PCR reaction large liquid drop, wherein the digital PCR pre-reaction liquid comprises a primer and a probe of a target gene to be detected, and a luminescent substance for detection is marked on the probe; s4, digital PCR reaction: And (3) controlling the power-on and power-off of the electrode of the digital micro-fluidic chip, tearing the large liquid drop of the digital PCR reaction in the step (S3) into a plurality of small liquid drops, and operating a temperature control program of the PCR reaction on the small liquid drops according to the small liquid drops to perform the digital PCR reaction.
  2. 2. The method of single-cell digital PCR according to claim 1, wherein the lysis condition is that the single-cell DNA droplet is heated to 60-70 ℃ and incubated for 10-20 min, then heated to 96-100 ℃ and incubated for 3-8min, and then the room temperature is restored.
  3. 3. The single cell digital PCR method according to claim 1, wherein the single cell droplets are obtained by half-tearing.
  4. 4. The single-cell digital PCR method according to claim 3, wherein the step S1 comprises the steps of injecting a cell suspension on a digital microfluidic chip, wherein the concentration of the cell suspension is 1X 10 5 -3×10 5 cells/ml, controlling the power-on and power-off of electrodes on the digital microfluidic chip to enable the liquid drops to be split in half by volume, repeating the split in half for a plurality of times on the split liquid drops, detecting and judging the cell number of the single-electrode liquid drops through a recognition system, marking the liquid drops containing only one cell, and continuously supplying power to the electrodes where the liquid drops containing only one cell are located to obtain the single-cell liquid drops to be detected.
  5. 5. The single cell digital PCR method according to claim 1, wherein the number of droplets in S4 is 8-32 droplets, more preferably 16 droplets.
  6. 6. The single-cell digital PCR method according to claim 5, wherein the amount of the single-cell DNA droplets to be mixed with the digital PCR pre-reaction solution is determined according to the number of the droplets in the step S4.
  7. 7. The single cell digital PCR method according to claim 1, wherein the target gene to be detected is one, two, three, or more.
  8. 8. The method according to claim 1, wherein the luminescent material is a fluorescent group, and wherein the types of fluorescent groups labeled in the probes of different genes to be detected are different when two or more genes to be detected are present.
  9. 9. Use of the digital microfluidic based single cell digital PCR method according to any one of claims 1 to 8 for quantitative detection of single cell gene copy number.
  10. 10. A method for detecting copy number of single-cell gene, characterized in that the quantitative detection method comprises the following steps: obtaining a target cell to be detected; Forming single-cell droplets to be detected on a digital microfluidic chip, and performing single-cell digital PCR amplification according to the single-cell digital PCR method based on digital microfluidic according to any one of claims 1 to 8; And detecting the luminescent substances, and calculating to obtain the copy number of the target genes of the single cells.

Description

Single-cell digital PCR (polymerase chain reaction) based on digital microfluidic and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to single-cell digital PCR (polymerase chain reaction) based on a digital microfluidic system and application thereof. Background Cells are basic units of life bodies, and along with the continuous development of modern biology, it is found that cell heterogeneity often contains important information such as early diagnosis and treatment of diseases. To meet the demand for cell analysis, digital PCR technology has been developed (DIGITAL PCR, DPCR). Digital PCR is an absolute quantitative technique for nucleic acid molecules. During dPCR, the sample is partitioned such that individual nucleic acid molecules within the sample are localized and concentrated within a number of separate areas (within droplets). The partitioning of the sample allows the number of different molecules to be estimated by assuming that the population of molecules obeys a poisson distribution. Thus, each partitioned sample will contain a "0" or "1" molecule, or a negative or positive reaction, respectively. Following PCR amplification, nucleic acids can be quantified by counting the positive reactions, the regions containing the PCR end products. Compared with the first generation (gel electrophoresis) and second generation (qPCR) PCR technology, the digital PCR technology has fewer required sample amounts, can directly obtain the copy number of target molecules, does not depend on a standard curve or a standard substance to determine the copy number of target genes, has extremely high sensitivity and accuracy when carrying out absolute quantification (Zou Y,Mason,M.G,Wang,Y.et al.Nucleic acid purification from plants,animals and microbes in under 30seconds[J].Plos Biology,2017,15(11):96-104.). on the target genes, and has important application values in the aspects of in-vitro diagnosis, gene detection, gene sequencing and the like. Microfluidic technology (microfluidics) is a technology that enables complex laboratory analysis based on the precise manipulation of liquids. With the gradual maturity of the microfluidic technology, the digital PCR technology based on the microfluidic system is rapidly developed, and is widely applied to the aspects of gene mutation detection, copy number variation detection, virus microorganism detection, transgenic food detection, sequencing and the like. The digital microfluidic platform based on the digital microfluidic chip (Digital microfluidics, DMF) can integrate micro-droplet generation, nucleic acid extraction, PCR amplification and fluorescence detection into one device, thereby improving the automation, sensitivity and accuracy of digital PCR. Chinese patent application CN115181789a discloses a method for analysis of single cell separation, cell lysis and detection of samples by using a fully automatic digital PCR all-in-one machine. The full-automatic digital PCR integrated machine is used for separating single cells, splitting cells and detecting and analyzing the samples, so that the application range of the digital PCR integrated machine is expanded, the digital PCR integrated machine can be used for detecting micro cells, and target genes in each single cell in the samples can be accurately detected. The single cell detection of the invention only separates the group cells into single cells, then prepares the single cells into a PCR reaction system, carries out the PCR reaction after carrying out the cell lysis on the single cell reaction system and carries out the fluorescence detection. The method is used for homogenizing and quantitatively detecting the whole cell population, and can not realize the characteristic quantitative detection of a specific single cell. The Chinese patent application CN106520997A discloses a quantitative analysis method for single cell gene expression, which comprises adding single cells into a reaction container, cracking the single cells, adding a reaction system into the reaction container, executing multi-site RT (reverse transcription), executing multi-site PCR amplification by taking cDNA obtained by the multi-site RT as a template, and finally executing specific fluorescent quantitative PCR by taking a sequence obtained by the multi-site PCR amplification as a substrate to analyze the expression quantity of specific genes in the single cells. The method utilizes a real-time fluorescent quantitative PCR method to quantify a target gene in a specific single cell, but the fluorescent quantitative method needs to establish a standard curve, and the standard curve is utilized to quantify target detection nucleic acid, so that the target detection nucleic acid cannot be directly quantified. The Chinese patent application CN116926174A discloses a multiple PCR detection method based on a digital microfluidic chip, which comprises the following steps of a) d