WO2019144907A1 - 数字pcr检测仪、数字pcr定量检测方法、不同体积数字pcr的定量分析方法、数字pcr检测方法、核酸检测微球、核酸检测微球制备方法、核酸检测微球试剂盒以及高通量核酸检测方法 - Google Patents

数字pcr检测仪、数字pcr定量检测方法、不同体积数字pcr的定量分析方法、数字pcr检测方法、核酸检测微球、核酸检测微球制备方法、核酸检测微球试剂盒以及高通量核酸检测方法 Download PDF

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WO2019144907A1
WO2019144907A1 PCT/CN2019/072974 CN2019072974W WO2019144907A1 WO 2019144907 A1 WO2019144907 A1 WO 2019144907A1 CN 2019072974 W CN2019072974 W CN 2019072974W WO 2019144907 A1 WO2019144907 A1 WO 2019144907A1
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Prior art keywords
nucleic acid
micro
microdroplet
microdroplets
fluorescence
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PCT/CN2019/072974
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English (en)
French (fr)
Inventor
盛广济
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北京光阱管理咨询合伙企业（有限合伙）
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Priority claimed from CN201810070377.2A external-priority patent/CN110066857B/zh
Priority claimed from CN201810932950.6A external-priority patent/CN110835645B/zh
Priority claimed from CN201811392278.2A external-priority patent/CN111206081B/zh
Application filed by 北京光阱管理咨询合伙企业（有限合伙） filed Critical 北京光阱管理咨询合伙企业（有限合伙）
Priority to US16/964,183 priority Critical patent/US20210032680A1/en
Priority to EP23206494.9A priority patent/EP4293673A3/en
Priority to ES19743502T priority patent/ES2967012T3/es
Priority to JP2020560539A priority patent/JP7094524B2/ja
Priority to EP19743502.7A priority patent/EP3739059B1/en
Priority to CA3089411A priority patent/CA3089411C/en
Publication of WO2019144907A1 publication Critical patent/WO2019144907A1/zh
Priority to JP2022096041A priority patent/JP2022120133A/ja

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q3/00—Condition responsive control processes

Definitions

  • the present invention relates to the field of nucleic acid detection and analysis, in particular to a digital PCR detector, a digital PCR quantitative detection method, a quantitative analysis method for different volume digital PCR, a digital PCR detection method, a nucleic acid detection microsphere, a nucleic acid detection microsphere preparation method, Nucleic acid detection microsphere kit and high-throughput nucleic acid detection method.
  • Digital PCR is an absolute quantification technique for nucleic acid molecules. Compared to qPCR, digital PCR allows you to directly count the number of DNA molecules, which is an absolute quantification of the number of copies of the starting sample. Quantitative PCR relies on standard curves or reference genes to determine the amount of nucleic acid, while digital PCR allows you to directly count the number of DNA molecules, which is an absolute quantification of the copy number of the starting sample.
  • digital PCR includes a droplet PCR detection method and a chip detection method.
  • the number of effective reaction chambers on a single chip in a chip-based inspection method is generally only a few thousand, much less than the droplet type. Therefore, the dynamic range of the chip-type digital PCR is narrower than that of the droplet type.
  • the droplet PCR method disperses the sample into a water-in-oil reaction unit, and then performs real-time or end-point fluorescence analysis on each reaction unit.
  • the number of effective reaction chambers of the conventional digital PCR instrument is small, resulting in a narrow dynamic range and low work efficiency of the current digital PCR.
  • the traditional droplet digital PCR endpoint detection method has limitations and low detection accuracy.
  • the present application provides a digital PCR detector including a microdroplet generating device, a temperature controlling device, a fluorescent signal detecting device, and a quantitative analyzing device.
  • the microdroplet generating device is configured to microdroplet the nucleic acid amplification reaction solution to form a plurality of microdroplets.
  • the temperature control device and the micro-droplet generating device are connected by a track for transferring the plurality of micro-droplets to the temperature control device to perform temperature cycling to realize nucleic acid amplification.
  • the fluorescent signal detecting device is disposed opposite to the temperature control device for performing photo-detection on the plurality of micro-droplets after nucleic acid amplification.
  • the fluorescent signal detecting device can perform imaging of a plurality of fluorescent channels on the microdroplets and perform bright field dark field imaging. Among them, multiple fluorescence channel imaging is used for the detection of microdroplet reaction signals, and brightfield darkfield imaging is used to detect the size information of the formed microdroplets and monitor the state of the droplets during the reaction.
  • the quantitative analysis device is connected to the fluorescent signal detecting device through a data line for realizing the transmission of the plurality of micro-droplet fluorescence information for quantitative analysis.
  • the digital PCR detector integrates the microdroplet generating device, the temperature control device, the fluorescent signal detecting device, and the quantitative analyzing device, so that the operator can implement the integrated digital PCR detector The automated operation increases the efficiency of the digital PCR detector.
  • the present application provides a digital PCR quantitative detection method comprising the following steps: S4110, acquiring a plurality of real-time fluorescence images of all micro-droplets, and obtaining micro-droplets for performing nucleic acid amplification according to the plurality of real-time fluorescence images.
  • a real-time fluorescence curve S4120, obtaining, according to the real-time fluorescence curve, a Ct value of all micro-droplets for performing nucleic acid amplification; S4130, according to the Ct value and a nucleic acid starting copy number of the micro-droplet for performing nucleic acid amplification Relationship, obtaining the initial copy number of the nucleic acid of all the microdroplets for nucleic acid amplification; S4140, obtaining the frequency of the initial copy number of the nucleic acid according to the initial copy number of the nucleic acid of all the microdroplets performing nucleic acid amplification Distribution; S4150, calculating a parameter ⁇ of the Poisson distribution according to the frequency distribution of the initial copy number of the nucleic acid.
  • the dynamic tracking of the plurality of micro-droplets can be realized by the digital PCR quantitative detection method, and the specific position corresponding to each micro-droplet can be found during the temperature cycling of the plurality of micro-droplets, and the nucleic acid amplification can be realized. Monitoring of the entire process.
  • the digital PCR quantitative detection method not only gets rid of the dependence on the standard curve, but also eliminates the uncertainty of the quantitative result caused by the standard curve, and solves the limitation of the droplet type digital PCR end point detection method, and breaks the use only.
  • the accuracy of the digital PCR quantitative detection is improved by processing the plurality of micro-droplet fluorescence curves and performing statistical correction independent of the uniformity hypothesis.
  • the present application provides a quantitative analysis method for different volume digital PCR.
  • the corresponding standard deviation ⁇ and confidence interval of ln(c) can be obtained by quantitative analysis methods of different volume digital PCR.
  • the nucleic acid concentration of the nucleic acid amplification reaction solution to be tested can be obtained by the corresponding standard deviation ⁇ and the confidence interval of ln(c), so that the number of initial copies of DNA contained in the amplification reaction solution for the nucleic acid to be tested can also be known.
  • the quantitative analysis method of the different volume digital PCR can realize the detection dynamic range of 5 orders of magnitude by using less than 200 micro droplets, and the detection dynamic range of the digital PCR detection instrument is improved. Moreover, its performance is comparable to a single-volume digital PCR with 12,000 microdroplets, saving instrument costs and reducing consumable costs.
  • the present application provides a digital PCR detection method, comprising: S10, preparing a nucleic acid amplification reaction solution to be tested; S20, microdropping the nucleic acid amplification reaction solution to form a microdroplet array; S30 And performing a polymerase chain reaction on the microdroplet array, and acquiring a fluorescence curve of each microdroplet in the microdroplet array and a melting curve of each microdroplet; S40, according to the microdroplet A fluorescence curve of each microdroplet in the array and a melting curve of each microdroplet are analyzed, and the microdroplet array is analyzed to obtain the nucleic acid to be tested.
  • the digital PCR detection method provided by the present application can realize genotyping, mutation scanning, methylation research, etc., with high resolution and sensitivity when preparing a nucleic acid amplification reaction solution to be tested. Reduce the cost of testing.
  • the digital PCR detection method the microdroplet array can be subjected to polymerase chain reaction on the same highly integrated digital PCR detector, and after the PCR amplification of the microdroplet array The PCR product was subjected to melting curve analysis.
  • the digital PCR detection method the fluorescence curve and the melting curve of the microdroplet array can be obtained, and the seamless connection of the real-time monitoring of the whole process of PCR amplification and the melting curve analysis of the PCR product can be completely realized.
  • the present application provides a nucleic acid detection microsphere, a preparation method, a kit, and a high-throughput nucleic acid detection method.
  • the wrapping layer encapsulates the core body to form the nucleic acid detecting microsphere.
  • the matrix is an aqueous polymer gel formed in a hydrophobic oil, has no fluidity, and is not easily altered in shape and volume.
  • the aqueous polymer gel is in a gel state at room temperature and melts at a temperature higher than room temperature without affecting the diffusion and activity of the enzyme, the reaction solution, and the like.
  • the primer dispersed in the matrix can be qualitatively analyzed and identified for the target detection nucleic acid.
  • the core body is a high temperature resistant material having a special labeling function, and each of the core bodies corresponds to one of the primers, and uniquely corresponds, so that the nucleic acid detecting microsphere can be labeled by the core body so that Tracking detection is possible.
  • PCR detection a plurality of nucleic acid detection microspheres of a plurality of types are mixed with a nucleic acid amplification reaction solution to be detected, and a nucleic acid detection liquid can be obtained.
  • Microdropping the nucleic acid detection solution can form a plurality of microdroplets, and a plurality of microdroplets are subjected to a PCR reaction.
  • the double-stranded DNA is denatured at 90 ° C to 95 ° C, and then rapidly cooled to 50 ° C to 60 ° C.
  • the primers are annealed and bound to the target sequence, and then rapidly heated to 70 ° C to 75 ° C to polymerize in Taq DNA.
  • the primer strand is extended along the template to amplify the nucleic acid in a suitable temperature range.
  • the wrap layer is melt-decomposed, and the primer carried in the wrap layer is released into the corresponding micro-droplet and the target contained in the micro-droplet
  • the nucleic acid molecule reacts, and finally, the nuclear body can be positioned and tracked, and the target nucleic acid molecule can be obtained by the primer corresponding to the nuclear body, thereby realizing high-throughput detection of the PCR.
  • a plurality of the plurality of nucleic acid detecting microspheres can be prepared in batches.
  • a plurality of the nucleic acid detection microspheres are mixed according to a requirement of actually detecting the target nucleic acid, and mixed with the nucleic acid amplification reaction solution to be detected, thereby obtaining a nucleic acid detection liquid, thereby detecting the target nucleic acid.
  • a plurality of target nucleic acid molecules can be detected at one time, without repeated multiple detections, and the workload is small, the detection time is short, and the sensitivity is high.
  • FIG. 1 is a schematic diagram of an overall structure of a digital PCR detector provided by the present application
  • FIG. 2 is a micro-droplet generating apparatus of a digital PCR detector provided by the present application
  • FIG. 3 is an outlet of a liquid-spraying gun head according to an embodiment of the present application.
  • FIG. 4 is a schematic diagram showing the change of the velocity of the outlet end of the ejection head according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram of the movement of the outlet end of the ejection head according to an embodiment of the present application;
  • FIG. 6 is a schematic structural view of a fluorescent signal detecting device of the present application;
  • FIG. 7 is a schematic structural view of a temperature control device of the present application;
  • FIG. 8 is a schematic structural view of a temperature control device of the present application
  • FIG. 10 is a schematic diagram of transient performance testing of the temperature control device of the present application
  • FIG. 11 is a schematic diagram of steady state performance testing of the temperature control device of the present application
  • FIG. 12 is a digital PCR detector of the present application
  • FIG. 13 is a flow chart of the microdroplet tiling method of the present application
  • FIG. 14 is a schematic diagram of the microdroplet stacking of the microdroplet container bottom plate of the present application
  • FIG. 16 is a flow chart of a partial sampling digital PCR quantitative detection method according to the present application
  • FIG. 17 is a comparison chart of a partial sampling digital PCR quantitative detection method and other methods CPD standard deviation;
  • FIG. 19 is a flow chart of the overall steps of the digital PCR detection method provided by the present application;
  • FIG. 20 is a schematic diagram of a micro-droplet array obtained by the digital PCR detection method provided by the present application; The schematic diagram of the fluorescence image of the microdroplet array obtained by the digital PCR detection method provided by the present application;
  • FIG. 22 is a schematic diagram of the real-time fluorescence curve obtained by the digital PCR detection method provided by the present application;
  • FIG. 23 is obtained by the digital PCR detection method provided by the present application.
  • FIG. 24 is a schematic diagram of the melting curve obtained by the digital PCR detection method provided by the present application
  • FIG. 25 is a schematic structural diagram of the nucleic acid detection microsphere provided by the present application.
  • FIG. 26 is a schematic structural view of a coating layer provided by the present application;
  • FIG. 27 is a core body provided by the present application;
  • Figure 28 is a schematic view showing the structure of a coating layer in an embodiment provided by the present application;
  • Figure 29 is a schematic view showing the structure of a nucleic acid detecting microsphere in an embodiment provided by the present application;
  • FIG. 31 is a schematic structural view of a different type of microfluidic chip provided by the present application;
  • FIG. 32 is a schematic structural view of a first effective microdroplet provided by the present application;
  • FIG. 34 is a schematic structural diagram of different types of microfluidic chips provided by the present application;
  • FIG. 35 is a schematic structural view of a second effective microdroplet provided by the present application.
  • a digital PCR detector is provided for the conventional digital PCR instrument, which has a small number of effective reaction units, high cost of consumables, narrow dynamic range, low work efficiency and low integration.
  • the present application provides a digital PCR detector 1 including: a microdroplet generating device 10, a temperature controlling device 20, a fluorescent signal detecting device 30, a quantitative analyzing device 40, and a controller. 50.
  • the microdroplet generating device 10 is configured to microdroplet a nucleic acid amplification reaction solution to form a plurality of microdroplets.
  • the temperature control device 20 and the micro-droplet generating device 10 are connected by a track for transferring the plurality of micro-droplets to the temperature control device 20 to perform temperature cycling to realize nucleic acid amplification.
  • the fluorescence signal detecting device 30 is disposed opposite to the temperature control device 20 for performing photo detection on the plurality of micro droplets after nucleic acid amplification.
  • the quantitative analysis device 40 and the fluorescent signal detecting device 30 are connected by a data line for realizing the transmission of the plurality of micro-droplet fluorescence information for quantitative analysis.
  • the controller 50 is connected to the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, respectively, for controlling the micro-droplet generating device 10, the temperature The control device 20, the fluorescence signal detecting device 30, and the quantitative analysis device 40.
  • the digital PCR detector 1 can integrate the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, thereby enabling an operator to perform an automatic operation. .
  • the digital PCR detector 1 has a high working efficiency.
  • the microdroplet generating device 10 may microdrop the nucleic acid amplification reaction solution to be formed to form a plurality of microdroplets.
  • the temperature control device 20 can perform nucleic acid amplification on the plurality of microdroplets.
  • the fluorescent signal detecting device 30 captures a fluorescence change picture of the plurality of micro-droplets in real time.
  • a fluorescence change curve of the plurality of microdroplets can be obtained by a fluorescence change picture of the plurality of microdroplets.
  • the Ct value of the plurality of microdroplets can be obtained, and the concentration of the initial DNA is quantitatively analyzed by the relationship between the Ct value and the initial copy number.
  • the Ct value refers to the number of cycles experienced when the fluorescence signal of each microdroplet reaches a set threshold.
  • the temperature control device 20 performs a nucleic acid amplification reaction on the plurality of micro droplets, and collects, by the fluorescence signal detecting device 30, product signals of the plurality of micro droplets after the nucleic acid amplification reaction, such as fluorescence, UV absorption, turbidity and other signals. Utilizing the difference in composition between the plurality of amplified and non-amplified microdroplets, the number of droplets obtained by obtaining the target sequence is analyzed, and finally quantitative analysis of the nucleic acid molecule is realized. By monitoring the fluorescence change pictures of the plurality of micro-droplets in real time, the detection result is direct, and the problem of false positives and false negatives among the plurality of micro-droplets can be solved.
  • the digital PCR detector 1 integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, so that the operator can realize automatic operation It does not improve the work efficiency, but also has the advantages of rapid response, good repeatability, high sensitivity, specificity and clear results.
  • a method and apparatus for generating microdroplets that are rapidly generated and have a high volume uniformity are provided.
  • the micro-droplet generating device 10 includes a ejector head 110, a fluid drive mechanism 120, a motion control mechanism 130, and a first controller 170.
  • the ejector tip 110 has an outlet end and an inlet end and is used to store the first liquid.
  • the microdroplet generating device 10 can be used in conjunction with a microdroplet container. A second liquid is stored in the microdroplet container, and an outlet end of the ejector tip 110 is inserted under the surface of the second liquid.
  • the first liquid and the second liquid are mutually incompatible or have an interfacial reaction.
  • the first liquid and the second liquid may be any two liquids which are immiscible.
  • the first liquid is an aqueous solution
  • the second liquid is an oily liquid that is immiscible with water, such as minerals. Oil (including n-tetradecane, etc.), vegetable oil, silicone oil, and perfluoroalkane oil, etc.
  • the resulting droplets are aqueous droplets.
  • the first liquid is a mineral oil such as an organic phase such as tetradecane and n-hexane
  • the second liquid is a perfluoroalkane oil which is immiscible with mineral oil.
  • the first liquid and the second liquid may be immiscible aqueous two phases.
  • the first liquid is an aqueous solution
  • the second liquid is an aqueous liquid that is immiscible with water.
  • the first liquid is a dextran solution
  • the second liquid is a polyethylene glycol (PEG) aqueous solution
  • the generated droplets are droplets of a dextran solution.
  • the first liquid and the second liquid may also be two liquids having an interfacial reaction.
  • the first liquid is an aqueous solution of sodium alginate
  • the second liquid is an aqueous solution of calcium oxide.
  • an aqueous calcium oxide solution having a mass concentration of 1% has an interfacial reaction
  • the resulting droplets are calcium alginate gel microspheres.
  • the application can also form a plurality of droplets of different components and volumes in the open container by replacing the components of the first liquid flowing out of the spit gun head or the spit gun head, which can be used for realizing large quantities.
  • Micro-volume high-throughput screening can also achieve multi-step ultra-micro biochemical reactions and detection, and has broad application prospects.
  • the fluid drive mechanism 120 is coupled to the inlet end of the ejector head 110 for discharging the first liquid stored inside the squirt head 110 from the outlet end of the squirt head 110 .
  • the motion control mechanism 130 is configured to control a relative movement between the outlet end of the ejector head 110 and the second liquid to generate a set trajectory or set a speed or set an acceleration, so as to discharge the sputum.
  • the first liquid at the outlet end of the tip 110 overcomes the surface tension and the adhesion of the ejector tip 110 thereto to form microdroplets.
  • the first controller 170 is connected to the fluid drive mechanism 120 and the motion control mechanism 130, respectively, for controlling the fluid drive mechanism 120 and the motion control mechanism 130 to work in coordination.
  • a microdroplet generation method that is stable to the microdroplet generation process is provided.
  • the outlet end 112 of the ejector head 110 can perform a motion including instantaneous acceleration under the second liquid level, and the acceleration is a 1 .
  • the first liquid is discharged from the outlet end 112 of the ejector head 110 to form a droplet 195 attached to the outlet end 112 of the squirt head 110.
  • the droplet 195 exits the outlet end 112 of the ejector tip 110 at the instant of instantaneous acceleration of the outlet end 112 of the ejector tip 110 to form microdroplets.
  • the forces applied by the microdroplets before exiting the outlet end 112 of the ejector tip 110 are gravity G, the buoyancy f 1 of the second liquid, the viscous resistance f 2 of the second liquid, and the ejector tip 110
  • Micro-droplets in the mass exit from the extruding liquid tip 110 before end 112 is m, is a magnitude of the acceleration a 2. According to Newton’s second law of motion,
  • the viscous resistance f 2 6 ⁇ rv received by the droplet 195 as it moves in the second liquid, where ⁇ is the viscosity coefficient of the second liquid and r is the radius of the droplet 195 , v is the speed of movement of the droplet 195.
  • the velocity of the droplet 195 is zero before the instantaneous end of the ejection tip 110 is instantaneously accelerated, so that the droplet 195 is viscous in the second liquid at the moment the tip end 112 of the ejection tip 110 is instantaneously accelerated.
  • f 2 is zero or very small.
  • the droplet diameter typically in the range of pi to 195 microliters of magnitude, and opposite to gravity and buoyancy G droplets 195 f 1 of the second liquid direction, the droplets of gravity G 195
  • the vector sum with the buoyancy f 1 of the second liquid is about zero. Since the viscous resistance f 2 is zero or very small, and the vector sum of gravity G and buoyancy f 1 is about zero, It can be known from Newton's second law of motion that the maximum acceleration that the droplet 195 can reach in the second liquid is a 2 ⁇ f 3 /m, where m is the droplet when the outlet end 112 of the ejector tip 110 is subjected to the instantaneous acceleration motion. The quality of 195.
  • the droplet 195 falls from the outlet end 112 of the ejector tip 110 to form a microdroplet.
  • the condition under which the droplet 195 exits the outlet end 112 of the ejector tip 110 i.e., produces a microdroplet
  • a 2 ⁇ (f 3 /m) ⁇ a 1 is approximately: a 2 ⁇ (f 3 /m) ⁇ a 1 .
  • the motion control mechanism 130 is capable of accurately controlling the magnitude of the instantaneous acceleration of the outlet end 112 of the ejector head 110. Therefore, by controlling the outlet end 112 of the ejector head 110 to have a large value of the instantaneous acceleration, the outlet end 112 of the ejector head 110 is instantaneously accelerated to generate the droplet 195 efficiently.
  • micro-droplet generation method comprising the following steps:
  • the outlet end 112 of the ejector head 110 performs a motion including instantaneous acceleration under the second liquid level, while the first liquid is discharged from the outlet end 112 of the ejector head 110, and the outlet end of the ejector head 110 is discharged.
  • the first liquid of 112 forms a droplet 195 attached to the outlet end 112 of the ejector tip 110, and the droplet 195 exits the outlet end of the squirt head 110 during the instantaneous acceleration movement of the outlet end 112 of the ejector tip 110.
  • 112 forms microdroplets under the second liquid level.
  • the outlet end 112 of the ejector head 110 performs a periodic motion including instantaneous acceleration under the second liquid level.
  • the outlet end 112 of the ejector tip 110 is periodically moved under the second liquid level, meaning that the displacement, velocity and acceleration of the outlet end 112 of the ejector tip 110 exhibit periodic changes.
  • the outlet end 112 of the ejector tip 110 performs a periodic motion including a momentary acceleration motion, and the first liquid is discharged from the outlet end 112 of the ejector head 110 at a constant flow rate to effect equal time interval generation of the microdroplets.
  • the flow rate of the outlet end 112 of the first liquid discharge jetting head 110 is varied, but during a period of motion of the outlet end 112 of the jetting head 110, the first liquid exits the outlet end 112 of the jetting head 110.
  • the volume remains the same. This ensures that the volume of the droplets 195 is the same each time the outlet end 112 of the ejection tip 110 is instantaneously accelerated to generate microdroplets of uniform size.
  • the surface free energy of the ejector head 110, the geometry of the ejector head 110, and the surface tension of the droplet 195 act as an outlet affecting the ejector head 110 without replacing the ejector tip 110 and the first liquid. factors maximal adhesive force f between two ends 112 and 195 is determined by the droplet. Thus, without replacing the tip 110 and the liquid discharge of the first liquid, the liquid discharge outlet tip end 110 of the maximum adhesion between the droplet 112 f 3 of 195 it is fixed. Driven by the fluid drive mechanism 120, the first liquid is capable of continuously discharging the outlet end 112 of the ejector head 110 at a uniform flow rate.
  • the motion control mechanism 130 can precisely control the timing at which the exit end 112 of the ejector tip 110 makes the instantaneous acceleration a 1 and the magnitude of the instantaneous acceleration a 1 .
  • Fluid drive mechanism 120 and the motion control mechanism 130 cooperate with each other can be easily achieved when the volume of the droplet 195 reaches a fixed value of the moment, the outlet tip 110 of the liquid discharge drive end 112 generates acceleration instantaneous acceleration a 1 to generate the size of the volume Consistent microdroplets.
  • the fluid drive mechanism 120 controls the first liquid to uniformly and continuously discharge the outlet end 112 of the ejector tip 110
  • only the motion control mechanism 130 drives the outlet end 112 of the ejector tip 110 to produce an instantaneous acceleration movement at equal intervals, i.e., Microdroplets of uniform size can be generated.
  • the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 act as an outlet end 112 and fluid that affect the ejector tip 110.
  • the two factors of maximum adhesion f 3 between drops 195 are varied.
  • batch processing can control the surface free energy of the ejector tip 110 and the geometry of the ejector tip 110 to vary within a certain interval.
  • the surface tension of the droplet 195 as impact extruding liquid tip outlet end 110 of the maximal adhesive force f 3 is another factor changes only in a small range between 112 and 195 drops.
  • the fluid drive mechanism 120 is capable of driving the first liquid to continuously discharge the outlet end 112 of the ejector head 110 at a uniform flow rate.
  • the motion control mechanism 130 can precisely control the timing at which the exit end 112 of the ejector tip 110 makes the instantaneous acceleration a 1 and the magnitude of the instantaneous acceleration a 1 .
  • Fluid drive mechanism 120 and the motion control mechanism 130 cooperate with each other can be easily achieved when the volume of the droplet 195 reaches a fixed value of the moment, the outlet tip 110 of the liquid discharge drive end 112 generates acceleration instantaneous acceleration a 1 to generate the size of the volume Consistent microdroplets. If the fluid drive mechanism 120 controls the first liquid to uniformly and continuously discharge the outlet end 112 of the ejector tip 110, only the motion control mechanism 130 drives the outlet end 112 of the ejector tip 110 to produce an instantaneous acceleration movement at equal intervals, i.e., Microdroplets of uniform size can be generated.
  • the fluid drive mechanism 120 when the first liquid is uniformly discharged from the outlet end 112 of the ejector head 110, cooperates with the motion control mechanism 130 to perform an instantaneous acceleration motion with a large acceleration value at the moment when the volume of the droplet 195 reaches a set value.
  • the micro-droplet generation method provided by the present application not only ensures that the same ejector tip 110 is used to generate the droplet 195 having a uniform size, and at the same time, the volume of the droplets simultaneously or sequentially generated by the plurality of ejector tips 110 can be ensured. Uniformity.
  • the micro-droplet generation method provided by the embodiment can ensure the generation efficiency of the micro-droplets by simultaneously generating the micro-droplets by the plurality of ejector tips 110 while ensuring the uniformity of the volume of the micro-droplets.
  • the outlet end 112 of the ejector head 110 includes multiple instantaneous acceleration motions in a periodic motion, and the accelerations of the multiple instantaneous acceleration motions are the same, and multiple instantaneous acceleration motions The moments are equally divided into one cycle of motion of the outlet end 112 of the jetting tip 110.
  • the exit end 112 of the ejector tip 110 includes a plurality of transient acceleration motions within a periodic motion to assist in generating a plurality of microdroplets during an exercise cycle at the outlet end 112 of the ejector tip 110.
  • the movement trajectory of the outlet end 112 of the ejector head 110 under the second liquid level includes a combination of one or more of a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • a plurality of trajectories such as a straight line segment, a circular arc segment, and a polygon.
  • the outlet end 112 of the ejector tip 110 When the outlet end 112 of the ejector tip 110 includes more than two instantaneous acceleration movements in a periodic motion, the outlet end 112 of the ejector tip 110 is traversed into a regular polygon in the second liquid, including an equilateral triangle, Square, regular pentagon, regular hexagon, etc.
  • step S203 during the periodic movement of the outlet end 112 of the ejector head 110 under the second liquid level, the velocity of the outlet end 112 of the ejector head 110 is a rectangular wave. Variety.
  • the velocity of the outlet end 112 of the ejector tip 110 changes in a rectangular wave.
  • the velocity is entered into a uniform phase, which facilitates the motion control mechanism 130 to achieve precise control of the motion state of the outlet end 112 of the ejector tip 110.
  • the high-order time and the low-order time of the rectangular wave indicating the change in the moving speed of the outlet end 112 of the ejector tip 110 may be equal or different.
  • step S203 during the periodic movement of the outlet end 112 of the ejector head 110 under the second liquid level, the velocity of the outlet end 112 of the ejector head 110 changes in a square wave.
  • the high-order time and the low-order time of the rectangular wave indicating the change in the moving speed of the outlet end 112 of the ejector head 110 are equal.
  • the velocity of the outlet end 112 of the ejection head 110 is zero or has a velocity in the opposite direction with respect to the high position. As shown in FIG.
  • the trajectory of the outlet end 112 of the ejector head 110 under the second liquid level is a straight line segment, and the outlet end 112 of the ejector head 110 is instantaneously accelerated from one end of the straight section.
  • the other end of the straight line segment performs the instantaneous acceleration motion in the opposite direction.
  • the acceleration of the two instantaneous acceleration motions is a 1 .
  • the trajectory of the outlet end 112 of the ejector tip 110 below the second liquid level is a circular arc segment or a polygon.
  • the frequency at which the outlet end 112 of the ejector head 110 periodically moves under the second liquid level is between 0.1 Hz and 200 Hz, which is easy to implement in engineering.
  • the fluid drive mechanism 120 controls the first liquid to exit the outlet end 112 of the ejector head 110 at a constant flow rate.
  • the motion control mechanism 130 controls the output end of the ejector head 110 to perform a periodic motion in which the motion trajectory is a straight line and the speed is a square wave.
  • the direction of velocity of the outlet end 112 of the ejector tip 110 changes, the instantaneous acceleration of the outlet end 112 of the ejector tip 110 reaches a maximum.
  • the droplet 195 attached to the outlet end 112 of the ejector tip 110 also exits the outlet end 112 of the ejector tip 110 when the instantaneous acceleration of the outlet end 112 of the ejector tip 110 reaches a maximum to form microdroplets 199. Since the first liquid is discharged at the outlet end 112 of the ejector head 110 at a constant flow rate, when the droplet 195 is detached from the outlet end 112 of the ejector head 110, the new droplet 195 enters the generated state. When the outlet end 112 of the ejector tip 110 is again accelerated in the reverse direction, the newly generated droplet 195 also falls from the outlet end 112 of the ejector tip 110 to form a new droplet 199.
  • two micro-droplets 199 can be generated in one motion cycle of the outlet end 112 of the ejector head 110, and the square wave is relatively easy to implement in engineering.
  • a droplet 199 is created during one cycle of the outlet end 112 of the ejector tip 110.
  • the outlet end 112 of the ejector head 110 performs a square wave motion of the trajectory in any direction in the second liquid 699, including: a plane perpendicular to the direction in which the squirting head 110 extends.
  • the square wave motion in which the trajectory is a straight line the square wave motion in which the trajectory is a straight line in a plane at an arbitrary angle with the extending direction of the ejector head 110, and the trajectory in a straight line along the extending direction of the ejector head 110 Wave movements, etc.
  • the trajectory of the outlet end 112 of the ejector head 110 is a circular arc segment or a polygon
  • the outlet end 112 of the ejector tip 110 is traversed in any direction in the second liquid 699.
  • the linear square wave motion includes: performing a square wave motion in which a trajectory is a straight line in a plane perpendicular to the extending direction of the ejector head 110, and making a trajectory in a plane at an arbitrary angle to the extending direction of the ejector head 110
  • the square wave motion of the straight line, the square wave motion in which the trajectory is a straight line along the extending direction of the ejector head 110 is performed by performing a square wave motion in which a trajectory is a straight line in a plane perpendicular to the extending direction of the ejector head 110, and making a trajectory in a plane at an arbitrary angle to the extending direction of the ejector head 110.
  • the microdroplet 199 is a nucleic acid amplification reaction solution to be tested, and the nucleic acid amplification reaction solution is microdropleted by the microdroplet generating device 10 to form a plurality of microdroplets.
  • the nucleic acid amplification reaction solution to be tested is passed through the micro-droplet generating device 10 of the integrated digital PCR detector 1 and converted into a plurality of micro-droplets 199 by micro-drip processing, so that the detected fragments in the sample to be tested are from a large amount.
  • the complex background is separated and placed in the microdroplet container 60 for inspection.
  • a plurality of droplets 199 of uniform size can be generated by the microdroplet generating device 10.
  • Each of the microdroplets 199 is on the order of micrometers, and each of the microdroplets 199 can be considered as a separate reactor, corresponding to a test tube commonly used in biochemical reactions.
  • the plurality of microdroplets 199 are placed in the microdroplet container 60 for easy inspection and observation.
  • a plurality of micro-droplets of different sizes can also be generated by the micro-droplet generating device 10 for medical clinical detection.
  • the plurality of microdroplets 199 are small in volume and large in number, and have the advantage that many conventional test tubes do not.
  • a large number of micro-droplets 199 can be generated by the micro-droplet generating device 10, so that the digital PCR detector 1 has the advantages of high flux, low consumable cost and low background noise, and has a good industrialization prospect.
  • a temperature control device having a fast temperature rise and fall rate and a high service life is provided.
  • the present application further provides a temperature control device 20 including a flexible circuit board 220, a heating substrate 240 spaced apart from the flexible circuit board 220, and a plurality of semiconductor galvanic pairs 230.
  • the heating substrate 240 includes a first surface 241 and a second surface 242 that are disposed opposite each other.
  • the plurality of semiconductor galvanic pairs 230 are disposed between the flexible circuit board 220 and the first surface 241, and the plurality of semiconductor galvanic pairs 230 are connected in series, in parallel, or in a mixed manner.
  • the temperature control device 20 is generally used in a high and low temperature cycle environment, and the temperature needs to be rapidly raised and lowered, so the temperature control device 20 is required to be extremely high.
  • the temperature control device 20 employs the flexible circuit board 220.
  • the flexible circuit board 220 has the characteristics of high wiring density, light weight, thin thickness, and good bending property.
  • the flexible circuit board 220 eliminates thermal stress by its own deformation during the temperature rise and fall process. The thermal stress existing in the temperature rise and fall process can be reduced by the flexible circuit board 220, thereby extending the service life of the temperature control device 20. At the same time, the problem of uneven temperature distribution is solved by the flexible circuit board 220.
  • the temperature control device 20 can realize instantaneous temperature rise and temperature decrease, and the process of temperature rise and temperature decrease is shortened, thereby achieving high and low temperature cycle, shortening the detection time of the digital PCR detector 1 and improving the detection efficiency.
  • the flexible printed circuit 220 can be a highly reliable and excellent flexible printed circuit board made of polyimide or polyester film.
  • the flexible circuit board has the characteristics of high wiring density, light weight, thin thickness and good bending property.
  • the flexible circuit board is light in weight and thin in thickness, and can effectively save product volume.
  • the semiconductor electric cooler (TEC) is made by utilizing the Peltier effect of a semiconductor material.
  • the so-called Peltier effect refers to the phenomenon that when a direct current passes through a galvanic couple composed of two kinds of semiconductor materials, one end absorbs heat and one end radiates heat. Replacing one of the conventional semiconductor refrigerators by the flexible circuit board 220 makes the semiconductor cooler more thermally conductive.
  • Thermal stress is also called temperature change stress.
  • the thermal stress is balanced with the zero external phase. It is a self-balancing stress caused by the thermal deformation. The compression occurs at a high temperature and the tensile deformation occurs at a low temperature. Under certain conditions, by controlling the stress to make it reasonably distributed, the mechanical properties and service life of the parts can be improved, and the damage can be improved.
  • the heating substrate 240 can be a superconducting aluminum substrate circuit.
  • the aluminum substrate is a metal-based copper clad plate with good heat dissipation function.
  • the single panel is composed of a three-layer structure, which is a circuit layer (copper foil), an insulating layer and a metal base layer.
  • the superconducting aluminum substrate circuit is a material of a circuit board which is an aluminum alloy and can conduct heat quickly.
  • the aluminum substrate minimizes thermal resistance and provides excellent thermal conductivity of the aluminum substrate, and its mechanical properties are excellent compared to thick film ceramic circuits.
  • the semiconductor galvanic pair 230 includes a P-type 236 and an N-type 232 spaced apart from the P-type 231.
  • the P-type coupler 231 and the N-type couple 232 are soldered between the flexible circuit board 220 and the substrate 240.
  • the semiconductor galvanic pair 230 includes a pair of galvanic couples formed by the P-type 232 and the N-type 232, and a plurality of pairs of the galvanic pairs 230 are connected by electrodes, and Sandwiched between the flexible circuit board 220 and the first surface 241.
  • a "hot" side and a "cold” side are produced. Whether it is cooling or heating, as well as the rate of cooling and heating, is determined by the direction and magnitude of the current passing through it.
  • the thermoelectric effect produced by a pair of said semiconductor couples 230 is small, so in practice, hundreds of pairs of said semiconductor couples 230 are connected in series, and the resulting thermoelectric effect is increased.
  • the first surface 241 includes a plurality of spaced apart first electrode sheets 243, one of the first electrode sheets 243 corresponding to one of the semiconductor couple pairs 230, the semiconductor couple pair 230
  • the P-type coupler 231 and the N-type couple 232 are connected in series through the first electrode piece 243.
  • the flexible circuit board 220 includes a plurality of second electrode sheets 221 spaced apart from each other and connected in series, and two adjacent semiconductor couple pairs 230 are connected in series by one of the second electrode sheets 221.
  • the temperature control device 20 further includes a thermally conductive enhancement layer 250 disposed on the second surface 242.
  • the thermally conductive reinforcing layer 250 has very good strength, flexibility, electrical conductivity, thermal conductivity, and optical properties.
  • the thermally conductive reinforcing layer 250 is directly in contact with the micro-droplet container 60, and the plurality of micro-droplets can be heated uniformly, thereby realizing nucleic acid amplification by controlling temperature.
  • the heat conduction enhancement layer 250 may be a graphite heat conduction layer or a silicone grease heat conduction layer to accelerate heat conduction, increase temperature uniformity of the second surface 242 of the heating substrate 240, thereby ensuring proximity to the microdroplet container 60.
  • the surface temperature is uniform so that the plurality of microdroplets are heated uniformly. Further, the nucleic acid amplification is completed, the detection efficiency is improved, and time is saved.
  • the material of the thermally conductive enhancement layer 250 includes graphene.
  • the graphene is a flat film which has very good heat conduction properties and can achieve uniform heat conduction in the lateral direction.
  • the temperature control device 20 further includes a second controller 210 electrically coupled to the flexible circuit board 220 for controlling the magnitude of the current.
  • the temperature control device 20 further includes a temperature sensor 260 disposed on the second surface 242 and electrically connected to the second controller 210 for detecting the temperature of the second surface 242. And sending the temperature to the second controller 210.
  • the temperature sensor 260 is disposed on the second surface 242 of the heat conduction enhancement layer 250 to detect the real-time temperature of the second surface 242, and then feed back temperature information to the second controller 210, thereby implementing Control of the heating temperature of the plurality of microdroplets.
  • the temperature sensor 260 is configured to measure the temperature of the micro-droplet container 60 by detecting a change in resistance of the metal, to detect the temperature change of the plurality of micro-droplets during the nucleic acid amplification process in real time, thereby Feedback to the second controller 210, thereby controlling the control circuit to perform temperature regulation, achieving temperature control, and better performing nucleic acid amplification.
  • the second controller 210 includes a temperature control unit 212 and a control circuit 214.
  • the temperature control unit 212 is coupled to the temperature sensor 260 for detecting the temperature of the second surface 242 in real time.
  • the control circuit 214 is coupled to the flexible circuit board 220 for regulating temperature changes of the plurality of semiconductor galvanic pairs 230.
  • the temperature control unit 212 and the control circuit 214 are disposed on a circuit board.
  • the relationship between the temperature control unit 212 and the control circuit 214 is a logical operation relationship of the internal algorithm, and a Packet Identifier closed-loop control algorithm, that is, a PID closed-loop control algorithm, may be used.
  • the temperature detected by the temperature control unit 212 is the temperature feedback of the nucleic acid amplification.
  • the control circuit 214 calculates the result as an output of the internal algorithm, thereby forming a closed loop relationship.
  • the temperature feedback of the circuit part is actually a sampling circuit.
  • the electrical signal on the platinum resistor is collected and converted to a temperature value that is passed to the input of the control circuit.
  • the temperature sensor 260 and the temperature control unit 212 are connected by a standard platinum resistance three-wire system.
  • the flexible circuit board 220 is provided with a first electrode 222 and a second electrode 223.
  • the plurality of second electrode sheets 221 are connected in series with the first electrode 222 and the second electrode 223 in series.
  • the first electrode 222 and the second electrode 223 are respectively connected to the control circuit.
  • connection between the control circuit 214 and the flexible circuit board 220 is two wires, and the first electrode 222 and the second electrode 223 are respectively connected.
  • the temperature control device 20 further includes a heat sink 270 that includes a substrate 271 and a heat sink 272 coupled to the substrate 271.
  • the flexible circuit board 220 is disposed on a surface of the substrate 271.
  • the heat exchange area is increased without increasing the area of the substrate 271, and the time during which the cool air acts on the surface of the substrate 271 is extended.
  • the heat dissipation air passage is also beneficial for accelerating heat exchange, and more heat is taken away from the surface of the substrate 271, thereby achieving a more ideal heat dissipation effect.
  • the temperature control device further includes a fan 273 disposed around the heat sink 273.
  • the heat sink 270 can be assisted by the fan 273 for heat dissipation.
  • the fan 273 is disposed around the heat sink 273, and a plurality of fans 273 can be disposed, so that a better heat dissipation effect can be achieved, and the temperature control device 20 can be warmed and cooled more quickly.
  • the temperature control device 20 is circulated with alternating current, and the current controller is adjusted by the second controller 210 to control whether the temperature control device 20 is cooled or heated, and cooled, heated. rate.
  • the temperature sensor 260 is used to detect the real-time heating temperature of the micro-droplet container 60, and then feed back the temperature information to the temperature control unit 212.
  • the temperature control unit 212 feeds back a temperature change condition to the control circuit 214 to control the temperature of the plurality of microdroplets.
  • the plurality of microdroplets can be subjected to nucleic acid amplification by the temperature control device 20. Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle.
  • the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C.
  • the primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA.
  • the primer strand is extended along the template to amplify the nucleic acid in a suitable temperature range.
  • the bottom of the micro-droplet container 60 is closely attached to the temperature control device 20, and there is no gap between the two, which improves the accuracy of the digital PCR detector 1.
  • the temperature control device 20 generally has two indexes for testing the temperature control performance, and observes the temperature rise and fall of the temperature control device 20 in the transient state and the steady state, respectively.
  • the temperature rise and fall rate can reach 13.34448 ° C / s and the control precision is 0.02722 ° C.
  • the rate of measurement of the temperature control device 20 when it is warmed to a steady state can be as fast as 18.953894 ° C / s. Therefore, the transient response of the temperature control device 20 is good, and the temperature control device 20 can realize instantaneous temperature rise and temperature reduction, save time, and improve detection efficiency.
  • the temperature control device 20 when the temperature control device 20 is in a steady state, that is, when the temperature is reached after stabilization is reached.
  • the temperature change is relatively stable and the temperature fluctuation is small. Therefore, the temperature control device 20 can achieve a rapid temperature rise and temperature cycle, and the temperature after the stabilization is small, which saves the time for the digital PCR to detect the sample sample and improves the work efficiency.
  • This rate of temperature rise and fall shortens the time required to complete nucleic acid amplification, improves nucleic acid amplification efficiency, and improves the accuracy of the digital PCR detection system.
  • the fluorescent signal detecting device 30 includes an excitation light source 340, a fluorescence detecting component 330, and a third controller 310.
  • the excitation light source 340 is disposed above the detection area of the micro-droplet container 60 and is irradiated at an oblique angle to the detection area of the micro-droplet container 60 to form an oblique light path.
  • the fluorescence detecting component 330 is disposed directly above the detection area of the micro-droplet container 60 for collecting a fluorescent image of the plurality of micro-droplets.
  • the third controller 310 is coupled to the excitation light source 340 and the fluorescence detecting component 330, respectively, for controlling the excitation light source 340 and the fluorescence detecting component 330.
  • the fluorescent signal detecting device can perform imaging of a plurality of fluorescent channels on the microdroplets and perform bright field dark field imaging. Among them, multiple fluorescent channel imaging is used for detecting the droplet reaction signal, and bright field dark field imaging is used to detect the size information of the formed microdroplet and monitor the state of the droplet during the reaction.
  • the third controller 310 can control the excitation light source 340 to move while the fluorescent detection assembly 330 and the micro-droplet container 60 are not moving. That is to say, at this time, the plurality of microdroplets are fluorescently detected by the excitation light source 340.
  • the third controller 310 can control the fluorescence detecting component 330 to move, and at this time, the microdroplet container 60 and the excitation light source 340 do not move, and the plurality of microdroplets are fluorescently detected.
  • the third controller 310 can control the movement of the micro-droplet container 60, the fluorescence detecting component 330 and the excitation light source 340 do not move, and perform fluorescence detection on the plurality of micro-droplets.
  • the positional movement of the excitation light source 340, the fluorescence detecting component 330, and the microdroplet container 60 can be adjusted by the third controller 310, thereby generating relative motion, thereby causing the microdroplets of the detection zone
  • the container 60 is aligned with the fluorescence detecting assembly 330 to take a picture to complete the entire fluorescence detection process.
  • the optical path emitted by the excitation light source 340 is obliquely irradiated to the plurality of micro-droplets such that the micro-droplets containing the fluorescent substance in the micro-droplet container 60 generate fluorescence.
  • Fluorescence information collection is performed on the micro-droplets containing the fluorescent substance by the fluorescence detecting component 330, and the fluorescent information of the fluorescent substance-containing micro-droplets is transmitted to the quantitative analysis device 40 in the form of a fluorescent image. Used for quantitative analysis.
  • An irradiation angle is applied to the microdroplet container 60 from above the microdroplet container 60.
  • the periodic detection of the plurality of micro-droplets by the fluorescence signal detecting device 30 is performed, and the photographing is performed in real time.
  • the oblique light path can effectively reduce the excitation light scattering background and improve the sensitivity of fluorescence detection.
  • the internal fluorescence of the plurality of microdroplets within the microdroplet container 60 is excited, and a fluorescent image of the plurality of microdroplets is acquired by the fluorescence detecting component 330.
  • the excitation source 340 provides the plurality of microdroplets with energy that is evaporated, atomized, or excited.
  • the excitation light source 340 has the characteristics of narrow spectral bandwidth, high spectral purity, good wavelength stability, high efficiency, long life, good reliability, good beam quality, etc., thereby ensuring the accuracy and stability of the detection result.
  • the excitation light source 340 includes a plurality of LED light sources 341 of different colors, a dichroic mirror 344, a fly-eye lens 345, and a focusing lens 346.
  • a collimator mirror 342 and a first filter 343 are sequentially disposed at the front end of each of the LED light sources 341.
  • the dichroic mirror 344 is obliquely disposed at the front end of the first filter 343 for refracting light emitted by each of the LED light sources 341 into an optical path.
  • the fly-eye lens 345 is used to improve the uniformity of the refracted light path.
  • the focus lens 346 is disposed at the front end of the fly-eye lens 345, for focusing imaging.
  • the plurality of LED light sources 341 of different colors are used as the excitation light source 340, and can generate fluorescence of different colors to increase the detection channel and realize the detection of the indispensable micro droplets.
  • the collimator mirror 342 and the first filter 343 are sequentially disposed at the front end of each of the LED light sources 341.
  • the collimating mirror 342 can be used in a beam delivery system to maintain the collimation of the beam between the laser cavity and the focusing optics.
  • the LED light source 341 can separate light of a desired wavelength band as excitation light by the first filter 343.
  • the excitation light is converted into a parallel or concentrated light beam, and irradiated onto a region of the chip containing a plurality of droplets to form an excitation. Area. Excitation light is excited by the plurality of microdroplets in the microdroplet container 60.
  • the excitation source 340 is integral with or separate from the fluorescence detection assembly 330.
  • the fluorescent signal detecting device 30 When the fluorescent signal detecting device 30 is irradiated to the micro-droplet container 60 at an oblique angle, the fluorescent signal detecting device 30 performs periodic two-dimensional scanning on the plurality of micro-droplets, and performs photographing in real time. .
  • the fluorescent signal detecting device 30 When the fluorescent signal detecting device 30 is irradiated to the micro-droplet container 60 at an oblique angle, the excitation light scattering background can be effectively reduced, and the sensitivity of fluorescence detection can be improved.
  • the internal fluorescence of the plurality of microdroplets in the microdroplet container 60 is excited, collected by the upper objective lens 332 through the second filter 333, and enters the camera 331, the camera 331 A fluorescent image of the plurality of microdroplets is acquired.
  • the optical path of the focusing lens 346 is obliquely irradiated to the plurality of micro-droplets such that the micro-droplets containing the fluorescent substance in the micro-droplet container 60 generate fluorescence. Fluorescence information collection is performed on the micro-droplets containing the fluorescent substance by the fluorescence detecting component 330, and the fluorescent information of the fluorescent substance-containing micro-droplets is transmitted to the quantitative analysis device 40 in the form of a fluorescent image. Used for quantitative analysis.
  • the third controller 310 can synchronously turn on the plurality of different colored LED light sources 341 and the camera 331.
  • the first filter 343 is used to select an optical device of a desired radiation band.
  • the first filter 343 is made of plastic or glass sheet and then added with a special dye.
  • the red filter can only pass red light, and so on.
  • the transmittance of the glass piece is originally similar to that of air. All colored light can pass, so it is transparent, but after dyeing the dye, the molecular structure changes, the refractive index also changes, and the passage of some color light changes. For example, a white light passes through a blue filter, and a blue light is emitted, while green light and red light are rare, and most of it is absorbed by the filter.
  • the dichroic mirror 344 is obliquely disposed at the front end of the first filter 343 for refracting light emitted by each of the LED light sources 341 into an optical path.
  • the fly-eye lens 345 is configured to improve the uniformity of the refracted optical path.
  • the focus lens 346 is disposed at the front end of the fly-eye lens 345, for focusing imaging.
  • the focus lens 346 belongs to a gradient index lens. It has the characteristics of end focusing and imaging, and its cylindrical shape, so it can be applied to many different micro optical systems.
  • the switching between the plurality of LED light sources 341 of different colors can be controlled by the third controller 310 to constitute different fluorescent detection channels.
  • the plurality of different color LED light sources 341 can be operated in turn without separately setting the runner.
  • the collimating mirror 342 is divided into a reflective collimating mirror and a transmissive collimating mirror.
  • Reflective collimating mirrors and transmissive collimating mirrors are used in the beam delivery system to maintain the collimation of the beam between the laser cavity and the focusing optics.
  • Reflective collimators typically use a copper full mirror, while transmissive collimators use a zinc selenide lens.
  • the fluorescence detecting component 330 includes an objective lens 332, a camera 331 and a second filter 333 disposed between the camera 331 and the second filter 333.
  • the second filter 333 uses a multi-band pass filter.
  • the multi-bandpass filter can simultaneously pass light in multiple bands, each band corresponding to a dye.
  • a characteristic band spectrum of excitation and emission fluorescence of a substance is separated and selected in a biomedical fluorescence assay system.
  • the molecule absorbs the excitation spectrum in the absorption band and then emits a long-wavelength radiation spectrum in the emission band, which forms a fluorescence spectrum.
  • the generation of the fluorescent image of the plurality of microdroplets is primarily accomplished by the camera 331.
  • the camera 331 is capable of converting an optical image into a digital signal.
  • the camera 331 is arranged with a plurality of neat capacitors that sense light and convert the image into a digital signal. Through the control of the external circuit, each small capacitor can transfer its charge to its adjacent capacitor.
  • the fluorescence collection of the plurality of micro-droplets is completed by using the camera 331 , which can provide an intuitive visualized fluorescent image, improve the speed of fluorescence detection, and make the detection result more accurate.
  • the fluorescent signal detecting device 30 can cause the plurality of micro-droplets to be fluorescently imaged, take a certain number of fluorescent images of the plurality of micro-droplets at a time, and then use image processing technology to perform droplet fluorescence in the image. Automatic identification to obtain fluorescence information of the droplets.
  • the fluorescent signal detecting device 30 can cause the plurality of micro-droplets to be fluorescently imaged, take a certain number of fluorescent images of the plurality of micro-droplets at a time, and then use image processing technology to perform droplet fluorescence in the image. Automatic identification to obtain fluorescence information of the droplets.
  • the fluorescent signal detecting device 30 is used to perform periodic two-dimensional scanning of the plurality of micro-droplets, and photographing is performed in real time.
  • the oblique light path can effectively reduce the excitation light scattering background and improve the sensitivity of fluorescence detection.
  • the internal fluorescence of the plurality of microdroplets in the microdroplet container 60 is excited, collected by the upper objective lens 332 through the second filter 333, and enters the camera 331, the camera 331 A fluorescent picture of the plurality of microdroplets is acquired.
  • the excitation source 340 provides the plurality of microdroplets with energy that is evaporated, atomized, or excited.
  • the excitation light source 340 has the characteristics of narrow spectral bandwidth, high spectral purity, good wavelength stability, high efficiency, long life, good reliability, good beam quality, etc., thereby ensuring the accuracy and stability of the detection result.
  • a computer is used to synchronize the opening of the LED light source 341 with the acquisition of the camera 331.
  • the LED light source 341 remains in the off state in the non-acquisition state.
  • the switching between the plurality of LED light sources 341 of different colors can be controlled by the third controller 310 to constitute different fluorescent detection channels.
  • the plurality of different color LED light sources 341 can be operated in turn without separately setting the runner.
  • a collimator mirror 342 and a first filter 343 are sequentially disposed at the front end of each of the LED light sources 341.
  • the collimating mirror 342 is divided into a reflective collimating mirror and a transmissive collimating mirror.
  • Reflective collimating mirrors and transmissive collimating mirrors are used in the beam delivery system to maintain the collimation of the beam between the laser cavity and the focusing optics.
  • Reflective collimators typically use a copper full mirror, while transmissive collimators use a zinc selenide lens.
  • the first filter 343 is used to select an optical device of a desired radiation band.
  • the filter is made of plastic or glass and then added with special dyes.
  • the red filter can only pass red light, and so on. Filter products are mainly classified according to spectral bands, spectral characteristics, film materials, and application characteristics.
  • the dichroic mirror 344 is obliquely disposed at the front end of the first filter 343 for refracting light emitted by each of the LED light sources 341 into an optical path.
  • the principle of the dichroic mirror 344 is to place a colorless calcite (ice stone) therein to decompose the light into two vertically oscillating lights, and observe the colors of the two rays through a dichroic mirror.
  • the fly-eye lens 345 is configured to improve the uniformity of the refracted optical path.
  • the fly-eye lens 345 is formed by a series of small lens combinations, and the application of the double-row fly-eye lens array to the illumination system can achieve high light energy utilization and large area uniform illumination.
  • the compound eye lens has broad application prospects in the field of microdisplay and projection display.
  • the double-row compound eye lens array can realize uniform illumination, improve the uniformity and illumination brightness of the LED light sources of a plurality of different colors, and can effectively calculate the orientation and distance of the object and the observed object, and can obtain more accurate fluorescence. image.
  • fly-eye lens array For the uniform illumination of the fly-eye lens array, two rows of fly-eye lens arrays are arranged in parallel, and the focus of each small unit lens in the first array of fly-eye lens arrays coincides with the center of the corresponding small unit lens in the fly-eye lens array of the second column.
  • the optical axes of the two rows of fly-eye lenses are parallel to each other.
  • the principle of the uniform illumination of the fly-eye lens array is that the light beam parallel to the optical axis passes through the first lens and is focused at the center of the second lens, and the first row of fly-eye lens intersects the light source to form a plurality of light source images for illumination.
  • Each lenslet of the two rows of fly-eye lenses images the small lens overlap of the first row of fly-eye lens pairs onto the illumination surface. Since the first row of fly-eye lenses divides the entire wide beam of the light source into a plurality of beamlets, and the tear unevenness in each beamlet range is superimposed by the beamlets at the symmetrical position, the teardrops of the beamlets are not The uniformity is compensated so that the light energy in the entire aperture is effectively and uniformly utilized.
  • the spot emerging from the second row of fly-eye lenses is focused on the illumination screen by a concentrating mirror, so that each spot of the spot on the illumination screen is illuminated by light from all points of the light source, and at the same time, the beams emitted from each point on the light source overlap. Go to the same field of view on the illumination spot, so get a uniform square spot.
  • the focus lens 346 is disposed at the front end of the fly-eye lens 345, for focusing imaging.
  • the focus lens 346 belongs to a gradient index lens. It has the characteristics of end focusing and imaging, and its cylindrical shape, so it can be applied to many different micro optical systems.
  • the fluorescence detecting component 330 includes an objective lens 332, a camera 331 and a second filter 333 disposed between the camera 331 and the second filter 333.
  • the third controller 310 can synchronously turn on the plurality of different colors of the LED light source 341 and the camera 331.
  • the generation of the fluorescent image of the plurality of microdroplets is primarily accomplished by the camera 331.
  • the camera 331 is capable of converting an optical image into a digital signal.
  • the camera 331 is arranged with a plurality of neat capacitors that sense light and convert the image into a digital signal. Through the control of the external circuit, each small capacitor can transfer its charge to its adjacent capacitor.
  • the fluorescence collection of the plurality of micro-droplets is completed by using the camera 331 , which can provide an intuitive visualized fluorescent image, improve the speed of fluorescence detection, and make the detection result more accurate.
  • the second filter 333 uses a multi-band pass filter.
  • the multi-bandpass filter can simultaneously pass light in multiple bands, each band corresponding to a dye.
  • a characteristic band spectrum of excitation and emission fluorescence of a substance is separated and selected in a biomedical fluorescence assay system.
  • the molecule absorbs the excitation spectrum in the absorption band and then emits a long-wavelength radiation spectrum in the emission band, which forms a fluorescence spectrum.
  • the excitation light source 340 includes five LED light sources 341 of different colors, five of the collimating mirrors 342, five of the first filters 343, and four A dichroic mirror 344, one of the fly-eye lenses 345, and one focus lens 346.
  • the five different colored LED light sources 341 can generate different colors of light to illuminate the plurality of microdroplets. By selecting the five differently colored LED light sources 341, illumination of different fluorescent colors can be obtained, and the five different colored LED light sources 341 can be operated in turn.
  • the collimating mirror, the first filter 343, and the dichroic mirror 344 are disposed in front of the optical path emitted by each of the LED light sources.
  • the collimating mirror 342 and the first filter 343 are disposed at a vertical angle to the optical path (90° angle setting).
  • the dichroic mirror 344 is disposed at an angle of 0 to 45 degrees with respect to the optical path.
  • An optical path formed by the dichroic mirror 344 is provided with the fly-eye lens 345 and the focus lens 346 in this order.
  • the fly-eye lens 345 and the focus lens 346 are disposed at a vertical angle to the optical path (90° angle setting).
  • the optical path of the focusing lens 346 is obliquely irradiated to the plurality of micro-droplets such that the micro-droplets containing the fluorescent substance in the micro-droplet container generate fluorescence. Fluorescence information collection is performed on the micro-droplets containing the fluorescent substance by the fluorescence detecting component 330, and the fluorescent information of the fluorescent substance-containing micro-droplets is transmitted to the computer as a fluorescent image for quantitative analysis. .
  • the LED light source 341, the collimating mirror 342, the first filter 343, the dichroic mirror 344, the fly-eye lens 345, and the The number of focus lenses 346 is not limited.
  • the excitation light source 340 is obliquely irradiated to the microdroplet container 60 for illuminating the plurality of microdroplets.
  • the oblique light path formed by the excitation light source 340 can effectively reduce the excitation light scattering background.
  • reducing the height of the side wall of the micro-droplet container 60 of the micro-droplet container 60 is advantageous for eliminating the shadow caused by the excitation light from the side, so that the camera 331 can acquire the fluorescence of all the micro-droplets.
  • the information improves the sensitivity of the fluorescent signal detecting device 30.
  • the quantitative analysis device 40 is a computer.
  • a fluorescent information photograph of the plurality of microdroplets can be obtained by the fluorescent signal detecting device 30.
  • the computer is provided with analysis software, such as matlab, microsoft office, origin, and analysis software such as Microsoft Office visual.c++, for performing quantitative analysis on the obtained fluorescence information of the plurality of microdroplets.
  • the controller 50 is connected to the first controller 170, the second controller 210, and the third controller 310, respectively, for controlling the micro-droplet generating device 10,
  • the temperature control device 20, the fluorescence signal detecting device 30, and the quantitative analysis device 40 operate.
  • the microdroplet generating device 10 microdrops the nucleic acid amplification reaction solution to be tested to form a plurality of microdroplets. Then, during the heating of the plurality of micro-droplets by the temperature control device 20, the fluorescence signal detecting device 30 is used to take a real-time measurement of the fluorescence change image of the plurality of micro-droplets. The fluorescence change image of the plurality of microdroplets is analyzed by the quantitative analysis device 40, the Ct value of the plurality of microdroplets is obtained, and the concentration of the initial nucleic acid is determined by the relationship between the Ct value and the initial copy number. Perform quantitative analysis.
  • the digital PCR detector 1 integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, so that the operator can pass the integrated
  • the digital PCR detector 1 realizes an automatic operation and improves the working efficiency of the digital PCR detector 1.
  • the microdroplet generating device 10 microdrops the nucleic acid amplification reaction solution to be tested to form a plurality of microdroplets.
  • the plurality of microdroplets are then subjected to nucleic acid amplification by the temperature control device 20.
  • the fluorescent signal detecting device 30 is used to take a picture of the fluorescence changes of the plurality of micro-droplets in real time.
  • a fluorescence change curve of the plurality of microdroplets is obtained by a fluorescence change picture of the plurality of microdroplets.
  • the Ct value of the plurality of microdroplets can be obtained, and the concentration of the initial DNA is quantitatively analyzed by the relationship between the Ct value and the initial copy number.
  • the Ct value refers to the number of cycles experienced when the fluorescence signal of each microdroplet reaches a set threshold.
  • the micro-droplet generating device 20 generates uniform-sized micro-droplets, and performs nucleic acid amplification reaction on the plurality of micro-droplets through the temperature control device 30, and collects product signals such as fluorescence, ultraviolet absorption, turbidity, and the like. signal. Utilizing the difference in composition between the plurality of amplified and non-amplified microdroplets, the number of droplets obtained by obtaining the target sequence is analyzed, and finally quantitative analysis of the nucleic acid molecule is realized. By monitoring the fluorescence change pictures of the plurality of micro-droplets in real time, the sequencing results are direct, and the problems of false positives and false negatives among the plurality of micro-droplets can be solved.
  • the digital PCR detector integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analysis device 40, so that the operator can pass an integrated digital
  • the PCR detector 1 realizes automatic operation, improves work efficiency, has fast response, good repeatability, high sensitivity, strong specificity and clear results.
  • the microdroplet generating device 10 microdrops the nucleic acid amplification reaction solution to form a plurality of microdroplets.
  • the micro-droplet generating device 10 generates micro-droplets of uniform size.
  • the plurality of microdroplets are then subjected to nucleic acid amplification by the temperature control device 20.
  • the fluorescent signal detecting device 30 is used to capture the fluorescence change image of the plurality of micro-droplets in real time.
  • a fluorescence change curve of the plurality of microdroplets is obtained by a fluorescence change image of the plurality of microdroplets. According to the fluorescence change curve, the Ct value of the plurality of microdroplets can be obtained, and the concentration of the initial nucleic acid is quantitatively analyzed by the relationship between the Ct value and the initial copy number.
  • C represents Cycle
  • t represents threshold
  • the meaning of the Ct value is: the number of cycles experienced when the fluorescent signal in each of the microdroplets reaches a set domain value.
  • the Ct value refers to the number of cycles experienced when the fluorescent signal within each of the microdroplets reaches a set threshold. That is to say, the meaning of the Ct value is the number of cycles experienced by the fluorescence signal of each microdroplet when it reaches the set domain value.
  • the PCR cycle When the PCR cycle reaches the number of cycles in which the Ct value is located, it just enters the true exponential amplification phase (log phase). At this time, the small error has not been amplified, so the reproducibility of the Ct value is excellent, that is, the same nucleic acid template is expanded at different times.
  • the Ct value obtained is increased or increased in different microdroplet containers at the same time, and the obtained Ct value is constant.
  • the fluorescence curve corresponding to the microdroplet is an amplification curve, it is indicated at this time that the microdroplet contains the target gene component.
  • the corresponding fluorescence curve of the microdroplet When the corresponding fluorescence curve of the microdroplet is a straight line, it indicates that the microdroplet does not contain the target gene component.
  • a Ct value can be obtained, and the Ct value of each micro-droplet is obtained when the real-time fluorescence curve is obtained, and the initial cycle number of the fluorescence curve of the slope of the real-time fluorescence curve is fixed. That is the required Ct value.
  • the micro-droplet generating device 10 generates uniform-sized micro-droplets, and performs nucleic acid amplification reaction on the plurality of micro-droplets through the temperature control device 20, and collects product signals such as fluorescence, ultraviolet absorption, turbidity, and the like. signal.
  • the digital PCR detector 1 integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, so that the operator can pass the integrated
  • the digital PCR detector 1 realizes automatic operation, improves work efficiency, has fast response, good repeatability, high sensitivity, strong specificity and clear results.
  • the detection process of the digital PCR detector 1 mainly comprises five steps: preparing a nucleic acid amplification reaction solution to be tested, microdropletization of the nucleic acid amplification reaction solution to be tested, nucleic acid amplification, collection of fluorescence information, and quantitative analysis. Referring to FIG.
  • a method for analyzing a digital PCR detector includes the following steps: S10, preparing a nucleic acid amplification reaction solution to be tested; S20, and amplifying the reaction liquid droplet of the nucleic acid to be tested Forming a plurality of micro-droplets; S30, performing nucleic acid amplification on the plurality of micro-droplets, and acquiring fluorescence information of the plurality of micro-droplets in real time; S40, according to fluorescence of the plurality of micro-droplets Information for quantitative analysis of the plurality of microdroplets.
  • the step S10 comprises: preparing a nucleic acid amplification reaction solution to be detected.
  • the nucleic acid amplification reaction solution contains a nucleic acid template to be detected, a reaction buffer aqueous solution, deoxyribonucleoside triphosphate, a primer, a polymerase, a product labeling substance, and the like.
  • the nucleic acid amplification reaction solution may be a nucleic acid amplification reaction solution (which may be referred to as a DNA amplification reaction solution) using deoxyribonucleic acid (DNA) as a template, or may be a reverse transcription using a ribonucleic acid (RNA) as a template.
  • the nucleic acid amplification reaction solution (which may be referred to as an RNA reverse transcription reaction solution) may also be another nucleic acid amplification reaction solution such as a loop-mediated isothermal amplification (LAMP) reaction solution.
  • LAMP loop-mediated isothermal amplification
  • the DNA amplification reaction solution is characterized by containing dNTPs, buffers, inorganic salt ions, polymerases, primers, DNA templates to be detected, fluorescent dyes or fluorescent probes required for DNA amplification.
  • the fluorescent dye or fluorescent probe in the reaction solution can indicate nucleic acid amplification, and may be a fluorescent dye that binds to DNA such as SYBR Green, or an oligosaccharide nucleotide probe that contains both a fluorescent group and a quenching group. Such as TaqMan fluorescent probes.
  • a kit of reagents and solutions dedicated to digital PCR is prepared to reduce or avoid potential contamination of the template DNA sample by exogenous DNA. All instruments and consumables used should be autoclaved and dried at high temperatures.
  • the nucleic acid amplification reaction solution component to be detected comprises: template DNA to be amplified, specific oligonucleotide primer for amplifying the template, heat-resistant DNA polymerase, and four deoxyribonucleotide substrates of triphosphate , a divalent metal cation Mg2+, a Taqman probe or a fluorescent dye, and a PCR buffer.
  • the nucleic acid amplification reaction solution to be tested when the nucleic acid amplification reaction solution to be tested is prepared, the nucleic acid amplification reaction solution to be tested is labeled with a Taqman probe. In one embodiment, when the nucleic acid amplification reaction solution to be tested is prepared, the nucleic acid amplification reaction solution to be tested is labeled with a SYBR fluorescent dye. In one embodiment, the step S20 microdropping the nucleic acid amplification reaction solution to be used to form a plurality of micro-droplets includes two micro-droplet generation methods: a transiently accelerated micro-droplet generation method and A method of generating a microdroplet in a shifting cycle.
  • the nucleic acid amplification reaction solution to be tested is subjected to a microdropping treatment, and a large number of microdroplets can be obtained for detection by the digital PCR detector 1.
  • the driving liquid is a liquid which is incompatible with the nucleic acid amplification reaction solution to be tested and does not affect each other.
  • the first liquid 190 is the nucleic acid amplification reaction solution to be tested, and the second liquid 699 is an oil phase mixture.
  • a large amount of microdroplets can be prepared by passing the prepared nucleic acid amplification reaction solution through the microdroplet generating device.
  • the plurality of microdroplets are placed in the microdroplet container for facilitating detection of the plurality of microdroplets.
  • a large number of microdroplets are generated in the second liquid by the microdroplet generating device 10, and the plurality of microdroplets can be kept from being fused.
  • the step S30 includes: S310: tiling the plurality of micro-droplets in the micro-droplet container; S320: performing nucleic acid amplification on the plurality of micro-droplets after tiling S330: Perform photo-detection of the plurality of micro-droplets in real time when the plurality of micro-droplets perform nucleic acid amplification.
  • the step S310 includes a microdroplet tiling method.
  • the microdroplet tiling method comprises: S311, providing a microdroplet container 60 having an opening 631, and the microdroplet container 60 contains a second liquid 699; S312, providing a first liquid 190, the first liquid 190 has a density greater than the second liquid 699 and is immiscible with the second liquid 699, and the first liquid 190 generates a plurality of micro-droplets stacked on the The micro-droplet container bottom plate 610; S313, the high-temperature cycle is performed on the plurality of micro-droplets until the plurality of micro-droplets are laid flat on the container bottom plate 610.
  • a plurality of microdroplets are generated in the microdroplet container 60, and the container bottom plate 610 that falls to the microdroplet container 60 is irregularly stacked.
  • the container bottom plate 610 When a large number of microdroplets are dropped onto the container bottom plate 610, multiple layers of microdroplets are formed in the container bottom plate 610.
  • the plurality of micro-droplets prepared by the micro-droplet generating device are concentrated in the middle portion of the micro-droplet container during the downward sedimentation process, and are gathered together, which is not conducive to observation.
  • a plurality of microdroplets are dropped into the microdroplet container 60, and the plurality of microdroplets are stacked on the microdroplet container bottom plate 610, that is, A plurality of microdroplets form a plurality of microdroplets on the microdroplet container bottom plate 610.
  • the microdroplet container 60 containing the plurality of microdroplets is subjected to high and low temperature circulation.
  • the plurality of microdroplets are subjected to high and low temperature cycles a plurality of times until the plurality of microdroplets are laid flat on the microdroplet container bottom plate 610 such that a large number of the microdroplets are tiled in the reaction In unit 612, large-scale parallel observation of massive droplets is facilitated.
  • the high temperature cycle is performed by the temperature control device 20, and the flattening is performed by the principle of thermal expansion and contraction.
  • the temperature of the object increases, the kinetic energy of the molecule increases, and the mean free path of the molecule increases, so it appears as thermal expansion.
  • the temperature of the object decreases, the kinetic energy of the molecule decreases, and the mean free path of the molecule decreases, so it appears as cold shrinkage.
  • the viscosity of the sample droplets becomes lower and the volume shrinks.
  • the higher the temperature the lower the viscosity.
  • the shape of the sample droplet is the softest. At this time, the shape is roughly hexagonal. However, at other temperatures, the shape of the droplet is more variability. Poor, it is not easy to achieve tiling in the droplet container.
  • the step of performing high and low temperature circulation by the temperature control device 20 is as follows: first, heating the plurality of micro droplets to a temperature of 90 ° C to 95 ° C, and heating for 5 min to 10 min; The plurality of microdroplets are cooled to 40° C. to 60° C. and annealed for 30 s to 60 s. Finally, the cells are sequentially circulated a plurality of times, and the temperature is lowered to 0° C. to 10° C., and the plurality of microdroplets are stored.
  • the step of performing the high and low temperature cycle by the temperature control device 20 further comprises: first, heating the plurality of microdroplets, heating the temperature to 95 ° C, and heating for 10 min; A plurality of microfluidics were heated to 95 ° C and heated for 10 min to thermally initiate the enzymes in the plurality of microdroplets. Then, after the plurality of micro-droplets complete the thermal start of the enzyme, the plurality of micro-droplets are denatured for 30 s; secondly, after the plurality of micro-liquid denaturation, the temperature is lowered to 55 ° C, and the annealing is extended for 45 s. The plurality of micro-droplets were photographed and subjected to 45 cycles; finally, after 45 cycles, the temperature was lowered to 4 ° C, and the plurality of micro-liquids were stored for a long time.
  • the nucleic acid amplification reaction solution to be tested generates a plurality of microdroplets through the microdroplet generating device 10 for detection.
  • the plurality of micro-droplets prepared by the micro-droplet generating device 10 are concentratedly collected in the intermediate portion of the micro-droplet container 60 during the downward sedimentation process, and are gathered together, which is disadvantageous for observation. Therefore, in order to more accurately acquire information on the nucleic acid amplification reaction of the plurality of microdroplets, it is necessary to tile the plurality of microdroplets in the microdroplet container.
  • the PCR reaction conditions are temperature, time, and number of cycles.
  • Temperature and time setting Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle.
  • the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C.
  • the primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA.
  • the primer strand is extended along the template by the action of the polymerase.
  • the two-temperature method can be used.
  • the annealing and extension temperatures can be combined into one. Generally, the denaturation at 94 °C is used, and the annealing and extension are performed at about 65 °C. Taq DNase still has high catalytic activity).
  • Denaturation temperature and time The denaturation temperature is low, and the incomplete melting is the most important cause of PCR failure. In general, 93 ° C ⁇ 94 ° Cmin is enough to denature the template DNA, if it is lower than 93 ° C, it needs to extend the time, but the temperature can not be too high, because the high temperature environment has an effect on the activity of the enzyme. If this step does not completely denature the target gene template or PCR product, PCR will fail.
  • Annealing temperature is an important factor affecting PCR specificity. After denaturation, the temperature is rapidly cooled to 40 ° C ⁇ 60 ° C, which can be combined with the template. Since the template DNA is much more complex than the primer, the collisional binding opportunity between the primer and the template is much higher than the collision between the complementary strands of the template.
  • the annealing temperature and time depend on the length of the primer, the base composition and its concentration, and the length of the target sequence. For 20 nucleotides, a primer with a G+C content of about 50%, 55 ° C is preferred as the starting point for selecting the optimum annealing temperature.
  • renaturation temperature can greatly reduce non-specific binding between the primer and the template, increasing the specificity of the PCR reaction.
  • the renaturation time is generally from 30 sec to 60 sec, sufficient to allow complete binding between the primer and the template.
  • Extension temperature and time biological activity of Taq DNA polymerase: 70-80 ° C 150 nucleotide / S / enzyme molecule; 70 ° C 60 nucleotide / S / enzyme molecule; 55 ° C 24 nucleotide / S / enzyme Molecules; above 90 ° C, DNA synthesis is almost impossible.
  • the extension temperature of the PCR reaction is generally selected between 70 and 75 ° C, and the usual temperature is 72 ° C.
  • the excessive extension temperature is not conducive to the combination of the primer and the template.
  • the time of the PCR extension reaction may depend on the length of the fragment to be amplified, and generally a DNA fragment within 1 Kb, and an extension time of 1 min is sufficient.
  • the target sequence of 3 to 4 kb takes 3 min to 4 min; the amplification of 10 Kb needs to be extended to 15 min. Excessive extension times can lead to the appearance of non-specific amplification bands. For the amplification of low concentration templates, the extension time is slightly longer.
  • the step S320 performs nucleic acid amplification on the plurality of microdroplets as follows: First, the microdroplet container 60 is placed on the heating substrate 240 of the temperature control device 20. And then heating the plurality of micro-droplets to a temperature of 95 ° C and heating for 10 min; heating the plurality of micro-liquids to 95 ° C, and heating for 10 min, to The enzyme in the microdroplets is hot-started. Secondly, after the plurality of micro-droplets complete the thermal initiation of the enzyme, the plurality of micro-droplets are denatured for 30 s; again, after the plurality of micro-liquid denaturation, the temperature is lowered to 55 ° C, and the annealing is extended for 45 s, and is performed. 45 cycles; finally, after 45 cycles, the temperature was lowered to 4 ° C, and the plurality of microliquids were stored for a long time.
  • the temperature control device 20 uses the flexible circuit board 220 and the heat conduction enhancement layer 250 to uniformly distribute the temperature of the microdroplet container 60, thereby accelerating the thermal conductivity of the semiconductor refrigerator.
  • a temperature sensor 260 disposed on a surface of the thermally conductive enhancement layer 250 is coupled to the second controller 210 to detect the microdroplets
  • the real-time temperature of the container 60 feeds back temperature information to the second controller 210 to effect control of the plurality of micro-droplet heating temperatures. It is possible to switch quickly in a matter of seconds.
  • the temperature control device 20 can realize instantaneous temperature rise and temperature decrease, and the process of temperature rise and temperature decrease is shortened, thereby achieving high and low temperature cycle, shortening the detection time of the digital PCR detector 1 and improving the detection efficiency.
  • the plurality of microdroplets can be subjected to nucleic acid amplification by the temperature control device 20.
  • Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle.
  • the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C.
  • the primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA. Under the action of the polymerase, the primer strand is extended along the template to amplify the nucleic acid in a suitable temperature range.
  • the micro-droplet container bottom plate 610 is closely attached to the heat-conductive reinforcing layer 250 of the temperature control device 20, and there is no gap between the two, and the heating is uniform, thereby improving the environment.
  • the accuracy of the digital PCR detector 1 is described.
  • the plurality of microdroplets are photodetected by the fluorescent signal detecting device 30 during nucleic acid amplification of the plurality of microdroplets.
  • the plurality of microdroplets are photographed by the fluorescent signal detecting device 30.
  • the excitation light source 340 provides the plurality of micro-droplets with energy for evaporation, atomization or excitation, and is irradiated onto the micro-droplet container 60 at an oblique angle from above the micro-droplet container 60.
  • the periodic detection of the plurality of micro-droplets by the fluorescence signal detecting device 30 is performed, and the photographing is performed in real time.
  • the internal fluorescence of the plurality of microdroplets in the microdroplet container 60 is excited, collected by the upper objective lens 332 through the second filter 333, and enters the camera 331, the camera 331 A fluorescent image of the plurality of microdroplets is acquired.
  • the third controller 310 can synchronously turn on the plurality of different colors of the LED light source 341 and the camera 331.
  • the fluorescent signal detecting device 30 can cause the plurality of micro-droplets to be fluorescently imaged, take a certain number of fluorescent images of the plurality of micro-droplets at a time, and then use image processing technology to perform droplet fluorescence in the image. Automatic identification to obtain fluorescence information of the droplets.
  • each microdroplet in the microdroplet container 60 can obtain 45 fluorescent images for quantitative analysis.
  • the step S330 when the plurality of micro-droplets are subjected to nucleic acid amplification, the steps of performing photo-detection on the plurality of micro-droplets in real time are as follows:
  • the plurality of micro-droplets are heated and heated, the temperature is heated to 95 ° C, and heated for 10 min;
  • the plurality of micro-droplets complete the thermal initiation of the enzyme, the plurality of micro-droplets are denatured for 30 s;
  • the temperature is lowered to 55 ° C, and the annealing is extended for 45 s, and the plurality of pico-liquids are photographed by the fluorescence signal detecting device, and 45 cycles are obtained, and 45 sheets are obtained.
  • a fluorescent image of a microdroplet
  • the optical path of the focusing lens 346 is obliquely irradiated to the plurality of micro-droplets such that the micro-droplets containing the fluorescent substance in the micro-droplet container 60 generate fluorescence. Fluorescence information collection is performed on the micro-droplets containing the fluorescent substance by the fluorescence detecting component 330, and the fluorescent information of the fluorescent substance-containing micro-droplets is transmitted to the computer as a fluorescent image for quantitative analysis. .
  • the fluorescence imaging method is used to capture a certain number of fluorescent images of the micro-droplets at a time, and then the image fluorescence is automatically recognized by image processing techniques to obtain fluorescence information of the droplets. Since the imaging range of the fluorescence imaging detection method is large, the requirement for the detection environment in which the microdroplets are placed is low at the time of detection.
  • the nucleic acid test sample is a sample to be tested containing DNA.
  • the uniform droplets generated by the micro-droplet generating device 10 perform nucleic acid amplification reaction on the plurality of micro-droplets through the temperature control device 20, and collect product signals such as fluorescence, ultraviolet absorption, turbidity and the like. . Utilizing the difference in composition between the plurality of amplified and non-amplified microdroplets, the number of droplets obtained by obtaining the target sequence is analyzed, and finally quantitative analysis of the nucleic acid molecule is realized.
  • the fluorescence change pictures of the plurality of micro-droplets are taken in real time, the Ct values of the plurality of micro-droplets are obtained, and the initial DNA is obtained by the relationship between the Ct value and the initial copy number.
  • the concentration was quantified.
  • the intensity of the fluorescent signal reaches a certain level, which is positive; if the microdroplet with zero DNA content can hardly detect the fluorescent signal, it is regarded as negative.
  • the expected value ⁇ and the variance ⁇ 2 of the Poisson distribution model it is understood that the expected value ⁇ is ⁇ and the variance ⁇ 2 is ⁇ . Therefore, it can be seen that the number of copies of the target DNA molecule contained in each microdroplet in the digital PCR is ⁇ , so that the obtained lambda value enables quantitative detection of the nucleic acid.
  • the concentration c (copy/ ⁇ L) of the nucleic acid amplification reaction solution to be tested is:
  • the real-time fluorescence quantitative PCR does not require an internal standard based on the reproducibility of the Ct value and the linear relationship between the Ct value and the initial DNA concentration.
  • the PCR cycle reaches the number of cycles in which the Ct value is located, it just enters the true exponential amplification phase (log phase).
  • the small error has not been amplified, so the reproducibility of the Ct value is excellent, that is, the same DNA template is expanded at different times.
  • the Ct value obtained is increased or increased in different microdroplet containers at the same time, and the obtained Ct value is constant.
  • the fluorescence curve corresponding to the microdroplet is an amplification curve, it is indicated at this time that the microdroplet contains the target gene component.
  • the corresponding fluorescence curve of the microdroplet is a straight line, it indicates that the microdroplet does not contain the target gene component.
  • a Ct value can be obtained, and the Ct value of each micro-droplet is obtained when the real-time fluorescence curve is obtained, and the initial cycle number of the fluorescence curve of the slope of the real-time fluorescence curve is fixed. That is the required Ct value.
  • a plurality of micro-droplets of uniform size can be generated by the micro-droplet generating device 10.
  • Each of the microdroplets is on the order of microns.
  • the plurality of microdroplets are quantitatively analyzed based on fluorescence information of the plurality of microdroplets.
  • the step S40 includes a digital PCR quantitative detection method.
  • the digital PCR quantitative detection method comprises the following steps:
  • S4110 acquiring a plurality of real-time fluorescence images of all the micro-droplets, and obtaining a real-time fluorescence curve of the micro-droplets for performing nucleic acid amplification according to the plurality of real-time fluorescence images;
  • the false positives and false negatives of the results were solved by the digital PCR quantitative detection method.
  • the high-throughput sample of the sequencing platform enables simultaneous detection of hundreds of samples.
  • different types of fluorescence can be used to detect multiple sites, speed up the detection, and reduce the experimental cost.
  • the digital PCR detector is used to separate the rare detection fragments from a large number of complex backgrounds, which greatly simplifies the operation steps, saves preparation time and detection time, and the results are intuitive and reliable, and can be stably implemented.
  • the detection sensitivity and accuracy are both accurate and quantitative, which improves the sensitivity and accuracy of the detection.
  • the S4110 includes:
  • S4115 Obtain a real-time fluorescence curve of all the microdroplets performing nucleic acid amplification according to the fluorescence curve of each microdroplet for nucleic acid amplification.
  • the step captures a fluorescent image of the plurality of microdroplets and performs image tracking.
  • each micro-droplet in each image needs to be separately positioned to obtain the fluorescence intensity of each micro-droplet.
  • the digital PCR detector it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained according to the previous.
  • NCAST image difference and clustering operations can be performed through the photos taken during each temperature cycle to identify the position of each micro-droplet.
  • the fluorescence intensity of the plurality of microdroplets is obtained.
  • the following method can be used to perform microdroplet tracking.
  • the image tracking step for each microdroplet is as follows:
  • the fluorescence curve for each microdroplet is obtained based on the fluorescence intensity value of each microdroplet during each temperature cycle.
  • the fluorescence intensity values at each particular moment of each microdroplet are summed by summing the fluorescence intensity values for each portion of each microdroplet during each temperature cycle.
  • the fluorescence intensity values at a particular moment of each micro-droplet are in a partial summation manner.
  • the change of the plurality of microdroplets during the entire cycle can be obtained by the fluorescence intensity values of the plurality of microdroplets during each temperature cycle, and the fluorescence curve of each microdroplet is obtained.
  • each microdroplet was subjected to 45 cycles and a total of 45 fluorescent pictures were obtained.
  • the fluorescence curve of each microdroplet was obtained by locating each microdroplet in 45 fluorescent images and acquiring 45 fluorescence intensity values for each microdroplet.
  • the S4120 includes:
  • the S4120 further includes:
  • C represents C Cycle and t represents threshold.
  • the meaning of Ct value is: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set domain value.
  • the Ct value refers to the number of cycles experienced when the fluorescent signal in each reaction tube reaches a set threshold.
  • the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal
  • the Ct value of each of the microdroplets in S4130 is linearly related to the logarithm of the DNA start copy number of each microdroplet.
  • x 0 is the amount of the initial template (DNA)
  • E x is the amplification efficiency
  • N is the amount of the amplification product when the fluorescence amplification signal reaches the threshold intensity.
  • a standard curve can be made using a standard of known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve.
  • x 0 is the starting copy number of the template (DNA).
  • the relationship between the Ct value and the starting DNA concentration is such that there is a linear relationship between the Ct value of each DNA template and the logarithm of the starting copy number of the DNA template. The more the starting copy number, the smaller the Ct value.
  • the S4140 includes:
  • S4143 Select a group distance and a group number according to the maximum value and the minimum value to obtain a frequency distribution of the starting copy number of the nucleic acid.
  • the frequency refers to the number of data falling in different intervals.
  • the sum of the frequencies of the different intervals is equal to the total number of data in this group.
  • the maximum likelihood estimation method when calculating the parameter ⁇ of the Poisson distribution in the S4150, the maximum likelihood estimation method is employed.
  • the initial copy number contained in a single droplet satisfies the Poisson distribution.
  • ⁇ is the average number of copies of the starting DNA contained in the microdroplets.
  • the average starting copy number contained in each droplet is represented by CPD (copies per droplet).
  • a digital PCR quantitative detection method includes the following steps:
  • S4210 Acquire a plurality of real-time fluorescence images of all the micro-droplets, and obtain a real-time fluorescence curve of the micro-droplets for nucleic acid amplification according to the plurality of real-time fluorescence images;
  • S4240 selecting a partial nucleic acid starting copy number according to the starting copy number of the nucleic acid of all the micro-droplets for nucleic acid amplification
  • S4260 Perform a point estimation on the Poisson distribution according to the frequency distribution of the starting copy number of the partial nucleic acid, and obtain a parameter ⁇ of the Poisson distribution.
  • the Poisson distribution is estimated by least squares based on incomplete sampling of the initial DNA concentration of all of the plurality of microdroplets.
  • the Poisson distribution can also be estimated by the Expectation Maximization Algorithm (EM) and the Markov chain Monte Carlo (MCMC) method.
  • EM Expectation Maximization Algorithm
  • MCMC Markov chain Monte Carlo
  • the Markov chain Monte Carlo (MCMC) method is one of the Bayesian methods.
  • the S4260 includes searching for ⁇ within an interval [ ⁇ min , ⁇ max ] such that the frequency-valued error squared sum err of the partial nucleic acid starting copy number is minimal.
  • the number of droplets containing more than 4 copies is small (or negligible).
  • the sample DNA concentration be no greater than 6 CPD.
  • the discrimination of Ct value becomes smaller. It is difficult to distinguish the initial copy number of a droplet from 4 or 5 according to the Ct value, so only x 0 , x 1 , x 2 , x are used. 3 Incomplete sampling to perform point estimation on the Poisson distribution. There are many algorithms for point estimation of Poisson based on incomplete sampling, and an operability algorithm-least squares method is introduced.
  • a given interval [ ⁇ min, ⁇ max], ⁇ in [ ⁇ min, ⁇ max] interval search, and the square calculation error select the appropriate [lambda] such that the minimum squared error.
  • the initial copy number of the DNA contained in each microdroplet is a random variable x
  • the frequency of the initial copy number of the DNA of the partial microdroplet corresponds to n k
  • N is the total number of the plurality of microdroplets.
  • the method for performing point estimation on the Poisson distribution in S4260 further includes a moment estimation method, a sequential statistic method, or a maximum likelihood method.
  • the method of point estimation also includes:
  • the moment estimation method uses the sample moment to estimate the corresponding parameter in the population.
  • the sequential statistic is a method of estimating the overall mathematical expectation of the population using the median of the sample.
  • the advantage of the sequential statistic estimation method is that it is easy to calculate and is not susceptible to individual abnormal data. If a set of sample values is abnormal (such as too small or too large), the abnormal data may be caused by the overall randomness, or it may be caused by external interference (such as staff carelessness and record errors). When the reason belongs to the latter, it is obviously affected to estimate E(x) with the sample mean value, but when estimating E(x) with the median of the sample, it is difficult to change the value of the median due to one (or even a few) abnormal data. , so the estimate is not easily affected.
  • Maximum likelihood method Maximum Likelihood (ML), also known as the most approximate likelihood estimation, also called maximum likelihood estimation, is a theoretical point estimation method.
  • the basic idea of the maximum likelihood method is that when n sets of sample observations are randomly selected from the model population, the most reasonable parameter estimator should make the probability of extracting the n sets of sample observations from the model the largest, rather than the minimum two.
  • the multiply estimation method aims to obtain a parameter estimate that allows the model to best fit the sample data.
  • the digital PCR quantitative detection method can accurately measure the initial DNA concentration of the plurality of microdroplets without depending on a standard curve.
  • the digital PCR detector 1 it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained.
  • the dynamic tracking of the plurality of micro-droplets can be realized by the digital PCR quantitative detection method, and the specific position corresponding to each micro-droplet can be found during the temperature cycling of the plurality of micro-droplets, and the nucleic acid amplification can be realized. Monitoring of the entire process. Therefore, the problem of the presence of a false positive in the plurality of microdroplets can be solved by the digital PCR quantitative detection method. At the same time, true absolute quantification is achieved by processing the plurality of microdroplet fluorescence curves and performing statistical corrections independent of the uniformity assumption.
  • Each fluorescence curve represents a change in the curve of useful information, participates in droplet sample information for real-time monitoring, and eliminates the interaction between adjacent droplets by a set algorithm.
  • the digital PCR quantitative detection method relies on an abstract mathematical model to achieve repeatability, high sensitivity, and a large dynamic range, which can be monitored by using a small number of droplets. Cover more information with a small amount of data.
  • the digital PCR quantitative detection method avoids the error of the previous Poisson distribution probability model, and achieves absolute quantification and is more intuitive. Combining all the data eliminates random errors.
  • the fluorescence curve of the droplet sample is obtained, and the change of the fluorescence brightness of the droplet sample is monitored in real time to remove the false positive; the interaction between adjacent droplets is eliminated, and a more accurate data source is provided for the subsequent quantitative analysis model.
  • the abscissa is the average copy number of copies (CPD) contained in each droplet.
  • the ordinate is the standard deviation (Std Dev, STD) of the average starting copy number contained in each droplet.
  • the average starting copy number contained in each droplet is represented by CPD (copies per droplet). It can be seen that the average starting copy number contained in each droplet obtained using the partial nucleic acid starting copy number is smaller than the standard deviation of the CPD by the standard deviation of the CPD from the average starting copy number obtained by other algorithms. Therefore, the average starting copy number contained in each droplet obtained by the algorithm is more accurate with the value of CPD.
  • the simulation results show that the accuracy of the experimental system of 200 droplets can be better than the traditional single point estimation algorithm (uCount algorithm) when using a digital PCR quantitative detection method.
  • the stability of the Poisson fitting algorithm the accuracy of the available dynamic range is much better than the traditional single point estimation algorithm.
  • the number of droplets required to use the Poisson fitting algorithm is two orders of magnitude lower than the number of droplets required by conventional single point estimation algorithms. Therefore, the detection accuracy of the digital PCR detector 1 can be improved, and the detection range can be expanded, and a plurality of different kinds of nucleic acids can be detected with a small number of droplets, thereby improving the use efficiency of the digital PCR detector 1.
  • the volume of the plurality of uniform micro-droplets generated by the micro-droplet generating device 10 has a special case, when the volume of the micro-droplets is changed, there is a volume unevenness. happening.
  • a plurality of micro-droplets of different volumes can also be generated by the micro-droplet generating device 10 for medical clinical detection.
  • the volume of the reaction unit tends to be highly consistent, which can be regarded as a single volume digital PCR technology.
  • the upper limit of quantitation of a single volume digital PCR depends primarily on the volume and number of reaction units, and the lower limit of detection is related to the total volume of the sample.
  • the resolution and dynamic range of single volume digital PCR technology cannot be independently adjusted.
  • the sample to be continuously diluted is diluted, although the dynamic range can be expanded, but the detection sensitivity cannot be improved.
  • the continuous dilution method increases the amount of reagents and the risk of cross-contamination, and the operation steps are cumbersome.
  • Multivolume digital PCR enables researchers to independently adjust dynamic range and resolution while avoiding the drawbacks of serial dilution.
  • the multi-volume digital PCR micro-droplet container contains a series of different volume of reaction units.
  • the small-volume reaction unit can quantify high-concentration samples, and the large-volume reaction unit uses a sufficient volume to achieve highly sensitive detection.
  • Multiple volume digital PCR does not require a large number of reaction units but can achieve the dynamic range of a single volume digital PCR, so that more sample analysis can be performed in the microdroplet container while the reagent consumption is effectively reduced.
  • the digital PCR detector it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained.
  • the sample solution is a nucleic acid amplification reaction solution and is applied to a quantitative analysis method of a digital PCR detector.
  • S4310 Obtain all micro-droplet volumes v 1 , v 2 , . . . v m , the volume is v 1 , v 2 , . . . v m sequentially corresponding to the number of micro-droplets n 1 , n 2 ,... , n m , and the volume of v 1 , v 2 , ... v m sequentially corresponding to the number of negative microdroplets after amplification of the microdroplet nucleic acid b 1 , b 2 , ..., b m ;
  • S4320 constructing according to relevant parameters v 1 , v 2 , . . . v m , n 1 , n 2 , . . . , n m , b 1 , b 2 , . . . , b m after amplification of all micro-droplet nucleic acids a combined binomial distribution function f(c) of the nucleic acid amplification reaction solution concentration c;
  • S4330 Determine, according to the joint binomial distribution function f(c), a value of c when the joint binomial distribution function f(c) takes an extreme value;
  • S4350 Obtain a standard deviation and a confidence interval of the concentration c of the nucleic acid amplification reaction solution according to the standard deviation of ln(c) and the confidence interval.
  • the intensity of the fluorescent signal reaches a certain level, which is positive; if the microdroplet with zero DNA content can hardly detect the fluorescent signal, it is regarded as negative.
  • the expected value ⁇ and the variance ⁇ 2 of the Poisson distribution model it is understood that the expected value ⁇ is ⁇ and the variance ⁇ 2 is ⁇ . Therefore, it can be seen that the number of copies of the target DNA molecule contained in each microdroplet in the digital PCR is ⁇ , so that the obtained lambda value enables quantitative detection of the nucleic acid.
  • the concentration c (copy/ ⁇ L) of the nucleic acid amplification reaction solution to be tested is:
  • the real-time fluorescence quantitative PCR does not require an internal standard based on the reproducibility of the Ct value and the linear relationship between the Ct value and the initial DNA concentration.
  • the PCR cycle reaches the number of cycles in which the Ct value is located, it just enters the true exponential amplification phase (log phase).
  • the small error has not been amplified, so the reproducibility of the Ct value is excellent, that is, the same DNA template is expanded at different times.
  • the Ct value obtained is increased or increased in different microdroplet containers at the same time, and the obtained Ct value is constant.
  • the fluorescence curve corresponding to the microdroplet is an amplification curve, it is indicated at this time that the microdroplet contains the target gene component.
  • the corresponding fluorescence curve of the microdroplet is a straight line, it indicates that the microdroplet does not contain the target gene component.
  • a Ct value can be obtained, and the Ct value of each micro-droplet is obtained when the real-time fluorescence curve is obtained, and the initial cycle number of the fluorescence curve of the slope of the real-time fluorescence curve is fixed. That is the required Ct value.
  • the S4310 includes:
  • S4311 microdropping a sample solution containing a target nucleic acid to obtain a plurality of microdroplets of different volumes v 1 , v 2 , . . . v m , the volume of the microdroplets being v 1 , v 2 ,... v m corresponds to the number of micro-droplets n 1 , n 2 , ..., n m ;
  • S4313 performing nucleic acid amplification on all the micro droplets, and taking a photo detection to obtain a fluorescence image of all the micro droplets;
  • S4315 The fluorescence images of all the micro-droplets to obtain the volume of all micro-droplets of v 1, v 2, ... v m after amplification the nucleic acid sequence corresponding to the number of negative droplet b 1, b 2 ,...,b m .
  • the S4310 further includes:
  • S4312 The sample solution containing the target nucleic acid is microdropped to form a plurality of microdroplets
  • S4314 performing nucleic acid amplification on the plurality of microdroplets, and taking a photo detection to obtain a fluorescence image after amplification of all microdroplet nucleic acids;
  • S4316 Obtain a volume after amplification of all the microdroplet nucleic acids according to the fluorescence image, wherein the volumes are v 1 , v 2 , . . . v m , respectively, and the volumes are v 1 , v 2 , respectively.
  • ...v m sequentially corresponding to the number of microdroplets after amplification of the nucleic acid n 1 , n 2 , ..., n m , and the nucleic acid corresponding to the volume v 1 , v 2 , ... v m
  • the number of negative microdroplets increased b 1 , b 2 ,..., b m .
  • the step of acquiring a fluorescent image of the plurality of microdroplets and performing image tracking In obtaining the real-time fluorescence curve of each micro-droplet, each micro-droplet in each image needs to be separately positioned to obtain the fluorescence intensity of each micro-droplet. In the digital PCR detector, it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained according to the previous.
  • NCAST image difference and clustering operations can be performed through the photos taken during each temperature cycle to identify the position of each micro-droplet.
  • the fluorescence intensity of the plurality of microdroplets is obtained.
  • the fluorescence curve for each microdroplet is obtained based on the fluorescence intensity value of each microdroplet during each temperature cycle.
  • Each fluorescence curve represents a change in the curve of useful information, participates in droplet sample information for real-time monitoring, and eliminates the interaction between adjacent droplets by a set algorithm.
  • the fluorescence intensity values at each particular moment of each microdroplet are summed by summing the fluorescence intensity values for each portion of each microdroplet during each temperature cycle.
  • the fluorescence intensity values at a particular moment of each microdroplet are in a partial summation manner.
  • the change of the plurality of microdroplets during the entire cycle can be obtained by the fluorescence intensity values of the plurality of microdroplets during each temperature cycle, and the fluorescence curve of each microdroplet is obtained.
  • each microdroplet was subjected to 45 cycles and a total of 45 fluorescent pictures were obtained.
  • the fluorescence curve of each microdroplet was obtained by locating each microdroplet in 45 fluorescent images and acquiring 45 fluorescence intensity values for each microdroplet.
  • the S4320 constructs a joint binomial distribution function f(c) for the concentration c of the nucleic acid amplification reaction solution to be tested as:
  • the volume of each microdroplet is v
  • the nucleic acid concentration of the nucleic acid amplification reaction solution to be tested is c
  • the average DNA number per microdroplet is vc, assuming each micro If the number of molecules contained in the droplet is k, the probability distribution P of k can be derived from the Poisson distribution probability model:
  • the probability of negative microdroplets can be estimated by the total number of microdroplets n and negative microdroplets b.
  • the S4330 includes:
  • the function value takes a maximum or minimum value. Since the binomial distribution has only one maximum value, the solution that makes the function's derivative function zero is the most likely concentration value. By taking the joint binomial distribution function f(c) to a maximum value, the maximum possible number of corresponding cs is obtained.
  • the joint binomial distribution function F( ⁇ ) for ln(c) in step S4340 is:
  • the P function is more symmetrical with respect to ln(c) than c, so the standard deviation ⁇ of ln(c) is more statistically significant. By strengthening the constraint with positive concentration, it has better accuracy in the analysis of low concentration samples. In order to simplify the calculation, when calculating the corresponding standard deviation ⁇ of ln(c), it is necessary to replace the corresponding variable.
  • the S4340 includes:
  • S4342 Find a first derivative of the function L( ⁇ ), and set a first derivative of the function L( ⁇ ) to 0;
  • the corresponding multiplication relationship can be made into an independent addition relationship, making the corresponding derivative function easier to handle.
  • the standard deviation ⁇ is obtained from the Fisher information amount I( ⁇ ) of ln(c) in the S4343.
  • the Fisher information amount I( ⁇ ) of ln(c) in the S4343 is:
  • the corresponding standard deviation ⁇ and the confidence interval of ln(c) are:
  • the nucleic acid concentration of the nucleic acid amplification reaction solution to be tested can be obtained from the corresponding standard deviation ⁇ of ln(c) and the confidence interval.
  • the corresponding value can be obtained by the standard normal distribution table, so that the confidence interval of ln(c) can be known, and then the nucleic acid concentration of the nucleic acid amplification reaction solution can be known as c, so that the nucleic acid to be detected can be obtained.
  • the number of initial copies of DNA contained in the reaction solution can be obtained from the corresponding standard deviation ⁇ of ln(c) and the confidence interval.
  • the corresponding value can be obtained by the standard normal distribution table, so that the confidence interval of ln(c) can be known, and then the nucleic acid concentration of the nucleic acid amplification reaction solution can be known as c, so that the nucleic acid to be detected can be obtained.
  • the number of initial copies of DNA contained in the reaction solution can be obtained from the corresponding standard deviation ⁇ of ln(
  • the confidence interval refers to the estimated interval of the overall parameters constructed by the sample statistic.
  • the Confidence Interval (CI) of a probability sample is an interval estimate for a population parameter of this sample.
  • the confidence interval shows the extent to which the true value of this parameter has a certain probability of falling around the measurement.
  • the confidence interval gives the degree of confidence of the measured value of the measured parameter, ie the "one probability" required previously.
  • the quantitative results of digital PCR usually need to be expressed in conjunction with confidence intervals and confidence levels.
  • the confidence interval shows the extent to which the true concentration of the sample falls within a range of the measurement result ⁇ with a certain probability. This probability is called the confidence level. Both ends of the confidence interval are called confidence limits.
  • different volume digital PCR can achieve 5 orders of magnitude detection dynamic range with less than 200 microdroplets, which is comparable to single-volume digital PCR with 12,000 microdroplets, saving instrumentation The cost of consumables is reduced.
  • the high-throughput sample of the sequencing platform enables simultaneous detection of hundreds of samples.
  • different types of fluorescence can be used to detect multiple sites, speed up the detection, and reduce the experimental cost.
  • the digital PCR detector is used to separate the rare detection fragments from a large number of complex backgrounds, which greatly simplifies the operation steps, saves preparation time and detection time, and the results are intuitive and reliable, and can be stably implemented.
  • the detection sensitivity and accuracy are both accurate and quantitative, which improves the sensitivity and accuracy of the detection.
  • the different volume digital PCR quantitative analysis method can accurately determine the initial DNA concentration of the plurality of microdroplets independently of the standard curve.
  • the digital PCR detector 1 it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained.
  • the dynamic tracking of the plurality of micro-droplets can be realized by the digital PCR quantitative detection method, and the specific position corresponding to each micro-droplet can be found during the temperature cycling of the plurality of micro-droplets, and the nucleic acid amplification can be realized. Monitoring of the entire process. Therefore, the problem of the presence of a false positive in the plurality of microdroplets can be solved by the digital PCR quantitative detection method. At the same time, true absolute quantification is achieved by processing the plurality of microdroplet fluorescence curves and performing statistical corrections independent of the uniformity assumption.
  • the digital PCR quantitative detection method relies on an abstract mathematical model to achieve repeatability, high sensitivity, and a large dynamic range, which can be monitored by using a small number of droplets. Cover more information with a small amount of data.
  • the digital PCR quantitative detection method avoids the error of the previous Poisson distribution probability model, and achieves absolute quantification and is more intuitive. Combining all the data eliminates random errors.
  • the fluorescence curve of the droplet sample is obtained, and the change of the fluorescence brightness of the droplet sample is monitored in real time to remove the false positive; the interaction between adjacent droplets is eliminated, and a more accurate data source is provided for the subsequent quantitative analysis model.
  • the detection DNA is: human cytomegalovirus DNA.
  • the real-time fluorescent quantitative PCR detection method uses a Taqman fluorescent probe.
  • a human cytomegalovirus nucleic acid quantitative detection kit is obtained, and the detection kit comprises a human cytomegalovirus DNA real-time fluorescence quantitative detection primer and a probe thereof.
  • the reagent configuration ratio of the sample to be tested is: 1 ul sample (2 ⁇ 10 ⁇ 6copies/mL added 2 ul sample), 1 ul DNA polymerase, 20 ul Buffer, and a total of 22 ul.
  • sample concentrations of 2 ⁇ 10 ⁇ 6copies/mL, 10 ⁇ 6copies/mL, 10 ⁇ 5copies/mL, 10 ⁇ 4copies/mL, and 0.5 ⁇ 10 ⁇ 4copies/mL were respectively obtained by the present application.
  • a digital PCR detector, a QX200 digital PCR detector and a qPCR digital PCR detector are used to detect, and the detection values of the initial copy number of the five nucleic acid amplification reaction solutions of each instrument in Table 1 are correlated with the true values.
  • the correlation coefficient R is a statistical indicator used to reflect the closeness of the correlation between variables, and is used to measure the linear relationship between the two variables. It can be seen from Table 1 that the correlation coefficient between the detection value of the initial copy number of the five nucleic acid amplification reaction liquids detected by a digital PCR detector provided by the present application is the largest, and is closest to 1. Therefore, the correlation coefficient between the detected value of the initial copy number of the five nucleic acid amplification reaction liquids detected by a digital PCR detector provided by the present application is the largest, and is closest to 1. Therefore, the digital PCR detector 1 provided by the present application has higher detection accuracy and higher accuracy.
  • the digital PCR detector 1 integrates the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, and causes the operator to pass through the controller 50.
  • the integrated digital PCR detector 1 can realize automatic operation, improve work efficiency, fast response, good repeatability, high sensitivity, specificity and clear results.
  • the present application provides a digital PCR detection method, including:
  • a saturated fluorescent dye can be used to classify different types of variations, with high resolution and sensitivity, and the cost of the digital PCR detector is reduced.
  • the microdroplet array can be completed on a same highly integrated digital PCR detector to complete a polymerase chain reaction (PCR), and in the microfluid
  • PCR polymerase chain reaction
  • the PCR product was subjected to melting curve analysis after PCR amplification of the droplet array. A fluorescence curve of each of the microdroplets is obtained during the polymerase chain reaction of the microdroplet array.
  • a high and low temperature cycle is performed to obtain a melting curve of each of the microdroplets during the cycle.
  • the fluorescence curve of the microdroplet array and the melting curve of the microdroplet array can be obtained, and the real-time monitoring of the whole process of PCR amplification and the melting curve analysis of the PCR product can be completely realized. Trace connection.
  • the qualitative and quantitative analysis of the microdroplet array is realized by the fluorescence curve and the melting curve of the microdroplet array, and the detection of the digital PCR is completed more comprehensively, conveniently and efficiently.
  • the nucleic acid amplification reaction solution contains the nucleic acid template to be detected, the reaction buffer solution, the deoxyribonucleoside triphosphate, the primer, the polymerase, the product labeling substance, and the like in the step S10.
  • the thermostable DNA polymerase may be FastStart Taq DNA polymerase, Ex Taq, Z-Taq, AccuPrime Taq DNA polymerase, HS Taq DNA polymerase or the like.
  • the nucleic acid amplification reaction solution may be a nucleic acid amplification reaction solution (which may be referred to as a DNA amplification reaction solution) using deoxyribonucleic acid (DNA) as a template, or may be a complementary deoxyribonucleic acid (cDNA) or ribonucleic acid.
  • the (RNA) is a template of a reverse transcription nucleic acid amplification reaction solution (which may be referred to as an RNA reverse transcription reaction solution), and may be another nucleic acid amplification reaction solution such as a loop-mediated isothermal amplification (LAMP) reaction solution.
  • LAMP loop-mediated isothermal amplification
  • the DNA amplification reaction solution is characterized by containing dNTPs, buffers, inorganic salt ions, polymerases, primers, DNA templates to be detected, and dyes required for DNA amplification.
  • the dye in the reaction solution can indicate nucleic acid amplification, and can be a fluorescent dye that binds to DNA such as SYBR Green.
  • a kit of reagents and solutions dedicated to digital PCR is prepared to reduce or avoid potential contamination of the template DNA sample by exogenous DNA. All instruments and consumables used should be autoclaved and dried at high temperatures.
  • the nucleic acid amplification reaction solution to be tested is labeled with a SYBR Green fluorescent dye.
  • SYBR Green fluorescent dyes are not selective for templates and are inexpensive due to their ability to bind to all double-stranded DNA, they are suitable for the detection of a variety of products.
  • SYBR Green is generally used as a fluorescent dye. Because SYBR Green dye is a non-specific dye, as long as there is amplification, the dye binds to double-stranded DNA and the fluorescence is greatly enhanced.
  • the melting curve is formed by plotting the change in fluorescence signal versus temperature to observe whether the amplified fluorescent fragment is the target gene to be detected during digital PCR detection. If the melting curve is only a single peak, the peak position is the annealing temperature, and the narrow peak, then the product is a specific product of PCR amplification. If the peak position is wrong or broad, it may be that the product is not specific or not. Design the corresponding product. If there is a small peak before the peak of the melting curve, it may be a primer dimer, and it is necessary to consider redesigning the primer.
  • the analysis of the dissolution curve of a large number of repeated micro-droplet arrays can automatically automate the nucleic acid through a dense curve analysis matching program. Sequence typing, etc.
  • the nucleic acid amplification reaction solution to be tested is microdropped in the step S20 to form a microdroplet array.
  • the array of microdroplets includes a plurality of microdroplets.
  • a digital PCR detector 1 includes a microdroplet generating device 10, a temperature controlling device 20, a fluorescent signal detecting device 30, a quantitative analyzing device 40, and a controller 50.
  • the microdroplet generating device 10 is configured to microdroplet the nucleic acid amplification reaction solution to form the microdroplet array, thereby forming a microdroplet array in the microdroplet container, and simultaneously detecting a plurality of target sequences.
  • the temperature control device 20 is configured to perform temperature cycling to achieve nucleic acid amplification.
  • the fluorescent signal detecting device 30 is disposed opposite to the temperature controlling device 20 for performing signal acquisition on the microdroplet array after nucleic acid amplification.
  • the quantitative analysis device 40 and the fluorescent signal detecting device 30 are connected by a data line for realizing the transmission of the micro-droplet array fluorescence information for quantitative analysis.
  • the controller 50 is connected to the micro-droplet generating device 10, the temperature control device 20, the fluorescent signal detecting device 30, and the quantitative analyzing device 40, respectively, for controlling the micro-droplet generating device 10, the temperature The control device 20, the fluorescence signal detecting device 30, and the quantitative analysis device 40.
  • the microdroplet generating device 10 may microdrop the nucleic acid amplification reaction solution to form the microdroplet array.
  • the array of microdroplets includes a plurality of large numbers of microdroplets.
  • the temperature control device 20 can perform nucleic acid amplification on the microdroplet array.
  • the fluorescent signal detecting device 30 acquires a fluorescence change image of the microdroplet array in real time.
  • the temperature control device 20 performs a nucleic acid amplification reaction on the microdroplet array, and collects a product signal of the microdroplet array after the nucleic acid amplification reaction by the fluorescence signal detecting device 30, such as fluorescence and ultraviolet absorption. , turbidity and other signals.
  • the number of droplets amplified by the target sequence is analyzed, and finally qualitative and quantitative analysis of the nucleic acid molecule is achieved.
  • the detection results are straightforward and can solve the problem of false positives and false negatives in the microdroplet array.
  • the microdroplet array is generated in the microdroplet container by the microdroplet generating device 10, and falls to the bottom of the microdroplet container, irregularly stacked together.
  • the micro-droplet array prepared by the micro-droplet generating device 10 is concentrated in the middle portion of the micro-droplet container during the downward sedimentation process, and is gathered together, in which case the micro-droplet array needs to be A plurality of the array of microdroplets are tiled at the bottom of the microdroplet container prior to signal acquisition.
  • high temperature is performed by the temperature control device 20, and a plurality of the micro-droplet arrays are tiled at the bottom of the micro-droplet container .
  • the array of microdroplets is warmed up.
  • the microdroplet array is cooled.
  • the array of microdroplets is cycled at high and low temperatures until the array of microdroplets is laid flat on the bottom plate of the microdroplet container.
  • the microdroplet array is tiled in a microdroplet container, and the tiled microdroplet array is subjected to PCR amplification and photodetection.
  • a plurality of micro-droplets of uniform size can be generated by the micro-droplet generating device 10.
  • Each of the microdroplets is on the order of microns.
  • a plurality of the microdroplets are quantitatively analyzed based on fluorescence information of the plurality of microdroplets.
  • step S30 includes:
  • PCR consists of three basic reaction steps: denaturation-annealing (refolding)-extension.
  • the denaturation of the template DNA means that the template DNA is double-stranded or double-stranded DNA formed by PCR amplification after being heated to 90 ° C to 95 ° C for a certain period of time, so that it becomes a single strand so that it binds to the primer.
  • Annealing (refolding) of the template DNA and the primer means that the template DNA is denatured into a single strand by heating, and the temperature is lowered to 50 to 60 ° C, and the primer is paired with the complementary sequence of the single strand of the template DNA.
  • the extension of the primer refers to the DNA template--primer conjugate under the action of DNA polymerase, at 70-75 ° C, using dNTP as the reaction material, the target sequence as a template, according to the principle of base pairing and semi-reserved replication, a new synthesis
  • the semi-reserved replication chain which is complementary to the template DNA strand, repeats the cyclic denaturation-annealing-extension process to obtain more "semi-reserved replication strands", and this new chain can be used as a template for the next cycle. It takes 2 to 4 minutes to complete each cycle, and the amplification of the gene to be amplified can be amplified several million times in 2 to 3 hours.
  • the annealing temperature is a more important factor affecting the specificity of PCR. After the denaturation, the temperature is rapidly cooled to 40 ° C ⁇ 60 ° C, which can be combined with the template. Since the template DNA is much more complex than the primer, the collisional binding opportunity between the primer and the template is much higher than the collision between the complementary strands of the template.
  • the annealing temperature and time depend on the length of the primer, the base composition and its concentration, and the length of the target sequence.
  • the microdroplet array is subjected to PCR by repeating the cycle of the denaturation, annealing and extension steps by about 30 to 50 times in the presence of the primer, the DNA sample to be detected, and the thermostable DNA polymerase.
  • the number of cycles is generally set to 30 to 50 times of the three-step process cycle of denaturation, annealing and extension.
  • the temperature parameter is generally set to 40 ° C ⁇ 95 ° C, the time parameter is determined according to each specific process.
  • the step S320 includes:
  • the step S321 includes:
  • the microdroplet array is heated to 95 ° C and heated for 4 min to thermally initiate the enzyme in the microdroplet array, after the microdroplet array completes the thermal initiation of the enzyme,
  • the droplet array is denatured for 1 min;
  • the temperature is lowered to 55 ° C, and the primer is combined with the DNA template to form a local double strand, and annealed (refolded) for 1 min.
  • the microfluid is detected by the fluorescent signal detecting device 30. Taking an image of the array to obtain a fluorescent image of the microdroplet array in the first cycle;
  • microdroplet array was heated to 70 ° C, extended for 7 min;
  • the cycle was repeated 45 times in accordance with the above-described denaturation-annealing (annealing)-extension three steps, and after 45 cycles, the temperature was lowered to 4 ° C, and the plurality of microliquids were stored.
  • the micro-droplet array is heated and cooled by the temperature control device 20, the number of cycles is set to 45 times, and thus each micro-droplet can acquire 45 fluorescences during 45 cycles. image.
  • Each of the microdroplets was subjected to 45 cycles, and a total of 45 fluorescence images were obtained.
  • a fluorescence curve for each of the microdroplets is obtained by locating each of the 45 microfluids and acquiring 45 fluorescence intensity values for each of the microdroplets.
  • a fluorescence intensity value of each of the microdroplets subjected to PCR amplification is obtained according to each of the fluorescence images. Then, a fluorescence curve of each microdroplet subjected to PCR amplification is obtained based on the fluorescence intensity values of each of the PCR-amplified microdroplets. Finally, according to the fluorescence curve of each of the micro-droplets subjected to PCR amplification, a fluorescence curve of all the micro-droplets subjected to PCR amplification, that is, a fluorescence curve of the micro-droplet array is obtained.
  • a fluorescent image of the array of microdroplets is acquired and image tracking is performed.
  • each of the microdroplets in each image needs to be separately positioned to obtain the fluorescence intensity of each of the microdroplets.
  • the digital PCR detector it is calibrated in the imaging system, what is the actual ratio of each pixel of the fluorescent image. According to the fluorescence image, how many pixels corresponding to the diameter of the micro-droplet are extracted, thereby obtaining how many micrometers the diameter corresponds to, and thus the diameter of the micro-droplet can be obtained.
  • NCAST image difference and clustering operations can also be performed by images acquired during each temperature cycle to identify each of the microdroplets. The position, in turn, the fluorescence intensity of the array of microdroplets.
  • the following method can be used for microdroplet tracking.
  • the image tracking step for each of the microdroplets is as follows:
  • the fluorescence curve of each of the microdroplets is obtained based on the fluorescence intensity value of each of the microdroplets during each temperature cycle.
  • the fluorescence intensity values at each particular moment of each of the microdroplets are summed by summing the fluorescence intensity values for each of the portions of the microdroplets during each temperature cycle.
  • the fluorescence intensity values at a particular moment of each of the microdroplets are in a partial summation manner.
  • the change of the microdroplet array during the entire cycle can be obtained by the fluorescence intensity value of the microdroplet array during each temperature cycle, and the fluorescence curve of each of the microdroplets is obtained.
  • the step S330 includes:
  • the micro-droplet array cooled down to 40 degrees Celsius is heated at a specific temperature interval to obtain a fluorescence image of the micro-droplet array corresponding to the temperature interval;
  • S333 Acquire, according to the fluorescence image of the micro-droplet array corresponding to the temperature interval, fluorescence information of each of the micro-droplets corresponding to the temperature interval;
  • a melting curve is generated by gradually increasing the temperature while monitoring the fluorescence signal of each step. Since different DNAs have different temperature melting lines, as the double-stranded DNA denatures in the reaction, the fluorescent dye returns to a free state, causing the fluorescent signal to decrease, and the fluorescence signal is changed to map with temperature.
  • a melting curve is required, because the melting curve is due to the poor specificity of the dye method, so whether the amplification product is the target product is examined by the melting curve.
  • There is a characteristic peak at the melting temperature and this characteristic peak can be used to separate specific products from other products such as primer dimer regions or non-specific products.
  • the PCR product is subjected to melting curve analysis after PCR amplification, so that the PCR amplification is seamlessly linked to the melting curve analysis of the PCR product.
  • the temperature is lowered to below 40 degrees Celsius by the temperature control device 20, and the temperature is sequentially raised to 95 ° C at the temperature interval of 0.1 ° C, and the fluorescence signal detecting device 30 is passed through the interval of 0.1 ° C. Photographs were taken once until the temperature was raised to 95 °C. Then, after the photographing is completed, the microdroplet array is cooled to 4 ° C, and the microdroplet array is stored.
  • the method of processing the fluorescent image when acquiring the fluorescence curve of each of the microdroplets is the same as the method of processing the fluorescent image when obtaining the melting curve of each of the microdroplets.
  • the fluorescence images obtained by collecting at intervals of 0.1 ° C are obtained, the fluorescence intensity corresponding to each of the micro droplets is obtained, a curve about temperature and fluorescence intensity is plotted, and a peak pattern containing peaks is obtained by one differential. .
  • the Dissociation curve is a curve showing the degree of degradation of the double helix structure of DNA as the temperature increases. Melting curve analysis can be used to determine different reaction products, including non-specific products.
  • the temperature at which the total DNA double helix is degraded by half is called the melting temperature (Tm), and the DNA of different sequences has different Tm values. That is to say, the melting curve of a DNA is a DNA fingerprint corresponding to a specific DNA. According to the melting curve, the temperature at which the peak is located represents the Tm value (melting point temperature) of the double-stranded DNA molecule.
  • the genotype can be judged based on the Tm value of the amplified product.
  • the Tm of a DNA fragment is dependent on its length, G+C composition, sequence, strand complementarity, concentration, and buffer components such as salts, dyes, and PCR enhancers.
  • Each melting curve represents a single condition of the product in each of the microdroplets. If a melting curve is a single peak and is within a reasonable temperature range (typically 80-90 ° C), it is considered normal if the melting curve is Bimodal may have non-specific amplification. From this, it can be judged whether the product is the gene of interest and whether it is single.
  • the internal reference has a line of internal parameters, and the same melting curve does not show peaks of two genes.
  • Single nucleotide polymorphisms and scan mutations can be detected for each high resolution melting curve analysis of the microdroplet array.
  • the micro-droplet array is image acquired by the fluorescent signal detecting device 30 during digital PCR detection.
  • Image acquisition is performed on the microdroplet array by the fluorescent signal detecting device 30.
  • the microdroplet container is irradiated at an oblique angle from above the microdroplet container.
  • the fluorescent signal detecting device 30 is used to perform periodic two-dimensional scanning on the micro-droplet array, and image acquisition is performed in real time.
  • the internal fluorescence of the array of microdroplets within the microdroplet container is excited, collected by the objective lens of the fluorescent signal detecting device 30, into the camera, and the camera captures a fluorescent image of the array of microdroplets.
  • the fluorescent signal detecting device 30 can cause the micro-droplet array to be fluorescently imaged, take a certain number of fluorescent images of the micro-droplets at a time, and then automatically recognize the droplet fluorescence in the image by using image processing technology. Thereby the fluorescence information of the droplets is obtained.
  • Fluorescence information collection is performed on the micro-droplet array containing the fluorescent substance by the fluorescence detecting component of the fluorescent signal detecting device, and the detected fluorescent information is transmitted to a computer in the form of a fluorescent image for quantitative analysis.
  • Fluorescence imaging detection method is used to capture a certain number of fluorescent images of the micro-droplets at a time, and then image fluorescence techniques are used to automatically identify droplet fluorescence in the image, thereby obtaining fluorescence information of the droplets. Since the imaging range of the fluorescence imaging detection method is large, the requirement for the detection environment in which the microdroplet array is placed is low at the time of detection.
  • step S40 includes:
  • S420 Acquire nucleic acid information of the microdroplet array according to a melting curve of the microdroplet array.
  • the step S410 includes:
  • the PCR cycle just enters the true exponential amplification period (log phase) when the number of cycles in which the Ct value is reached, and the micro error has not been amplified yet, so the reproducibility of the Ct value is excellent. That is, the same DNA template is amplified at different times or amplified in different microdroplet containers at the same time, and the obtained Ct value is constant.
  • the fluorescence curve corresponding to the microdroplet is an amplification curve, it is indicated at this time that the microdroplet contains the target gene component.
  • the corresponding fluorescence curve of the microdroplet is a straight line, it indicates that the microdroplet does not contain the target gene component.
  • a Ct value can be obtained, and the Ct value of each of the micro-droplets is obtained when the fluorescence curve is obtained, and the initial cycle number of the fluorescence curve of the fluorescence curve is fixed That is the required Ct value.
  • the fluorescence curve of each of the micro-droplets subjected to PCR amplification is derivatized, and the slope of the fluorescence curve of each micro-droplet subjected to PCR amplification is obtained.
  • a value in which the slope of the fluorescence curve of each of the PCR-amplified microdroplets is fixed is obtained.
  • the corresponding number of initial cycles is obtained, and the number of the starting cycles is the Ct value of each microdroplet subjected to PCR amplification.
  • the Ct values of all the PCR-amplified microdroplets are obtained.
  • a defect value of a fluorescence domain value of each of the PCR-amplified microdroplets is obtained according to a fluorescence curve of each of the PCR-amplified microdroplets.
  • the corresponding number of cycles is obtained, which is the Ct value of each microdroplet subjected to PCR amplification.
  • the Ct values of all of the PCR-amplified microdroplets are obtained based on the Ct values of each of the PCR-amplified microdroplets.
  • C represents C Cycle and t represents threshold.
  • the meaning of Ct value is: the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set domain value.
  • the Ct value refers to the number of cycles experienced when the fluorescent signal in each reaction tube reaches a set threshold.
  • the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal
  • step S412 clustering is performed according to the Ct value of the fluorescence curve of each of the micro-droplets, and sequentially sorted from large to small, obtaining x 2 , . . . , x n categories.
  • the dark droplets in the microdroplet array correspond to a category of x 1
  • the droplets in the microdroplet array that do not contain the initial copy number of the nucleic acid correspond to a category of x 1 . Since the Ct value is smaller as the starting copy number of the nucleic acid is larger, the Ct value corresponding to the dark droplet (negative droplet) at this time is infinite, that is, the Ct value corresponding to the x 1 category is infinite.
  • the x 2 class corresponds to a nucleic acid starting copy number of 1
  • the x 3 class corresponding nucleic acid starting copy number is 2
  • the x 4 class corresponding nucleic acid starting copy number is 3
  • the starting copy number of the nucleic acid corresponding to the x 5 class is 4 or the like.
  • step S415 when the number y 1 of the micro-droplets of the x 1 category is greater than or equal to the feature value m, a Poisson distribution fitting is performed according to the frequency distribution, The parameter ⁇ of the Poisson distribution is obtained to obtain the starting copy number of the nucleic acid of the microdroplet array.
  • characteristic value m ranges from 0.5% to 10% of the total number of the microdroplets in the microdroplet array.
  • the characteristic value m can be 5% of the total number of microdroplets.
  • the number y 1 of the micro-droplets of the x 1 class is greater than or equal to the feature value m
  • the number y 1 of dark droplets in the micro-droplet array is greater than or equal to the feature value m.
  • the number of dark droplets in the microdroplet array plays a certain role in calculating the initial copy number of the nucleic acid of the microdroplet array as a whole, so that the number of the microdroplets y 1 , y 2
  • the ..., y n frequency distribution is fitted to the Poisson distribution to obtain the parameter ⁇ of the corresponding Poisson distribution.
  • the expected value ⁇ and the variance ⁇ 2 of the Poisson distribution model it is understood that the expected value ⁇ is ⁇ and the variance ⁇ 2 is ⁇ . Therefore, it can be seen that the number of copies of the target DNA molecule contained in each microdroplet in the digital PCR is ⁇ , so that the obtained lambda value enables quantitative detection of the nucleic acid.
  • the initial copy number contained in a single droplet satisfies the Poisson distribution.
  • ⁇ is the average number of copies of the starting DNA contained in the microdroplets.
  • the average starting copy number contained in each droplet is represented by CPD (copies per droplet).
  • the initial copy number of the nucleic acid of the microdroplet array is ⁇ multiplied by the number of the microdroplets of the microdroplet array.
  • the concentration c (copy/ ⁇ L) of the nucleic acid amplification reaction solution to be tested is:
  • the method when the number y 1 of the micro-droplets of the x 1 category is smaller than the feature value m, the method includes:
  • the number y 1 of the micro-droplets of the x 1 class is less than the feature value m
  • the number of dark droplets in the micro-droplet array at this time is calculated as a whole for the initial copy of the nucleic acid of the micro-droplet array The number has no effect and can be ignored.
  • the sample DNA concentration be no greater than 6 CPD.
  • the discrimination of Ct value becomes smaller, it is difficult to distinguish the initial copy number of one droplet according to the Ct value to 4 or 5, so only x 2 ,...,x n can be used.
  • the categories were subjected to incomplete sampling to fit the Poisson distribution.
  • the square of the calculated error and the err are the smallest, and the optimal ⁇ optimal is selected to minimize the sum of squared errors.
  • the parameter ⁇ is estimated using a maximum likelihood estimation method when the parameter ⁇ of the Poisson distribution is obtained.
  • the method of estimating the parameter ⁇ may also be a moment estimation method, a sequential statistic method, or a maximum likelihood method.
  • This method does not require the number of negative dark droplets to be guaranteed, and the accuracy and stability of the estimation is much higher than using a single frequency point alone.
  • the nucleic acid starting copy number of the microdroplet array is the optimal ⁇ optimal multiplied by the number of the microdroplets of the microdroplet array.
  • the point ⁇ of the Poisson distribution is estimated by least squares based on incomplete sampling.
  • the digital PCR quantitative detection method can accurately determine the nucleic acid starting copy number of the microdroplet array without depending on a standard curve.
  • the problem of false positives in the microdroplet array can be solved by a real-time fluorescence curve.
  • True absolute quantification is achieved by processing the fluorescence curve of the microdroplet array and performing statistical corrections independent of the uniformity assumption.
  • Each fluorescence curve represents a change in the curve of useful information, participates in droplet sample information to achieve real-time monitoring, and sets the algorithm to eliminate the interaction between adjacent droplets.
  • the digital PCR detection method relies on an abstract mathematical model to achieve repeatability, high sensitivity, and a large dynamic range, which can be monitored with a small number of droplets. Cover more information with a small amount of data.
  • the digital PCR quantitative detection method avoids the error of the previous Poisson distribution probability model, and achieves absolute quantification and is more intuitive. Moreover, all of the data can be combined by the digital PCR quantitative detection method, so that the generation of random errors can be avoided.
  • the fluorescence curve of the droplet sample is obtained, and the change of the fluorescence brightness of the droplet sample is monitored in real time to remove false positives and eliminate the interaction between adjacent droplets, thereby providing a more accurate data source for the subsequent quantitative analysis model.
  • the abscissa is the average copy number of copies (CPD) contained in each of the microdroplets.
  • the ordinate is the standard deviation (Std Dev, STD) of the average starting copy number contained in each of the microdroplets.
  • the average starting copy number contained in each of the microdroplets is represented by CPD (copies per droplet). It can be seen that the average starting copy number contained in each of the microdroplets obtained using a part of the starting copy number of the nucleic acid is smaller than the standard deviation of the CPD by the standard deviation of the standard starting copy number obtained by the CPD.
  • the average starting copy number contained in each of the microdroplets obtained by the digital PCR detecting method is more accurate with the value of CPD.
  • the results of 1000 simulations of 20,000 droplets showed.
  • a single point estimation method can only cover a limited concentration range, and the estimation accuracy sharply deteriorates as the sample concentration increases.
  • the estimation accuracy does not deteriorate significantly with the increase of the sample concentration, and the concentration of the nucleic acid amplification reaction solution can be doubled.
  • the incomplete Poisson distribution fitting algorithm partial sampling Poisson distribution fitting algorithm
  • the false positives and false negatives of the results were solved by the digital PCR quantitative detection method.
  • the high-throughput sample of the sequencing platform enables simultaneous detection of hundreds of samples.
  • different types of fluorescence can be used to detect multiple sites, speed up the detection, and reduce the experimental cost.
  • the digital PCR detector is used to separate the rare detection fragments from a large number of complex backgrounds, which greatly simplifies the operation steps, saves preparation time and detection time, and the results are intuitive and reliable, and can be stably implemented.
  • the detection sensitivity and accuracy are both accurate and quantitative, which improves the sensitivity and accuracy of the detection.
  • step S420 includes:
  • S422 classifying the microdroplet array according to the melting temperature, acquiring nucleic acid information of the microdroplet array, and further obtaining nucleic acid information of the nucleic acid to be tested.
  • the melting curve of a DNA is a DNA fingerprint corresponding to a specific DNA. According to the melting curve, the temperature at which the peak is located represents the Tm value (melting point temperature) of the double-stranded DNA molecule.
  • the genotype can be judged based on the Tm value of the amplified product. By classifying the same melting curve, the genotyping or categorization of melting curves of different shapes and comparison with the melting curve of the target gene can remove non-specific false positives and exclude sequences different from the target gene.
  • micro-droplet array when the micro-droplet array is classified by a melting curve, a decision tree, a Bayesian, an artificial neural network, a K-nearest neighbor, a support vector machine, and an association rule-based classification, Bagging, and Boosting may be employed. Algorithm.
  • the digital PCR detection method further includes:
  • S50 Obtain a high-resolution melting curve of the micro-droplet array, classify the micro-droplet array, and obtain nucleic acid information such as genotyping and mutation detection of the micro-droplet array.
  • the specificity of nucleic acid amplification of the microdroplet array, and whether there is a primer dimer phenomenon or the like during nucleic acid layer expansion can be obtained.
  • a high-resolution melting curve of the micro-droplet array is obtained according to a real-time detection of a change in a fluorescence signal value during dsDNA melting of the microdroplet array.
  • the high-resolution melting curve is a new gene analysis technique based on the melting temperature of single nucleotides to form different morphological melting curves. It has extremely high sensitivity, can detect single base differences, and has low cost and flux. High, fast, accurate results, and limited by detection sites, realizing a closed tube operation. HRM analysis techniques play an important role in mutation scanning, single nucleotide polymorphism analysis, methylation studies, genotyping, and sequence matching.
  • thermostability of double-stranded nucleotides is affected by their length and base composition, and sequence changes can lead to changes in the melting behavior of dsDNA during warming.
  • the fluorescent dye used can only be embedded and bound to dsDNA
  • real-time PCR technology can be used to detect the difference in the fluorescence signal value during the dsDNA melting process, and the difference in the PCR product can be generated by generating different shape melting curves. Show it intuitively.
  • genotyping or categorization based on different shape melting curves can be realized for the test population.
  • a transiently accelerated microdroplet generation method or a variable cycle microdroplet generation method may be employed.
  • the polymerase chain reaction product is analyzed using a saturated dye in the nucleic acid amplification reaction solution to be tested in the step S10.
  • high resolution melting analysis when qualitatively classifying a plurality of the microdroplets, high resolution melting analysis (HRM) can also be used without using a sequence-specific probe, but using a The product of the PCR reaction was analyzed by a saturated dye.
  • the thermal stability of double strand DNA (dsDNA) is affected by its length and base composition. Sequence changes can lead to changes in the melting behavior of dsDNA during temperature rise.
  • real-time PCR technology can be used to detect the difference in the fluorescence signal value during the dsDNA melting process, and the difference in the PCR product can be generated by generating different shape melting curves. Show it intuitively.
  • genotyping or categorization based on different shape melting curves can be realized for the test population.
  • the primer design has three basic principles. First, the primers are closely complementary to the sequence of the template. Second, avoid formation of a stable dimer or hairpin structure between the primer and the primer. Again, the primers are unable to initiate DNA polymerization (ie, mismatches) at non-target sites of the template.
  • the realization of these three basic principles requires consideration of many factors, such as primer length, product length, sequence Tm value, internal stability of primers and templates to form double strands, formation of primer dimers, and energy values of hairpin structures, in mismatches. The efficiency of initiation of the sites, the GC content of the primers and products, and the like.
  • the primers can be modified for specific detection, such as increasing restriction enzyme sites, introducing mutations, and the like.
  • the Tm value of the template position sequence corresponding to the primer is about 72 ° C to optimize the renaturation conditions.
  • a melting curve of the microdroplet array obtained by the digital PCR detecting method is plotted with a negative first derivative of a fluorescent signal and a temperature, and a temperature at which the peak is located Represents the Tm value (melting point temperature) of a double-stranded DNA molecule.
  • the genotype can be judged based on the Tm value of the amplified product. It can be seen that there are two melting temperatures Tm1 and Tm2.
  • the melting temperature Tm1 and the melting temperature Tm2 correspond to two different classes of DNA, respectively, whereby the target DNA in the microdroplet array can be distinguished.
  • a nucleic acid detection microsphere for high-throughput nucleic acid detection and analysis can be realized with high throughput, high sensitivity and short detection time. , preparation methods, kits and high-throughput nucleic acid detection methods.
  • the present application provides a nucleic acid detection microsphere 700 for high throughput nucleic acid detection analysis.
  • the nucleic acid detecting microspheres 700 include a core body 730 and a coating layer 710.
  • the core body 730 has fluorescent coded information.
  • the cladding layer 710 encloses the core body 730.
  • the cladding layer 710 includes a substrate 711 and a primer 712 dispersed on the substrate 711, and the primer 712 uniquely corresponds to the core body 730.
  • the wrapping layer 710 wraps the core body 730 to form the nucleic acid detecting microspheres 700.
  • the substrate 711 is an aqueous polymer gel formed in a hydrophobic oil, has no fluidity, and is not easily changed in shape and volume.
  • the aqueous polymer gel is in a gel state at room temperature and melts at a temperature higher than room temperature without affecting the diffusion and activity of the enzyme, the reaction solution, and the like.
  • the primer 712 dispersed on the substrate 711 can perform qualitative analysis and recognition on the target detection nucleic acid.
  • the core body 730 is a high temperature resistant material having fluorescent coded information.
  • the fluorescently encoded information is displayed by the fluorescently encoded signal of the core 730, thereby effecting a special marking function by the fluorescently encoded signal.
  • each of the core bodies 730 corresponds to one of the primers 712 and uniquely corresponding, so that the nucleic acid detecting microspheres 700 can be marked by the core body 730 so that tracking detection can be performed.
  • a plurality of types of the nucleic acid detection microspheres 700 are mixed with a nucleic acid amplification reaction solution to be detected, and a nucleic acid detection liquid can be obtained.
  • Microdropping the nucleic acid detection solution can form a plurality of microdroplets, and a plurality of microdroplets are subjected to a PCR reaction.
  • the double-stranded DNA is denatured at 90 ° C to 95 ° C, and then rapidly cooled to 50 ° C to 60 ° C.
  • the primers are annealed and bound to the target sequence, and then rapidly heated to 70 ° C to 75 ° C to polymerize in Taq DNA.
  • the primer strand is extended along the template to amplify the nucleic acid in a suitable temperature range.
  • the encapsulation layer 710 is melt-decomposed, and the primer 712 carried in the encapsulation layer 710 is released into the corresponding micro-droplet, and is in the micro-droplet.
  • the target nucleic acid molecule is reacted, and finally, the nuclear body 730 can be positioned and tracked, and the target nucleic acid molecule can be obtained by the primer 712 corresponding to the nuclear body 730, thereby realizing high-throughput detection of the PCR.
  • a plurality of the plurality of nucleic acid detecting microspheres 700 can be prepared in batches. And a plurality of the nucleic acid detecting microspheres 700 are mixed in a certain ratio according to the actual need of detecting the target nucleic acid, and mixed with the nucleic acid amplification reaction solution to be detected, thereby obtaining a nucleic acid detecting liquid, thereby performing the target nucleic acid. Detection, a plurality of target nucleic acid molecules can be detected at one time, without repeated multiple detections, and the workload is small, the detection time is short, and the sensitivity is high.
  • the cladding layer 710 further includes a probe 713, and the probe 713 and the primer 712 are dispersed on the substrate 711 and the core body. 730 only corresponds.
  • the probe 713 may be a fluorescent probe capable of indicating nucleic acid amplification, and may be an oligosaccharide nucleotide probe containing a fluorescent group and a quenching group, such as a TaqMan fluorescent probe.
  • the probe 713 is dispersed in the matrix 711 to perform qualitative analysis and identification on the target detection nucleic acid molecule.
  • the coating layer 710 is melted and decomposed, and the primer 712 carried in the wrapping layer 710 and the probe 713 are released into the corresponding micro-droplets.
  • the nuclear body 730 can be positioned and tracked, and the target nucleic acid can be obtained by the primer 712 and the probe 713 corresponding to the core 730. Molecules, in order to achieve high-throughput PCR detection.
  • the primers 712 can be configured as different types of primers, and when batch detection is performed, a plurality of the primers 712 can be set as different types of primers, which can be used to detect different types of target nucleic acid molecules. Meanwhile, one of the primers 712 corresponds to one of the core bodies 730, that is, each of the primers 712 has its corresponding representative number, that is, the core body 730, so that the core body 730 can pass through. Detection to identify.
  • 100 of the nucleic acid detecting microspheres 700 correspond to 100 different kinds of the primers 712, that is, 100 of the nucleic acid detecting microspheres 700.
  • 100 different kinds of said core bodies 730 correspond to 100 different kinds of the primers 712.
  • the plurality of microdroplets containing 100 of the nucleic acid detecting microspheres 700 are subjected to a PCR warming process, and the wrapping layer 710 in each of the nucleic acid detecting microspheres 700 is melt-decomposed to carry the wrapping layer 710.
  • the primer 712 is released into the corresponding microdroplet for PCR amplification.
  • a part of the micro-droplets contains one of the core bodies 730, and the primers 712 contained in the micro-droplets are known by the core body 730, and the micro-droplets are known by the primers 712.
  • the target nucleic acid molecule in the droplet is subjected to PCR detection.
  • the matrix is an agarose gel.
  • the agarose gel is a gel prepared by using agarose as a supporting medium.
  • the melting point of the agarose is between 62 ° C and 65 ° C. After melting, it can be maintained at 37 ° C for several hours, and at 30 ° C to solidify into a gel.
  • the encapsulation layer 710 is melted and decomposed, and the primer 712 and the probe 713 carried in the encapsulation layer 710 are released into the corresponding micro-droplets.
  • the primer 712 is subjected to a PCR reaction with a target nucleic acid molecule contained in the microdroplet, and the fluorescent dye or the probe 713 is used to indicate whether the nucleic acid is amplified.
  • the core body 730 is a solid sphere containing a fluorescent dye.
  • the material of the core body 730 may be a high temperature resistant material, and may be polyimide, polytetrafluoroethylene, polyphenylene sulfide or polyamide.
  • the core body 730 contains a fluorescent dye and can emit a fluorescent signal.
  • the core body 730 can be encoded by different kinds of fluorescent dyes and the intensity of fluorescence, and a large number of different types of the core bodies 730 are obtained, so that the core bodies 730 have fluorescent coded signals, thereby achieving multiple The nucleic acid detection microspheres 700 are encoded.
  • Each of the nucleic acid detecting microspheres 700 corresponds to one of the core bodies 730, and one of the core bodies 730 corresponds to one of the primers 712, and one of the core bodies 730 corresponds to a type of Probe 713.
  • the plurality of the nucleic acid detection microspheres 700 can realize PCR detection of a plurality of different kinds of target nucleic acid molecules at one time, and does not require repeated multiple detections, and has small workload, short detection time, high throughput, and high sensitivity. specialty.
  • the core body 730 has a diameter of from 10 micrometers to 100 micrometers.
  • the coating layer has a thickness of from 10 micrometers to 100 micrometers.
  • the nucleic acid detecting microspheres 700 generally have a diameter ranging from 20 micrometers to 150 micrometers, which allows sufficient droplets to be collected during image acquisition.
  • the core body 730 may have a diameter of 10 micrometers to 100 micrometers, and the cladding layer may have a thickness of 10 micrometers to 100 micrometers.
  • the nucleic acid detecting microspheres 700 should not be too large or too small, and if it is too small, it is not easy to be recognized. If it is too large, it is easy to block the outlet end of the micro-droplet generating device when generating a plurality of micro-droplets, thereby hindering multiple Generation of microdroplets.
  • the diameter of the nucleic acid detecting microspheres 700 it is possible to reproduce a plurality of microdroplets that can be recognized by the fluorescent signal detecting device 30, and can cover as many microdroplets as possible to facilitate the image. It is not easy to prevent the generation of a plurality of micro-droplets at the outlet end of the micro-droplet generating device.
  • a method for preparing a nucleic acid detecting microsphere comprises:
  • the plurality of core bodies 730 are the same kind of physical spheres containing fluorescent dyes, thereby preparing the nucleic acid detecting microspheres 700, such that the nucleic acid detecting microspheres 700 and one kind
  • the core bodies 730 uniquely correspond, and one of the primers 712 uniquely corresponds to one of the core bodies 730.
  • the primer solution contains the primer 712.
  • the dry powdery primer 712 is diluted with sterilized ultrapure water, it is diluted to a primer concentration of 100 ⁇ M, that is, 100 ⁇ mol/L. Then, 100 ⁇ L of a primer solution having a concentration of 100 ⁇ M was placed in the coating layer preparation liquid 900 ul, and the primer concentration was set to 10 ⁇ M ( ⁇ mol/L).
  • the gel powder may be a substance such as agar powder or ethylene glycol diacrylate which can be gel-formed.
  • the coating layer preparation liquid may be an agar powder solution, and agar powder of 1.5% to 4.5% by mass and 10 ml of double distilled water are provided, and the agar powder is added to the double distilled water to dissolve at a high temperature to clarify. To obtain the coating preparation liquid.
  • the coating layer preparation liquid is also an agar powder solution.
  • the plurality of the primers 712 contained in the primer solution are the same type of primers, and the plurality of the core bodies 730 are the same fluorescent type of the core body.
  • the gel melting temperature is the temperature at which the gel is converted to a liquid solution.
  • the agarose has a melting point of between 62 ° C and 65 ° C and solidifies into a gel at 30 ° C. Therefore, a plurality of the primers 712 and a plurality of the core bodies 730 are added to the coating layer preparation liquid at a high temperature environment of 62 ° C to 65 ° C, and the coating layer preparation liquid is also an agar powder solution, thereby obtaining The nucleic acid detection microspheres prepare a solution.
  • the size is generally selected according to the size of the nucleic acid detecting microsphere 700.
  • the concentration of the core 730 is generally selected according to the size of the nucleic acid detecting microsphere 700.
  • a plurality of the nucleic acid detecting microsphere droplets are formed in a hydrophobic oil by a microfluidic chip, a microfluidizer or a microdroplet generating device in a high temperature environment. .
  • the step S140 includes:
  • the outlet end of the ejector head is subjected to an instantaneous acceleration movement or a shifting cycle under the liquid surface of the hydrophobic oil, and the nucleic acid detecting microsphere preparation solution is taken from the outlet of the ejector head The end is discharged, and the plurality of nucleic acid detecting microsphere droplets are formed under the liquid surface of the hydrophobic oil.
  • the present application provides a micro-droplet generating device including a ejector gun head, a fluid drive mechanism, and a motion control mechanism.
  • the ejector head has an outlet end and an inlet end, and the fluid driving mechanism drives the ejector head to suck the nucleic acid detecting microsphere preparation solution into the ejector head through the inlet end, and the sputum is discharged
  • the outlet end of the tip is inserted into a container in which the oily liquid is stored such that the outlet end of the ejector head enters the level of the oily liquid.
  • the movement control mechanism performs an instantaneous acceleration movement or a shifting cycle under the liquid surface of the oily liquid, so that the nucleic acid detecting microsphere preparation solution is discharged from the outlet end of the ejector head.
  • the plurality of nucleic acid detecting microsphere droplets are formed under the liquid surface of the oily liquid.
  • the oily liquid and the nucleic acid detecting microsphere preparation solution are mutually incompatible or have an interfacial reaction
  • the oily liquid may be mineral oil (including n-tetradecane, etc.), vegetable oil, silicone oil and Perfluoroalkane oil, etc.
  • a plurality of the nucleic acid detecting microsphere droplets are cooled to a normal temperature of about 30 ° C, at which time a plurality of the solidified gels are gelatinized, and a plurality of the nucleic acid detecting microspheres are obtained by flow sorting. 700.
  • the flow sorting detects a microsphere droplet by irradiating a plurality of the nucleic acids in a high-speed flow state with a high-energy laser.
  • the core body 730 is a solid sphere containing a fluorescent dye, the generated scattered light and The intensity of the fluorescence is emitted to perform screening to obtain nucleic acid detecting microspheres 700 containing only a single of said core bodies 730.
  • the plurality of the nucleic acid detecting microspheres 700 after cooling are in a gel state, which can be conveniently stored and transported under normal temperature environment, and is convenient for bulk transportation for PCR detection.
  • a method for preparing a nucleic acid detecting microsphere comprises:
  • the nucleic acid detecting microsphere preparation solution is microdropped to form a plurality of nucleic acid detecting microsphere droplets
  • the probe solution comprises the probe 713 for detecting whether the nucleic acid is amplified, and may be an oligosaccharide nucleotide probe containing both a fluorescent group and a quenching group, such as TaqMan fluorescent probes, etc.
  • the coating layer 710 is melted and decomposed, and the primer 712 carried in the wrapping layer 710 and the probe 713 are released into the corresponding micro-droplets.
  • the nuclear body 730 can be positioned and tracked, and the target nucleic acid can be obtained by the primer 712 and the probe 713 corresponding to the core 730. Molecules, in order to achieve high-throughput PCR detection.
  • the plurality of core bodies 730 are the same type of solid spheres containing fluorescent dyes, thereby preparing the nucleic acid detecting microspheres 700 such that one of the nucleic acid detecting microspheres 700 and one of the core bodies 730 Uniquely corresponding, and one of the primers 712 uniquely corresponds to one of the core bodies 730, and one of the probes 713 uniquely corresponds to one of the primers 712.
  • the coating layer preparation liquid may be the same as the preparation method in the step 120.
  • a method of preparing a plurality of the nucleic acid detecting microsphere droplets may be the same as the step S140 method.
  • the method of obtaining a plurality of the plurality of nucleic acid detecting microspheres 700 may be the same as the step 150 method.
  • the nucleic acid detecting microsphere is a nucleic acid detecting microsphere containing a single of the nucleus.
  • the gel powder is agar powder or polyethylene glycol diacrylate or the like.
  • kits for high throughput nucleic acid detection analysis comprises the nucleic acid detecting microsphere and nucleic acid reaction solution as described in any of the above embodiments.
  • the nucleic acid reaction solution contains an enzyme, a dNTP, a fluorescent dye, an ion, and the like required for PCR amplification. If the probe 713 is included in the nucleic acid detecting microsphere 700, a fluorescent dye may not be included in the nucleic acid reaction solution.
  • the kit can be used to store and carry a plurality of different nucleic acid detection microspheres 700.
  • the nucleic acid detecting microspheres 700 can be stored in glycerin.
  • a kit of reagents and solutions dedicated to digital PCR is prepared to reduce or avoid potential contamination of the template DNA sample by exogenous DNA. All instruments and consumables used should be autoclaved and dried at elevated temperatures.
  • a high-throughput nucleic acid detection method includes:
  • nucleic acid detection microspheres 700 include a core body 730 and a cladding layer 710, the core body 730 having encoded information, the package
  • the cladding layer 710 encloses the core body 730, the cladding layer 710 includes a base body 711 and a primer 712 dispersed in the base body 711, and the primer 712 uniquely corresponds to the core body 730, and the core body 730 is a solid sphere containing a fluorescent dye;
  • the nucleic acid detection solution is microdropped to form a plurality of microdroplets 800;
  • the plurality of micro-droplets 800 are subjected to nucleic acid amplification to obtain the plurality of micro-droplets 800 after completion of amplification;
  • the nucleic acid amplification reaction solution is a nucleic acid amplification reaction solution using deoxyribonucleic acid as a template, a reverse transcription nucleic acid amplification reaction solution using ribonucleic acid as a template, or a loop-mediated isothermal amplification reaction solution.
  • the nucleic acid amplification reaction solution contains a fluorescent dye.
  • the nucleic acid amplification reaction solution includes a nucleic acid template, a reaction buffer, deoxyribonucleoside triphosphate, a polymerase, a divalent metal cation, and the like, and if the probe 713 is absent in the coating layer 710, A fluorescent dye is contained in the reaction buffer.
  • the nucleic acid amplification reaction solution may be a nucleic acid amplification reaction solution (which may be referred to as a DNA amplification reaction solution) using deoxyribonucleic acid (DNA) as a template, or may be a template of ribonucleic acid (RNA).
  • the reverse transcription nucleic acid amplification reaction solution (which may be referred to as an RNA reverse transcription reaction solution) may also be another nucleic acid amplification reaction solution such as a loop-mediated isothermal amplification (LAMP) reaction solution.
  • LAMP loop-mediated isothermal amplification
  • the DNA amplification reaction solution is characterized by containing dNTPs, reaction buffers, inorganic salt ions, polymerases, DNA templates to be detected, and fluorescent dyes required for DNA amplification.
  • the fluorescent dye may be a fluorescent dye that binds to DNA such as SYBR Green.
  • the SYBR Green fluorescent dye is added to emit a weak fluorescence in the free state, but once bound to the double-stranded DNA, the fluorescence is greatly enhanced, and a fluorescent signal is emitted, thereby ensuring an increase in the fluorescent signal and the PCR product. Increase full sync.
  • the fluorescent signal emitted by the SYBR Green fluorescent dye can be detected to obtain a fluorescent signal after the nucleic acid amplification reaction, thereby obtaining whether the corresponding first nucleic acid molecule 810 has a corresponding target nucleic acid molecule.
  • the size, shape, and inclusion of the primer 712 of the nucleic acid detecting microspheres 700 in the plurality of different types of the nucleic acid detecting microspheres 700 may be the same or different.
  • a plurality of the nucleic acid detecting microspheres 700 may contain different types of the primers 712 for detecting different kinds of the target nucleic acid molecules.
  • the nucleic acid in the nucleic acid detecting solution may be adjusted.
  • the concentration of the microspheres 700 is detected such that the number of single packages is maximized when the plurality of microdroplets 800 are generated, and the distribution of the microspheres conforms to the Poisson distribution theoretical model.
  • the present application provides a nucleic acid high-throughput detector comprising a micro-droplet generating device, a temperature controlling device, and a fluorescent signal.
  • the microdroplet generating device is configured to microdroplet the nucleic acid detecting solution to form the plurality of microdroplets 800.
  • the temperature control device and the micro-droplet generating device are connected by a track for transferring the plurality of micro-droplets to the temperature control device, and performing temperature cycling through the temperature control device to realize nucleic acid amplification .
  • fluorescence detection is performed on the plurality of microdroplets after the nucleic acid amplification is completed by the fluorescence signal detecting device.
  • the controller is respectively connected to the microdroplet generating device, the temperature controlling device and the fluorescent signal detecting device for controlling the microdroplet generating device, the temperature controlling device and the fluorescent signal detecting Device.
  • the micro-droplet generating device comprises a squirting gun head, a fluid driving mechanism and a motion control mechanism.
  • the ejector head has an outlet end and an inlet end, and the fluid driving mechanism drives the ejector head to suck the nucleic acid detecting liquid into the ejector head through the inlet end, and the ejector gun
  • the outlet end of the head is inserted into a container in which the oily liquid is stored such that the outlet end of the ejector head enters the level of the oily liquid.
  • the movement control mechanism performs an instantaneous acceleration movement or a shifting cycle under the liquid surface of the oily liquid, so that the nucleic acid detection liquid is discharged from the outlet end of the ejector head, in the oily
  • the plurality of microdroplets 800 are formed under the liquid surface of the liquid.
  • the oily liquid and the nucleic acid detecting liquid are two liquids which are incompatible with each other or have an interfacial reaction, and the oily liquid may be mineral oil (including n-tetradecane, etc.), vegetable oil, silicone oil and perfluoroalkane. Oil, etc.
  • the nucleic acid detecting solution is microdropped in the step S330, and a microfluidic chip, a microfluidizer or a micro can be used to form a plurality of microdroplets 800.
  • the method of preparing the plurality of micro-droplets 800 is not limited to the above-described device, and may be other devices for preparing the plurality of micro-droplets 800.
  • each of the micro-droplets 800 may include zero, one or more of the nucleic acid detecting microspheres 700, and each of the micro-droplets 800 contains the nucleic acid amplification reaction liquid for performing Nucleic acid amplification.
  • the size of the plurality of microdroplets 800 formed by microdropping the nucleic acid detection solution in the step S330 may be the same or different.
  • a microfluidic chip may be employed when the nucleic acid detection solution is microdropped.
  • each of the microdroplets 800 may include zero, one or more of the nucleic acid detection microspheres 700.
  • the wrap layer 710 is melted to release the primer 712, thereby causing the primer 712 and the microfluid
  • the nucleic acid molecules in the drop 800 are simultaneously subjected to PCR amplification.
  • the type of the primer 712 can be known by identifying the corresponding core body 730 in the microdroplet 800, thereby obtaining the target nucleic acid molecule.
  • the first effective micro-droplet 810 includes one of the core bodies 730.
  • the microdroplets 800 containing zero or more than one are considered to be invalid microdroplets.
  • the first effective micro-droplet 810 contains a fluorescent dye and the primer 712. If the fluorescent dye binds to the double-stranded DNA during the PCR amplification process of the nucleic acid molecule in the primer 712 and the first effective micro-droplet 810, the fluorescence is greatly enhanced, and a strong fluorescent signal can be emitted.
  • the first effective micro-droplet 810 has a strong fluorescent signal, so that the corresponding target nucleic acid molecule in the first-effect micro-droplet 810 can be obtained according to the core body 730 and the primer 712. kind.
  • the step S360 includes:
  • S361 providing a fluorescent signal detecting device, the fluorescent signal detecting device comprising a fluorescent channel and a fluorescent dye detecting channel, and identifying a fluorescent signal of the core body 730 in the effective microdroplet according to the encoded fluorescent channel;
  • the core body 730 is a solid sphere containing a fluorescent dye, and the core body 730 can be labeled by using different kinds of fluorescent dyes and the intensity of fluorescence.
  • Each of the phosphors corresponds to one of the core bodies 730, and each of the core bodies 730 corresponds to one of the primers 712, which corresponds to each of the said 712 having its corresponding representative number, ie, the core body. 730.
  • step S360 qualitative detection is performed by detecting the presence or absence of the target nucleic acid molecule based on the presence or absence of the reported fluorescent signal.
  • the first effective micro-droplets 810 containing the core body 730 of the same category may be grouped into one group during the screening process.
  • the ratio of the number of microdroplets in the first effective microdroplet 810 in the nucleus 730 of this class to the total number of microdroplets in the class can be obtained, thereby
  • the concentration of the target nucleic acid molecule corresponding to the core body 730 of the same class is calculated from the Poisson distribution.
  • a high-throughput nucleic acid detection method includes:
  • the nucleic acid detection microspheres 700 include a core body 730 and a cladding layer 710, the core body 730 having encoded information, the package
  • the cladding layer 710 encloses the core body 730, the cladding layer 710 includes a substrate 711 and a primer 712 and a probe 713 dispersed in the substrate 711, and the primer 712 and the probe 713 and the core body 730 uniquely corresponding, the core body 730 is a physical sphere having fluorescent coded information;
  • the nucleic acid detection solution is microdropped to form a plurality of microdroplets 800;
  • the plurality of micro-droplets 800 are subjected to nucleic acid amplification to obtain the plurality of micro-droplets 800 after completion of amplification;
  • each of the microdroplets 800 may include zero, one or more of the nucleic acid detection microspheres 700.
  • the wrap layer 710 is melted to release the primer 712 and the probe 713.
  • the primer 712 and the probe 713 and the nucleic acid molecule in the micro-droplet 800 are simultaneously subjected to PCR amplification, and at this time, the corresponding nuclear body 730 in the micro-droplet 800 can be identified.
  • the primer 712 and the probe 713 are known, and the kind of the primer 740 can be known, thereby obtaining the target nucleic acid molecule.
  • the second effective micro-droplet 820 includes one of the core bodies 730.
  • the microdroplets 800 containing zero or more than one of the core bodies 730 are considered to be ineffective microdroplets.
  • the second effective micro-droplet 820 further includes the probe 713 and the primer 712.
  • the probe 713 When the probe 713 is contained in the second effective micro-droplet 820, the second effective micro-droplet 820 may not contain a fluorescent dye, and the probe 713 functions as a fluorescent calibration.
  • the probe 713 binds to double-stranded DNA so that the first fragment containing the corresponding target nucleic acid molecule can be identified.
  • Two effective microdroplets 820 are two effective microdroplets 820.
  • the probe 713 can be identified by judging the probe 713. Whether the second effective micro-droplet 820 contains a corresponding target nucleic acid molecule.
  • the type of the corresponding target nucleic acid molecule in the first effect microdroplet 810 can be obtained according to the core body 730 and the primer 712.
  • the method of microdropping the nucleic acid detection solution in the step S330 is the same in the step S430.
  • the present application provides a fluorescent signal detecting device 30.
  • the fluorescent signal detecting device 30 includes an excitation light source 340, a fluorescence detecting component 330, and a third controller 310.
  • the excitation light source 340 is disposed above the detection area of the plurality of micro-droplets 800 and is irradiated at an oblique angle to the detection area of the plurality of micro-droplets 800 to form an oblique optical path.
  • the fluorescence detecting component 330 is disposed directly above the detection area of the plurality of micro-droplets 800 for acquiring a fluorescent image of the plurality of micro-droplets 800.
  • the third controller 310 is coupled to the excitation light source 340 and the fluorescence detecting component 330, respectively, for controlling the excitation light source 340 and the fluorescence detecting component 330.
  • the fluorescent signal detecting device 30 can perform imaging of a plurality of fluorescent channels on the microdroplets and perform bright field dark field imaging. Among them, multiple fluorescent channel imaging is used for detecting the droplet reaction signal, and bright field dark field imaging is used to detect the size information of the formed microdroplet and monitor the state of the droplet during the reaction.
  • the excitation light source 340 includes an LED light source 341 of different colors, a collimating mirror 342, a first color filter 343, a dichroic mirror 344, a fly-eye lens 345, and a focusing lens 346.
  • the different colored LED light sources 341 can generate different colors of light that illuminate the plurality of micro-droplets 800. Irradiation of different fluorescent colors can be obtained by selecting the LED light sources 341 of different colors, which can be operated in turn.
  • the collimating mirror 342, the first filter 343, and the dichroic mirror 344 are disposed in front of the optical path emitted by each of the LED light sources.
  • the collimating mirror 342 and the first filter 343 are disposed at a vertical angle to the optical path (90° angle setting).
  • the dichroic mirror 344 is disposed at an angle of 0 to 45 degrees with respect to the optical path.
  • An optical path formed by the dichroic mirror 344 is provided with the fly-eye lens 345 and the focus lens 346 in this order.
  • the fly-eye lens 345 and the focus lens 346 are disposed at a vertical angle to the optical path (90° angle setting).
  • the internal fluorescence of the plurality of micro-droplets 800 is excited, collected by the upper objective lens 332 through the second filter 333, and enters the camera 331, and the camera 331 collects fluorescence of the plurality of micro-droplets image.
  • the optical path emitted by the excitation light source 340 is obliquely irradiated to the plurality of micro-droplets 800 such that the micro-droplets containing the fluorescent substance in the plurality of micro-droplets 800 generate fluorescence.
  • Fluorescence information collection is performed on the micro-droplet containing the fluorescent substance by the fluorescence detecting component 330, and the fluorescent information of the fluorescent substance-containing micro-droplet is transmitted to the analyzing device (computer) in the form of a fluorescent image. For analysis.
  • the second controller 310 is configured to switch different filters to form different fluorescence detecting channels.
  • the fluorescent signal detecting device includes a fluorescent channel, a fluorescent dye detecting channel, a fluorescent probe detecting channel, a microdroplet recognition channel, a plurality of alternate channels, and the like.
  • a ROX internal reference dye is added to the nucleic acid detecting solution.
  • the ROX internal reference dye does not participate in the PCR reaction and can be used to identify information such as the specific position, contour and number of the plurality of microdroplets 800.
  • the microdroplet recognition channel is used to identify the fluorescence of the ROX internal reference dye to accurately locate each of the microdroplets 800.
  • the coded fluorescent channel is used to identify the fluorescent signal and the fluorescence signal intensity of the core body 730 for acquiring the first effective micro-droplet 810 containing one of the core bodies 730.
  • the fluorescent dye detection channel or the fluorescent probe detection channel is configured to identify a reporter fluorescent signal after the nucleic acid amplification reaction in the first effective microdroplet 810, thereby determining the primer 712 or the solution according to the reported fluorescent signal. Whether the primer 712 and the probe 713 and the target nucleic acid molecule are subjected to PCR amplification are described.
  • step S460 qualitative detection is performed by detecting the presence or absence of the target nucleic acid molecule based on the presence or absence of the reported fluorescent signal.
  • the second effective micro-droplets 820 containing the core body 730 of the same category may be grouped into one group during the screening process.
  • the ratio of the number of microdroplets in the second effective microdroplet 820 in the nucleus 730 of this class to the total number of microdroplets in the class can be obtained, thereby
  • the concentration of the target nucleic acid molecule corresponding to the core body 730 of the same class is calculated from the Poisson distribution.
  • the first effective micro-droplet 810 can be screened from a plurality of the micro-droplets 800 by detecting the fluorescent signal of the core body 730 in the plurality of micro-droplets 800 by the encoded fluorescent channel. Wherein, the first effective micro-droplet 810 contains one of the core bodies 730. Thereby, the first effective micro-droplet 810 is detected by the fluorescent dye detecting channel, and the reported fluorescent signal after the nucleic acid amplification reaction is read to determine whether the first effective micro-droplet 810 contains a corresponding target. Nucleic acid molecule.
  • the corresponding target nucleic acid can be obtained according to the primer 712 corresponding to the core body 730 in the first effective micro-droplet 810.
  • the type of molecule can be obtained according to the primer 712 corresponding to the core body 730 in the first effective micro-droplet 810.
  • the fluorescent signal of the plurality of the micro-droplets 800 in the plurality of micro-droplets 800 can be detected by the encoded fluorescent channel, and the second effective micro-droplets can be screened from the plurality of the micro-droplets 800. 810.
  • the second effective micro-droplet 820 contains one of the core bodies 730. Therefore, the second effective micro-droplet 820 is detected by the fluorescent probe detecting channel, and the reported fluorescent signal after the nucleic acid amplification reaction is read to determine whether the second effective micro-droplet 820 contains a corresponding one. Target nucleic acid molecule.
  • the primer 712 or the probe 713 corresponding to the core body 730 in the second effective micro-droplet 820 may be used.
  • the type of the corresponding target nucleic acid molecule is known.
  • the fluorescent signal detecting device includes a plurality of the encoded fluorescent channels, and the plurality of encoded fluorescent channels can be used to identify the plurality of different fluorescently labeled cores 730.
  • the first coded fluorescent channel is set to the fluorescence A
  • the concentration of 10 gradients is set
  • the second coded fluorescence channel is set to the fluorescence B
  • the concentration of 10 gradients is also set
  • a large number of different kinds of the core bodies 730 can be obtained, which can be used to label a large number of different kinds of the nucleic acid detecting microspheres 700.
  • the nucleic acid detecting microspheres 700 are composed of the core body 730 having fluorescent coded information, the primer 712, and the coating layer 710 of the probe 713. a plurality of the nucleic acid detecting microspheres 700 are randomly distributed into the nucleic acid amplification reaction solution, and are mixed to obtain the nucleic acid detecting liquid, and the nucleic acid detecting liquid is further micro dropletized to generate the plurality of micro droplets. 800. When the temperature rises above 60 degrees Celsius, the coating layer 710 melts, and the primer 712 and the probe 713 are released into the microdroplet 800 to constitute a complete nucleic acid amplification reaction system. A core 730 remains in the microdroplet 800 as a fluorescent label that marks the microdroplet 800.
  • the fluorescent dye or the probe 713 will bind to the double-stranded DNA during the amplification process, the fluorescent signal is enhanced, and a fluorescent signal is reported. produce.
  • the micro-droplet 800 of the core body 730 having one and only one piece of fluorescence-encoded information is screened as effective
  • the microdroplets enter the subsequent analysis. Detecting the core body 730 in the effective micro-droplet based on the obtained effective micro-droplet (the effective micro-droplet is the first effective micro-droplet 810 or the second effective micro-droplet 820 described in the above embodiment)
  • the fluorescence signal is obtained to obtain the corresponding type of the primer 712 and the probe 713.
  • the nucleic acid detecting microspheres 700, the preparation method, the kit, and the high-throughput nucleic acid detecting method can realize the detection of the presence or absence of a plurality of target nucleic acid molecules at a time by adding a plurality of the nucleic acid detecting microspheres 700 at a time, and The concentration of each detection target nucleic acid can be obtained according to the Poisson distribution.
  • a plurality of target nucleic acids can be detected at a time, without repeated multiple detections, and the workload is small. Save time and sensitivity.
  • first feature "on” or “below” the second feature may be a direct contact of the first feature and the second feature, or the first and second features are indirectly contacted by an intermediate medium, unless otherwise explicitly stated and defined.
  • first feature “above”, “above” and “above” the second feature may be that the first feature is directly above or above the second feature, or merely that the first feature level is higher than the second feature.
  • first feature “below”, “below” and “below” the second feature may be that the first feature is directly below or obliquely below the second feature, or merely that the first feature level is less than the second feature.

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Abstract

提供一种数字PCR检测仪、数字PCR定量检测方法、不同体积数字PCR的定量分析方法、数字PCR检测方法、核酸检测微球、核酸检测微球制备方法、试剂盒以及高通量核酸检测方法。

Description

数字PCR检测仪、数字PCR定量检测方法、不同体积数字PCR的定量分析方法、数字PCR检测方法、核酸检测微球、核酸检测微球制备方法、核酸检测微球试剂盒以及高通量核酸检测方法
相关申请
本申请要求2018年1月24日申请的，申请号为201810070377.2，名称为“数字PCR定量检测方法”和2018年8月16日申请的，申请号为201810932950.6，名称为“数字PCR检测方法”和2018年11月21日申请的，申请号为201811392278.2，名称为“核酸检测微球、制备方法、试剂盒及高通量核酸检测方法”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本申请涉及核酸检测分析领域，特别是涉及一种数字PCR检测仪、数字PCR定量检测方法、不同体积数字PCR的定量分析方法、数字PCR检测方法、核酸检测微球、核酸检测微球制备方法、核酸检测微球试剂盒以及高通量核酸检测方法。
背景技术
数字PCR(Digital PCR，dPCR)是一种核酸分子绝对定量技术。相较于qPCR，数字PCR可让你能够直接数出DNA分子的个数，是对起始样品拷贝数的绝对定量。定量PCR是依靠标准曲线或参照基因来测定核酸量，而数字PCR则让你能够直接数出DNA分子的个数，是对起始样品拷贝数的绝对定量。
目前，数字PCR包括液滴式PCR检测方法和芯片式检测方法。芯片式检测方法中单个芯片上的有效反应腔数量一般只有数千个，远少于液滴式。所以，芯片式数字PCR的动态范围相对于液滴式较窄。液滴式PCR检测方法将样本分散成油包水的反应单元，之后对每个反应单元进行实时或终点荧光分析。但是，传统的数字PCR仪器的有效反应腔数量少，从而导致目前的数字PCR的动态范围较窄，工作效率低。传统液滴数字PCR终点检测方法有局限性，检测精度低。在数字PCR检测分析过程中，对于成千上万个纳升级的微液滴阵列，传统的数字PCR检测方法如果想同时检测多种目标序列，则需要设计多种引物依次进行检测，不断反复的多次检测增大了工作量，且耗费时间工作效率低。并且，传统的PCR检测技术，只能检测有限的目标序列。PCR检测如果要检测十几种、几十种或者上百种目标序列，则需要重复多次检测，增大了工作量，耗费大量样本，且时间工作效率低。
申请内容
有鉴于此，本申请提供一种数字PCR检测仪包括微液滴生成装置、温控装置、荧光信号检测装置以及定量分析装置。所述微液滴生成装置用以将核酸扩增反应液微滴化，形成多个微液滴。所述温控装置与所述微液滴生成装置通过轨道连接，用以将所述多个微液滴转移至所述温控装置，进行温度循环，实现核酸扩增。所述荧光信号检测装置与所述温控装置相对设置，用以对核酸扩增后的所述多个微液滴进行拍照检测。所述荧光信号检测装置可以对微液滴进行多个荧光通道成像以及进行明场暗场成像。其中多个荧光通道成像用 于微液滴反应信号的探测，明场暗场成像用于检测形成微液滴的尺寸信息以及在反应过程中监测液滴的状态。定量分析装置，所述定量分析装置与所述荧光信号检测装置通过数据线连接，用以实现所述多个微液滴荧光信息的传输，进行定量分析。控制器，所述控制器分别与所述微液滴生成装置、所述温控装置、荧光信号检测装置以及定量分析装置连接，用以控制所述微液滴生成装置、所述温控装置、所述荧光信号检测装置以及所述定量分析装置。所述数字PCR检测仪将所述微液滴生成装置、所述温控装置、所述荧光信号检测装置以及所述定量分析装置集成化，使得所述操作人员可以通过一体式数字PCR检测机实现自动化操作，提高了所述数字PCR检测仪的工作效率。
有鉴于此，本申请提供一种数字PCR定量检测方法包括以下步骤：S4110，获取所有微液滴的多个实时荧光图像，根据所述多个实时荧光图像获得进行核酸扩增的微液滴的实时荧光曲线；S4120，根据所述实时荧光曲线，获得所有进行核酸扩增的微液滴的Ct值；S4130，根据所述Ct值与进行核酸扩增的微液滴的核酸起始拷贝数的关系，获得所有进行核酸扩增的微液滴的核酸起始拷贝数；S4140，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，获得所述核酸起始拷贝数的频数分布；S4150，根据所述核酸起始拷贝数的频数分布，计算泊松分布的参数λ。通过数字PCR定量检测方法可以实现所述多个微液滴的动态跟踪，在所述多个微液滴进行温度循环过程中都可以找到每个微液滴对应的具体位置，可以实现核酸扩增的全部过程的监测。通过所述数字PCR定量检测方法不仅摆脱了对标准曲线的依赖，排除了由标准曲线引起的定量结果不确定的问题，并且解决了液滴式数字PCR终点检测方式的限制，打破了只采用了一个p(x＝0)的数据对待测样本整体进行参数估计的局限性。同时，通过对所述多个微液滴荧光曲线进行处理，并进行不依赖于均匀性假设进行的统计修正，提高了数字PCR定量检测的准确性。
有鉴于此，本申请提供一种不同体积数字PCR的定量分析方法。通过不同体积数字PCR的定量分析方法，可以获得的ln(c)相应的标准差σ以及置信区间。通过ln(c)相应的标准差σ以及置信区间可以得到待测核酸扩增反应液的核酸浓度为c，因此也可以获知所述待测核酸扩增反应液中含有的DNA起始拷贝数目。所述不同体积数字PCR的定量分析方法可以利用少于200个微液滴实现5个数量级的检测动态范围，提高了数字PCR检测仪器的检测动态范围。并且，其性能可以和拥有12000个微液滴的单一体积数字PCR相媲美，节省了仪器的成本，耗材成本降低。
有鉴于此，本申请提供一种数字PCR检测方法，包括：S10，制备待测核酸扩增反应液；S20，将所述待测核酸扩增反应液微滴化，形成微液滴阵列；S30，将所述微液滴阵列进行聚合酶链式反应，并获取所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线；S40，根据所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线，对所述微液滴阵列进行分析，以获得所述待测核酸信息。本申请提供的所述数字PCR检测方法，在制备待测核酸扩增反应液时，可以使用一种染料即可实现基因分型、突变扫描、甲基化研究等，具有高分辨率与灵敏度，降低了检测的成本。并且，通过所述数字PCR检测方法，可以使得所述微液滴阵列在同一个高度集成化的数字PCR检测仪上完成聚合酶链 式反应，并在所述微液滴阵列PCR扩增后对PCR产物进行熔解曲线分析。同时，通过所述数字PCR检测方法，可以获得所述微液滴阵列的荧光曲线与熔解曲线，完全可以实现PCR扩增整个过程的实时监测与PCR产物的熔解曲线分析的无痕连接。从而，通过所述微液滴阵列的荧光曲线与熔解曲线，实现了基于不同形状熔解曲线的基因分型或归类，从而实现对所述微液滴阵列的定性以及定量分析，更加全面、简便、高效的完成数字PCR的检测。
有鉴于此，本申请提供一种核酸检测微球、制备方法、试剂盒及高通量核酸检测方法。其中，所述包裹层将所述核体包裹，形成所述所述核酸检测微球。所述基体为在疏水性油中形成的含水聚合物凝胶，没有流动性，且形状和体积不易改变。含水聚合物凝胶在室温下为凝胶状态，并在高于室温的温度下熔融，且不影响酶、反应液等的扩散及活性。同时，分散于所述基体的所述引物可以对目标检测核酸进行定性分析识别。所述核体为耐高温材料，具有特殊标记功能，并且每个所述核体对应着一种所述引物，且唯一对应，从而可以通过所述核体来标记所述核酸检测微球，以便可以进行追踪检测。在进行PCR检测时，将多个且多种类的所述核酸检测微球与需要检测的核酸扩增反应液混合，可以获得核酸检测液。将所述核酸检测液微滴化可以形成多个微液滴，并将多个微液滴进行PCR反应。在PCR反应过程中，双链DNA在90℃～95℃变性，再迅速冷却至50℃～60℃，引物退火并结合到靶序列上，然后快速升温至70℃～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸，在适合的温度范围内对核酸进行扩增。在多个微液滴进行PCR温控过程中，所述包裹层熔融分解，将所述包裹层中携带的所述引物释放到所对应的微液滴中，并与微液滴中含有的目标核酸分子反应，最终可以对所述核体进行定位追踪识别，并通过所述核体对应的所述引物来获知目标核酸分子，以此来实现PCR高通量检测。在实际应用过程中，可以批量制备多个多种所述核酸检测微球。并将多个多种所述核酸检测微球按照实际检测目标核酸的需要按照一定比例混合，并与需要检测的核酸扩增反应液混合，可以获得核酸检测液，以此来对目标核酸进行检测，一次即可检测多种目标核酸分子，不需要反复的多次检测，并且工作量小、检测时间短、灵敏度高。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本申请提供的数字PCR检测仪的整体结构示意图；图2为本申请提供的数字PCR检测仪的微液滴生成装置；图3为本申请一实施例提供的吐液枪头的出口端运动时液滴的受力示意图；图4为本申请一实施例提供的吐液枪头的出口端的速度变化示意图；图5为本申请一实施例提供的吐液枪头的出口端运动时微液滴生成过程示意图；图6为本申请荧光信号检测装置结构示意图；图7为本申请温控装置结构示意图；图8为本申请温控装置结构切面结构示意图；图9为本申请温控装置的半导体电偶对电极连接结构示意图； 图10为本申请温控装置的瞬态性能测试示意图；图11为本申请温控装置的稳态性能测试示意图；图12为本申请数字PCR检测仪的分析方法流程图；图13为本申请微液滴平铺方法流程图；图14为本申请微液滴容器底板微液滴堆积示意图；图15为本申请完全抽样的数字PCR定量检测方法流程图；图16为本申请部分抽样的数字PCR定量检测方法流程图；图17为部分抽样的数字PCR定量检测方法与其他方法CPD的标准偏差进行对比图；图18为不同体积数字PCR的定量分析方法流程图；图19为本申请提供的数字PCR检测方法的整体步骤流程图；图20为本申请提供的数字PCR检测方法获得的微液滴阵列示意图；图21为本申请提供的数字PCR检测方法获得的微液滴阵列的荧光图像示意图；图22为本申请提供的数字PCR检测方法获得的实时荧光曲线示意图；图23为本申请提供的数字PCR检测方法获得的部分抽样的数字PCR定量检测方法与其他方法CPD的标准偏差进行对比图；图24为本申请提供的数字PCR检测方法获得的熔解曲线示意图；图25为本申请提供的核酸检测微球的结构示意图；图26为本申请提供的包覆层的结构示意图；图27为本申请提供的核体的结构示意图；图28为本申请提供的一个实施例中包覆层的结构示意图；图29为本申请提供的一个实施例中核酸检测微球的结构示意图；图30为本申请提供的微液滴生成装置的结构示意图；图31为本申请提供的不同类型的微流控芯片的结构示意图；图32为本申请提供的第一有效微液滴的结构示意图；图33为本申请提供的微液滴生成装置的结构示意图；图34为本申请提供的不同类型的微流控芯片的结构示意图；图35为本申请提供的第二有效微液滴的结构示意图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
为了使本申请的目的、技术方案及优点更加清楚明白，以下通过实施例，并结合附图，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
针对传统的数字PCR仪器存的有效反应单元数量少，耗材成本高、动态范围较窄，工作效率低以及集成化程度不高的问题，提供一种数字PCR检测仪。
请参见图1，本申请提供一种数字PCR检测仪1，所述数字PCR检测仪1包括：微液滴生成装置10、温控装置20、荧光信号检测装置30、定量分析装置40以及控制器50。所述微液滴生成装置10用以将核酸扩增反应液微滴化，形成多个微液滴。所述温控装置20与所述微液滴生成装置10通过轨道连接，用以将所述多个微液滴转移至所述温控装置20，进行温度循环，实现核酸扩增。所述荧光信号检测装置30与所述温控装置20相对设置，用以对核酸扩增后的所述多个微液滴进行拍照检测。所述定量分析装置40与所述荧光信号检测装置30通过数据线连接，用以实现所述多个微液滴荧光信息的传输，进行定量分析。所述控制器50分别与所述微液滴生成装置10、所述温控装置20、荧光信号检测 装置30以及定量分析装置40连接，用以控制所述微液滴生成装置10、所述温控装置20、荧光信号检测装置30以及定量分析装置40。
所述数字PCR检测仪1可以将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，从而使得操作人员可以实现自动化操作。所述数字PCR检测仪1具有较高的工作效率。
所述数字PCR检测仪1在工作时，所述微液滴生成装置10可以将所述待测核酸扩增反应液进行微滴化，从而形成多个微液滴。所述温控装置20可以对所述多个微液滴进行核酸扩增。所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图片。通过所述多个微液滴的荧光变化图片，可以获取所述多个微液滴的荧光变化曲线。根据所述荧光变化曲线，可以获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始DNA的浓度进行定量分析。其中，Ct值是指每个微液滴的荧光信号达到设定的阈值时所经历的循环数。
所述温控装置20对所述多个微液滴进行核酸扩增反应，并通过所述荧光信号检测装置30采集核酸扩增反应后的所述多个微液滴的产物信号，如荧光、紫外吸收、浊度等信号。利用所述多个扩增与非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定量分析。通过实时监测所述多个微液滴的荧光变化图片，检测结果具有直接性，可以解决所述多个微液滴中的假阳性和假阴性的问题。
所述数字PCR检测仪1将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，使得所述操作人员可以实现自动化操作，不进提高了工作效率，还具有反应快速、重复性好、灵敏度高、特异性强和结果清晰的优点。
在一个实施例中，提供一种微液滴快速生成且体积大小均一性高的微液滴生成方法及装置。
请参见图2，在一个实施例中，所述微液滴生成装置10包括吐液枪头110、流体驱动机构120、运动控制机构130以及第一控制器170。所述吐液枪头110具有出口端及入口端，并用于储存第一液体。微液滴生成装置10可以与微液滴容器配合使用。所述微液滴容器中储存有第二液体，所述吐液枪头110的出口端插入所述第二液体的液面下。
所述第一液体与所述第二液体之间互不相溶或具有界面反应。第一液体和第二液体可以为任意不互溶的两种液体，在本申请的一个实施例中，所述第一液体为水溶液，所述第二液体为与水不互溶的油性液体，如矿物油(包括正十四烷等)、植物油、硅油和全氟烷烃油等，生成的液滴为水溶液液滴。或者，所述第一液体为矿物油，如十四烷和正己烷等有机相，所述第二液体为与矿物油不互溶的全氟烷烃油。所述第一液体和第二液体可以为不互溶的双水相，在本申请的另一个实施例中，所述第一液体为水溶液，所述第二液体为与水不互溶的水性液体，如第一液体为右旋糖酐溶液，第二液体为聚乙二醇(PEG)水溶液，生成的液滴为右旋糖酐溶液液滴。
所述第一液体和第二液体也可以为具有界面反应的两种液体，在本申请的一个实施例 中，所述第一液体为海藻酸纳水溶液，所述第二液体为氧化钙水溶液，如质量浓度为1％的氧化钙水溶液，两者存在界面反应，生成的液滴为海藻酸钙凝胶微球。本申请还可以通过更换吐液枪头或吐液枪头内流出第一液体的组分，顺次在开口容器中形成多个不同组分和体积的液滴，既可以用于实现大批量的微体积高通量筛选，也可以实现多步骤的超微量生化反应和检测，具有广阔的应用前景。
所述流体驱动机构120与所述吐液枪头110的入口端连接，用于将储存在所述吐液枪头110内部的所述第一液体从所述吐液枪头110的出口端排出。所述运动控制机构130用于控制所述吐液枪头110的出口端与所述第二液体之间产生设定轨迹或设定速度或设定加速度的相对运动，以使排出所述吐液枪头110的出口端的第一液体克服表面张力及所述吐液枪头110对其的附着力形成微液滴。所述第一控制器170分别与所述流体驱动机构120以及所述运动控制机构130连接，用以控制所述流体驱动机构120以及所述运动控制机构130协调工作。
在一个实施例中，提供一种微液滴生成过程稳定的微液滴生成方法。
如图3所示，在本申请一实施例中，在运动控制机构130的带动下，吐液枪头110的出口端112可以在第二液体液面下做包含瞬时加速的运动，加速度大小为a 1。第一液体从吐液枪头110的出口端112排出后形成附着在吐液枪头110的出口端112的液滴195。液滴195在吐液枪头110的出口端112瞬时加速的瞬间脱离吐液枪头110的出口端112形成微液滴。微液滴在脱离吐液枪头110的出口端112之前的所受到的作用力分别为重力G、第二液体的浮力f 1、第二液体的粘滞阻力f 2以及吐液枪头110的出口端112与液滴195之间的最大附着力f 3。微液滴在脱离吐液枪头110的出口端112之前的质量为m、加速度大小为a 2。根据牛顿第二运动定律，得出
Figure PCTCN2019072974-appb-000001
吐液枪头110的出口端112与液滴195之间附着力的最大值f 3与吐液枪头110的表面自由能、液滴195的表面张力以及吐液枪头110的几何尺寸有关。吐液枪头110的出口端112做瞬时加速运动时，吐液枪头110的出口端112对液滴195附着力的方向与加速度的方向相同。将附着在吐液枪头110的出口端112的液滴195简化为球状。由斯托克斯(Stokes)公式可知，液滴195在第二液体中运动时所受到的粘滞阻力f 2＝6πηrv，其中η为第二液体的粘滞系数，r为液滴195的半径，v为液滴195的运动速度。在吐液枪头110的出口端112做瞬时加速之前液滴195的速度为零，因此液滴195在吐液枪头110的出口端112瞬时加速的瞬间在第二液体中受到的粘滞阻力f 2为零或极小。在微液滴生成的过程中，一般液滴195的直径范围在皮升至微升的数量级，且液滴195的重力G和第二液体的浮力f 1方向相反，因此液滴195的重力G与第二液体的浮力f 1的矢量和约为零。由于粘滞阻力f 2为零或极小，以及重力G与浮力f 1的矢量和约为零，所以
Figure PCTCN2019072974-appb-000002
由牛顿第二运动定律可知，吐液枪头110的出口端112做瞬时加速运动时，液滴195在第二液体中能达到的最大加速度为a 2≈f 3/m，其中m为液滴195的质量。当液滴195的加速度a 2小于吐液枪头110的出口端112的加速度a 1时，液滴195从吐液枪头110的出口端112掉落形成微液滴。因此，液滴195脱离吐液枪头110的出口端112(即生成一个微液滴)的条件近 似为：a 2≈(f 3/m)＜a 1。
运动控制机构130能够精确控制吐液枪头110的出口端112瞬时加速度的大小。因此，通过控制吐液枪头110的出口端112每次瞬时加速度的值均较大，吐液枪头110的出口端112做瞬时加速运动能够有效的生成液滴195。
基于此，本申请还提供一种微液滴生成方法，包括以下步骤：
S201，提供具有出口端112的吐液枪头110，吐液枪头110内储存有第一液体；提供储存有第二液体的微液滴容器，微液滴容器具有开口，其中第一液体与第二液体为任意互不相溶的两种液体或具有界面反应的两种液体；
S202，吐液枪头110的出口端112由微液滴容器的开口插入第二液体的液面下；
S203，吐液枪头110的出口端112在第二液体液面下做包含瞬时加速的运动，同时第一液体由吐液枪头110的出口端112排出，排出吐液枪头110的出口端112的第一液体形成附着在吐液枪头110的出口端112的液滴195，液滴195在吐液枪头110的出口端112的瞬时加速运动过程中脱离吐液枪头110的出口端112在第二液体液面下形成微液滴。
在本申请一实施例中，步骤S203中，吐液枪头110的出口端112在第二液体液面下做包含瞬时加速的周期运动。吐液枪头110的出口端112在第二液体液面下做周期运动，意即吐液枪头110的出口端112的位移、速度及加速度均呈现出周期性的变化。吐液枪头110的出口端112做包含瞬时加速运动的周期运动，配合第一液体由吐液枪头110的出口端112以恒定的流速排出，实现了微液滴的等时间间隔生成。或者第一液体排出吐液枪头110的出口端112的流速是变化的，但在吐液枪头110的出口端112的一个运动周期内，第一液体排出吐液枪头110的出口端112的体积保持相同。以此保证每次吐液枪头110的出口端112瞬时加速前液滴195的体积是相同的，以生成体积大小一致的微液滴。
在不更换吐液枪头110及第一液体的情况下，吐液枪头110的表面自由能、吐液枪头110的几何尺寸及液滴195的表面张力作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是确定的。因此，在不更换吐液枪头110及第一液体的情况下，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3是固定的。在流体驱动机构120的带动下，第一液体能够实现以均匀的流速连续排出吐液枪头110的出口端112。运动控制机构130能够精确控制吐液枪头110的出口端112做瞬时加速度a 1运动的时刻及瞬时加速度a 1的大小。流体驱动机构120与运动控制机构130相互配合能够容易的实现当液滴195的体积达到固定值的瞬间，驱动吐液枪头110的出口端112产生加速度为a 1的瞬时加速度，以生成体积大小一致的微液滴。如果流体驱动机构120控制第一液体均匀、连续的排出吐液枪头110的出口端112，只需运动控制机构130驱动吐液枪头110的出口端112产生等时间间隔的瞬时加速运动，即可生成体积大小一致的微液滴。
当使用多个吐液枪头110同时或者顺次生成微液滴时，吐液枪头110的表面自由能及吐液枪头110的几何尺寸作为影响吐液枪头110的出口端112与液滴195之间最大附着力f 3的两个因素是变化的。但批量加工能够控制吐液枪头110的表面自由能及吐液枪头110的几何尺寸在一定的区间内变化。液滴195的表面张力作为影响吐液枪头110的出口端112 与液滴195之间最大附着力f 3的另一个因素也只是在很小的范围内变化。因此，吐液枪头110的出口端112与液滴195之间附着力的最大值f 3只在很小的区间内波动。流体驱动机构120能够驱动第一液体以均匀的流速连续排出吐液枪头110的出口端112。运动控制机构130能够精确控制吐液枪头110的出口端112做瞬时加速度a 1运动的时刻及瞬时加速度a 1的大小。流体驱动机构120与运动控制机构130相互配合能够容易的实现当液滴195的体积达到固定值的瞬间，驱动吐液枪头110的出口端112产生加速度为a 1的瞬时加速度，以生成体积大小一致的微液滴。如果流体驱动机构120控制第一液体均匀、连续的排出吐液枪头110的出口端112，只需运动控制机构130驱动吐液枪头110的出口端112产生等时间间隔的瞬时加速运动，即可生成体积大小一致的微液滴。
流体驱动机构120在将第一液体匀速排出吐液枪头110的出口端112的同时，配合运动控制机构130在液滴195的体积达到设定值的瞬间做加速度值较大的瞬时加速运动。本申请提供的微液滴生成方法不仅保证了使用同一根吐液枪头110生成体积大小均一的液滴195，同时能够保证多根吐液枪头110同时或者顺次生成的微液滴体积大小的均一性。本实施例提供的微液滴生成方法在保证微液滴体积大小均一性的同时，可通过多根吐液枪头110同时生成微液滴提高微液滴的生成效率。
进一步的，在运动控制机构130的控制下，吐液枪头110的出口端112在一个周期性运动内包括多次瞬时加速运动，多次瞬时加速运动的加速度大小相同，且多次瞬时加速运动的时刻均分吐液枪头110的出口端112的一个运动周期。吐液枪头110的出口端112在一个周期性运动内包括多次瞬时加速运动有助于在吐液枪头110的出口端112在一个运动周期内生成多个微液滴。可选的，步骤S203中，吐液枪头110的出口端112在第二液体液面下的运动轨迹包括直线段、圆弧段、多边形等多种轨迹中的一种或多种的组合。作为一个可实现的方式，当吐液枪头110的出口端112在一个周期性运动内包括两次瞬时加速运动时，吐液枪头110的运动轨迹为直线或者圆弧。当吐液枪头110的出口端112在一个周期性运动内包括两次以上的瞬时加速运动时，吐液枪头110的出口端112在第二液体内做轨迹为正多边形，包括正三角形、正方形、正五边形、正六边形等。
作为一种可实现的方式，在步骤S203中，吐液枪头110的出口端112在第二液体液面下的周期运动过程中，吐液枪头110的出口端112的速度大小呈矩形波变化。吐液枪头110的出口端112的速度大小呈矩形波变化，加速阶段结束以后即进入匀速阶段，有利于运动控制机构130实现对吐液枪头110的出口端112的运动状态的精确控制。可选的，表示吐液枪头110的出口端112运动速度大小变化的矩形波的高位时间和低位时间可以是相等的也可以是相异的。进一步，在步骤S203中，吐液枪头110的出口端112在第二液体液面下的周期运动过程中，吐液枪头110的出口端112的速度大小呈方波变化。表示吐液枪头110的出口端112运动速度大小变化的矩形波的高位时间和低位时间是相等的。表示吐液枪头110的出口端112运动速度大小变化的矩形波处于低位时，吐液枪头110的出口端112的速度为零或具有相对于高位时反方向的速度。如图4所示，更进一步的，所述吐液枪头110的出口端112周期运动的前半周期与后半周期内，所述吐液枪头110的出口端 112的速度大小相同，方向相反。在吐液枪头110的出口端112的一个运动周期内包含两次方向相反的瞬时加速运动。
在本实施例中，吐液枪头110的出口端112在第二液体液面下的运动轨迹是直线段，吐液枪头110的出口端112从直线段的一个端点做瞬时加速运动，从直线段的另一个端点做反方向的瞬时加速运动。两次瞬时加速运动的的加速度大小均为a 1。在其他的实施例中，吐液枪头110的出口端112在第二液体液面下的运动轨迹是圆弧段或者多边形。进一步，步骤S203中，吐液枪头110的出口端112在第二液体液面下周期运动的频率介于0.1赫兹至200赫兹之间，在工程上容易实现。
如图4及图5所示，在本申请一个具体的实施例中，流体驱动机构120控制第一液体以恒定的流速排出吐液枪头110的出口端112。运动控制机构130控制吐液枪头110的输出端做运动轨迹为直线、速度呈方波变化的周期运动。当吐液枪头110的出口端112的速度方向发生改变时，吐液枪头110的出口端112的瞬时加速度达到最大值。附着在吐液枪头110的出口端112的液滴195也在吐液枪头110的出口端112的瞬时加速度达到最大值时脱离吐液枪头110的出口端112而形成微液滴199。由于第一液体是以恒定流速排出吐液枪头110的出口端112，当液滴195从吐液枪头110的出口端112脱落时，新的液滴195进入生成状态。当吐液枪头110的出口端112再次反向加速时，新生成的液滴195也从吐液枪头110的出口端112掉落形成新的微液滴199。
本实施例中，吐液枪头110的出口端112的一个运动周期内可生成两个微液滴199，且方波在工程上较易实现。在其他的实施例中，吐液枪头110的出口端112的一个运动周期内生成一个微液滴199。可选的，在实施例中吐液枪头110的出口端112在第二液体699中沿任意方向做轨迹为直线的方波运动，包括：在与吐液枪头110的延伸方向垂直的平面内做轨迹为直线的方波运动、在与吐液枪头110的延伸方向成任意角度的平面内做轨迹为直线的方波运动、沿吐液枪头110的延伸方向做轨迹为直线的方波运动等。在本申请的其他实施例中，吐液枪头110的出口端112的运动轨迹为圆弧段或多边形时，吐液枪头110的出口端112在第二液体699中沿任意方向做轨迹为直线的方波运动，包括：在与吐液枪头110的延伸方向垂直的平面内做轨迹为直线的方波运动、在与吐液枪头110的延伸方向成任意角度的平面内做轨迹为直线的方波运动、沿吐液枪头110的延伸方向做轨迹为直线的方波运动等。
在一个实施例中，所述微液滴199为待测核酸扩增反应液，通过所述微液滴生成装置10将所述待测核酸扩增反应液微滴化，形成多个微液滴，用以通过所述数字PCR检测仪1进行检测。将所述待测核酸扩增反应液通过一体式数字PCR检测仪1的微液滴生成装置10，通过微滴化处理转化为多个微液滴199，使得待测样本中的检测片段从大量的复杂背景中分离出来，并放置于微液滴容器60中，等待检测。通过微液滴生成装置10可以生成多个大小均一的微液滴199。每个所述微液滴199大小在微米级，并且每个所述微液滴199可以视作一个独立的反应器，相当于生化反应中常用的试管。所述多个微液滴199放置于微液滴容器60中，便于检测观察。同时，通过所述微液滴生成装置10也可以生成多个体 积不同的微液滴，用以进行医学临床检测。所述多个微液滴199体积小、数量多，具有许多常规试管没有的优势。通过所述微液滴生成装置10可以生成大量微液滴199，使得所述数字PCR检测仪1具有通量高、耗材成本低和背景噪声低的优点，具有很好的工业化前景。
在一个实施例中，提供一种升降温速率快、使用寿命高的温控装置。
如图7-8所示，本申请还提供一种温控装置20，其包括柔性电路板220、与所述柔性电路板220间隔设置的加热基板240以及多个半导体电偶对230。所述加热基板240包括相对设置的第一表面241和第二表面242。所述多个半导体电偶对230设置于所述柔性电路板220与所述第一表面241之间，所述多个半导体电偶对230相互串联、并联或者混合连接。
所述温控装置20通常应用于高低温循环环境中，温度需要快速升降，所以对所述温控装置20要求极高。为了满足所述温控装置20的应用需求，所述温控装置20采用所述柔性电路板220。所述柔性电路板220具有配线密度高、重量轻、厚度薄、弯折性好的特点。所述柔性电路板220在升降温过程中以自身的变形消除热应力。通过所述柔性电路板220可以降低升降温过程中存在的热应力，从而延长了所述温控装置20的使用寿命。同时，通过所述柔性电路板220，解决了温度分布不均匀的问题。当所述多个微液滴在不同温度范围内进行核酸扩增时，通过所述柔性电路板220、加热基板240以及多个半导体电偶对230可以实现迅速在几秒时间内进行切换。所述温控装置20可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环，将所述数字PCR检测仪1的检测时间缩短了，提高了检测效率。
所述柔性电路板220(Flexible Printed Circuit，FPC)可以是以聚酰亚胺或聚酯薄膜为基材制成的一种具有高度可靠性，绝佳的可挠性印刷电路板。所述柔性电路板具有配线密度高、重量轻、厚度薄、弯折性好的特点。所述柔性电路板重量轻、厚度薄，可以有效节省产品体积。其中，所述半导体致冷器(Thermo Electric Cooler，TEC)是利用半导体材料的珀尔帖效应制成的。所谓珀尔帖效应，是指当直流电流通过两种半导体材料组成的电偶时，其一端吸热，一端放热的现象。通过所述柔性电路板220替换传统半导体致冷器中的一个基板，使得所述半导体致冷器导热性能更好。
当温度改变时，物体由于外在约束以及内部各部分之间的相互约束，使其不能完全自由胀缩而产生的应力。热应力又称变温应力。热应力与零外载相平衡，是由热变形受约束引起的自平衡应力，在温度高处发生压缩，温度低处发生拉伸形变。在一定条件下控制应力使之合理分布，就可以提高零件的机械性能和使用寿命，变害为利。
在一个实施例中，所述加热基板240可以为超导铝基板电路。
铝基板是一种具有良好散热功能的金属基覆铜板，一般单面板由三层结构所组成，分别是电路层(铜箔)、绝缘层和金属基层。所述超导铝基板电路是线路板的材料是铝合金，能够导热快。铝基板能够将热阻降至最低，使铝基板具有极好的热传导性能，与厚膜陶瓷电路相比，它的机械性能又极为优良。
如图9所述，在一个实施例中，所述半导体电偶对230包括一个P型电偶231和一个 与所述P型电偶231间隔设置的N型电偶232。
所述P型电偶231与所述N型电偶232焊接在所述柔性电路板220和所述基板240之间。所述半导体电偶对230包括一些由所述P型电偶231和所述N型电偶232形成的一对电偶，多对所述半导体电偶对230之间通过电极连在一起，并且夹在所述柔性电路板220与所述第一表面241之间。当有电流流过时，会产生″热″侧和″冷″侧。是致冷还是加热，以及致冷、加热的速率，由通过它的电流方向和大小来决定。一对所述半导体电偶对230产生的热电效应很小，所以在实际中都将上百对所述半导体电偶对230串联在一起，这样产生的热电效应就会增大。
在一个实施例中，所述第一表面241包括多个间隔设置的第一电极片243，一个所述第一电极片243与一个所述半导体电偶对230对应，所述半导体电偶对230中的所述P型电偶231与所述N型电偶232通过所所述第一电极片243串联。
在一个实施例中，所述柔性电路板220包括多个间隔设置并相互串联的第二电极片221，相邻的两个所述半导体电偶对230通过一个所述第二电极片221串联。
当所述P型电偶231和所述N型电偶232联结成的所述半导体电偶对230中有电流通过时，两端之间就会产生热量转移，热量就会从一端转移到另一端，从而产生温差形成冷热端。但是所述P型电偶231和所述N型电偶232自身存在电阻当电流经过所述P型电偶231和所述N型电偶232时就会产生热量，从而会影响热传递。而且所述柔性电路板220与所述加热基板240之间的热量也会通过空气和所述P型电偶231和所述N型电偶232材料自身进行逆向热传递。当冷热端达到一定温差，这两种热传递的量相等时，就会达到一个平衡点，正逆向热传递相互抵消。此时冷热端的温度就不会继续发生变化。为了达到更低的温度，可以采取散热等方式降低热端的温度来实现。
在一个实施例中，所述温控装置20还包括导热增强层250，设置于所述第二表面242。
所述导热增强层250具有十分良好的强度、柔韧、导电、导热、光学特性。所述导热增强层250直接与所述微液滴容器60接触，可以使得所述多个微液滴受热均匀，从而通过对温度的控制实现核酸扩增。所述导热增强层250可以为石墨导热层或者硅脂导热层，加快导热，增加了所述加热基板240的所述第二表面242的温度均匀性，进而保证靠近所述微液滴容器60的表面温度均匀，从而使得所述多个微液滴受热均匀。进而完成核酸扩增，提高了检测效率，节省了时间。
在一个实施例中，所述导热增强层250的材料中包括石墨烯。所述石墨烯是一种平面薄膜具有非常好的热传导性能，横向可以实现均匀导热。
在一个实施例中，所述温控装置20还包括第二控制器210，与所述柔性电路板220电连接，用于控制电流大小。
在一个实施例中，所述温控装置20进一步包括温度传感器260，设置于所述第二表面242，并与所述第二控制器210电连接，用于检测所述第二表面242的温度并将该温度发送给所述第二控制器210。
所述温度传感器260设置于所述导热增强层250的所述第二表面242，用以检测所述 第二表面242的实时温度，进而将温度信息反馈给所述第二控制器210，从而实现对所述多个微液滴加热温度的控制。所述温度传感器260用于通过检测金属的电阻变化来测量所述微液滴容器60的温度，用以实时检测所述多个微液滴进行核酸扩增过程中的温度变化，从而将温度信息反馈给所述第二控制器210，进而控制所述控制电路进行温度的调控，实现温度控制，更好进行核酸扩增。
在一个实施例中，所述第二控制器210包括温度控制单元212以及控制电路214。所述温度控制单元212与所述温度传感器260连接，用以实时检测所述第二表面242的温度。所述控制电路214与所述柔性电路板220连接，用以调控所述多个半导体电偶对230的温度变化。
所述温度控制单元212与所述控制电路214设置于一块电路板上。所述温度控制单元212与所述控制电路214的关系为内部算法的逻辑运算关系，可以采用Packet Identifier闭环控制算法，亦即PID闭环控制算法。所述温度控制单元212检测到的温度是核酸扩增的温度反馈，作为内部算法的输入，所述控制电路214计算后的结果作为内部算法的输出，从而形成闭环关系。电路部分的温度反馈实际上是一个采样电路。采集的就是铂电阻上的电信号，转换为温度值传递给控制电路的输入端。所述温度传感器260与所述温度控制单元212通过标准的铂电阻三线制连接。
在一个实施例中，所述柔性电路板220设置有第一电极222以及第二电极223。所述多个第二电极片221串联后与所述第一电极222和所述第二电极223串联。所述第一电极222与所述第二电极223分别与所述控制电路连接。
所述控制电路214与所述柔性电路板220之间的连接为两根线，分别连接所述第一电极222以及所述第二电极223。
在一个实施例中，所述温控装置20还包括散热装置270，所述散热装置270包括基板271以及与所述基板271连接的散热片272。所述柔性电路板220设置于所述基板271的表面。
由于所述散热片272设置于所述基板271表面，在没有减少所述基板271面积的情况下，更增加了热交换面积，延长凉风作用于所述基板271表面的时间，形成的多股散热风道，也有利于加快热交换，把更多的热量从所述基板271表面上带走，从而达到更加理想的散热效果。
在一个实施例中，所述温控装置还包括风扇273设置于所述散热片273周围。
通过所述风扇273可以协助所述散热装置270进行散热。其中，所述风扇273设置于所述散热片273的周围，可以设置多个，从而可以达到更好的散热效果，进而使得所述温控装置20升温降温更加快速。
在一个实施例中，对所述温控装置20通以交流电，并通过所述第二控制器210对电流大小调节来控制所述温控装置20是致冷还是加热，以及致冷、加热的速率。同时，通过所述温度传感器260用以检测所述微液滴容器60的实时加热温度，进而将温度信息反馈给所述温度控制单元212。所述温度控制单元212将温度变化情况反馈给所述控制电路 214，从而控制所述多个微液滴的温度。通过所述温控装置20可以使得所述多个微液滴进行核酸扩增。基于PCR原理三步骤而设置变性-退火-延伸的三个温度点。在标准反应中采用三温度点法，双链DNA在90～95℃变性，再迅速冷却至40～60℃，引物退火并结合到靶序列上，然后快速升温至70～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸，在适合的温度范围内对核酸进行扩增。同时，在核酸扩增过程中，所述微液滴容器60底部与所述温控装置20紧密帖合，两者之间不会有间隙，提高了所述数字PCR检测仪1的准确性。
请参见图10，所述温控装置20一般情况下测试温控性能主要有两个指标，分别在瞬时状态和稳定状态下观测所述温控装置20的升降温度变化情况。通过对所述多个微液滴加热过程的监控，所述温控装置20对所述多个微液滴进行温度升降时，升降温速率最大可以达到13.34448℃/s，控制精度为0.02722℃。并且，有时所述温控装置20升温到稳态的时候测量的速率最快可以到18.953894℃/s。因此，所述温控装置20的瞬态响应好，通过所述温控装置20可以实现瞬时升温降温，节省了时间，提高了检测效率。
请参见图11，当所述温控装置20处于稳定状态时，也就是在达到稳定后温度浮动的情况。当所述温控装置20处于稳定状态时，温度变化比较平稳，温度浮动较小。因此，所述温控装置20可以达到快速的升温降温循环，且稳定后温服浮动较小，节省了数字PCR检测溶液样本的时间，提高了工作效率。通过这种升降温速率会缩短完成核酸扩增所需的时间，提高了核酸扩增效率，并且提高了数字PCR检测系统的准确性。
请参见图6，在一个实施例中，所述荧光信号检测装置30包括激发光源340、荧光探测组件330以及第三控制器310。所述激发光源340设置于所述微液滴容器60检测区域上方，并与所述微液滴容器60检测区域呈倾斜角度进行照射，形成斜射光路。所述荧光探测组件330设置于所述微液滴容器60检测区域正上方，用以采集所述多个微液滴的荧光图像。所述第三控制器310分别与所述激发光源340和所述荧光探测组件330连接，用以控制所述激发光源340与所述荧光探测组件330。所述荧光信号检测装置可以对微液滴进行多个荧光通道成像以及进行明场暗场成像。其中多个荧光通道成像用于微液滴反应信号的探测，明场暗场成像用于检测形成微液滴的尺寸信息以及在反应过程中监测液滴的状态。
在一个实施例中，所述第三控制器310可以控制所述激发光源340移动，而此时所述荧光探测组件330与所述微液滴容器60的不移动。也就是说，此时通过所述激发光源340移动对所述多个微液滴进行荧光检测。或者，所述第三控制器310可以控制所述荧光探测组件330移动，而此时所述微液滴容器60与所述激发光源340不移动，对所述多个微液滴进行荧光检测。又或者，所述第三控制器310可以控制所述微液滴容器60移动，所述荧光探测组件330与所述激发光源340不移动，对所述多个微液滴进行荧光检测。通过所述第三控制器310可以调节所述激发光源340、所述荧光探测组件330以及所述微液滴容器60的位置移动，进而可以产生相对运动，从而使得检测区的所述微液滴容器60与所述荧光探测组件330对准，进行拍照，完成整个荧光检测的过程。
通过所述激发光源340发射的光路倾斜照射于所述多个微液滴，使得所述微液滴容器60中含有荧光物质的微液滴产生荧光。通过所述荧光探测组件330对所述含有荧光物质的微液滴进行荧光信息采集，并将所述含有荧光物质的微液滴进行荧光信息以荧光图像的形式传输至所述定量分析装置40，用以进行定量分析。
从所述微液滴容器60上方采取倾斜角度照射在所述微液滴容器60上。采用所述荧光信号检测装置30实现对所述多个微液滴进行周期性的二维扫描，并实时进行拍照。斜射光路可有效降低激发光散射背景，提高荧光检测的灵敏度。所述微液滴容器60内的所述多个微液滴的内部荧光被激发，通过所述荧光探测组件330采集所述多个微液滴的荧光图像。
所述激发光源340给所述多个微液滴提供蒸发、原子化或激发的能量。所述激发光源340具有光谱带宽窄、频谱纯度高、波长稳定性好、效率高、寿命长、可靠性好、光束质量好等特点，从而保证了检测结果的准确性与稳定性。
在一个实施例中，所述激发光源340包括：多个不同颜色的LED光源341、二向色镜344、复眼透镜345以及聚焦透镜346。每个所述LED光源341前端依次设有准直镜342和第一滤光片343。所述二向色镜344倾斜设置于所述第一滤光片343前端，用以将每个所述LED光源341发出的光折射成一条光路。所述复眼透镜345用以提高经折射后的所述光路的均匀性。所述聚焦透镜346设置于所述复眼透镜345的前端，用以聚焦成像。
所述多个不同颜色的LED光源341作为激发光源340，可以产生不同颜色的荧光，用以增大检测通道，实现不容种类的微液滴的检测。每个所述LED光源341的前端依次设有所述准直镜342和所述第一滤光片343。所述准直镜342可以用在光束传递系统中，以维持激光谐振腔和聚焦光学元件之间的光束的准直性。并且，所述LED光源341通过所述第一滤光片343可以将所需波段的光分离出来作为激发光。利用所述二向色镜344、所述复眼透镜345以及所述聚焦透镜346等光学镜片，将激发光变成平行或是汇聚的光束，照射到芯片上含有多个液滴的区域，形成激发区。并将激发光激发所述微液滴容器60中的所述多个微液滴。
在一个实施例中，所述激发光源340与所述荧光探测组件330是一体的或者是分体的。
将所述荧光信号检测装置30可以倾斜的角度照射到所述微液滴容器60时，所述荧光信号检测装置30对所述多个微液滴进行周期性的二维扫描，并实时进行拍照。通过将所述荧光信号检测装置30以倾斜的角度照射到所述微液滴容器60时，可有效降低激发光散射背景，提高荧光检测的灵敏度。所述微液滴容器60内的所述多个微液滴的内部荧光被激发，通过所述第二滤光片333被上方的所述物镜332收集，进入所述相机331，所述相机331采集所述多个微液滴的荧光图像。
通过所述聚焦透镜346的光路倾斜照射于所述多个微液滴，使得所述微液滴容器60中含有荧光物质的微液滴产生荧光。通过所述荧光探测组件330对所述含有荧光物质的微液滴进行荧光信息采集，并将所述含有荧光物质的微液滴进行荧光信息以荧光图像的形式传输至所述定量分析装置40，用以进行定量分析。
在一个实施例中，所述第三控制器310可以同步开启所述多个不同颜色的LED光源341和所述相机331。
所述第一滤光片343用来选取所需辐射波段的光学器件。所述第一滤光片343是塑料或玻璃片再加入特种染料做成的，红色滤光片只能让红光通过，如此类推。玻璃片的透射率原本与空气差不多，所有有色光都可以通过，所以是透明的，但是染了染料后，分子结构变化，折射率也发生变化，对某些色光的通过就有变化了。比如一束白光通过蓝色滤光片，射出的是一束蓝光，而绿光、红光极少，大多数被滤光片吸收了。所述二向色镜344倾斜设置于所述第一滤光片343前端，用以将每个所述LED光源341发出的光折射成一条光路。所述复眼透镜345，用以提高经折射后的所述光路的均匀性。所述聚焦透镜346设置于所述复眼透镜345的前端，用以聚焦成像。所述聚焦透镜346属于梯度折射率透镜。具有端面聚焦和成像的特性，以及其具有圆柱状的外形特点，因而可以应用于多种不同的微型光学系统中。
通过所述第三控制器310可以控制所述多个不同颜色的LED光源341之间的切换，从而构成不同的荧光检测通道。所述多个不同颜色的LED光源341可以轮流工作，不需要单独设置转轮。
所述准直镜342分为反射式准直镜和透射式准直镜。反射式准直镜和透射式准直镜被用在光束传递系统中，以维持激光谐振腔和聚焦光学元件之间的光束的准直性。反射式准直镜一般使用的是铜制全反镜，而透射式准直镜则使用硒化锌透镜。
在一个实施例中，所述荧光探测组件330包括物镜332、相机331以及第二滤光片333，所述物镜332设置于所述相机331与所述第二滤光片333之间。
所述第二滤光片333采用多带通滤光片。所述多带通滤光片同时可以通过多个波段的光，每一个波段对应一种染料。在生物医学荧光检验分析系统中分离和选择物质的激发光与发射荧光的特征波段光谱。分子在吸收带吸收激发光谱，然后在发射带发射长波的辐射光谱，即形成了荧光光谱。
所述多个微液滴的荧光图像的生成主要是通过所述相机331完成。所述相机331能够把光学影像转化为数字信号。所述相机331中排列有许多整齐的电容，能感应光线，并将影像转变成数字信号。经由外部电路的控制，每个小电容能将其所带的电荷转给它相邻的电容。采用所述相机331完成对所述多个微液滴的荧光采集，能够提供直观可视化的荧光图像，提高了荧光检测的速度，使得检测结果更加准确。
通过所述荧光信号检测装置30可以使得所述多个微液滴荧光成像，一次拍摄一定数量的所述多个微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。
通过所述荧光信号检测装置30可以使得所述多个微液滴荧光成像，一次拍摄一定数量的所述多个微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。
从所述微液滴容器60上方采取倾斜角度照射在所述微液滴容器60上。采用所述荧光 信号检测装置30实现对所述多个微液滴进行周期性的二维扫描，并实时进行拍照。斜射光路可有效降低激发光散射背景，提高荧光检测的灵敏度。所述微液滴容器60内的所述多个微液滴的内部荧光被激发，通过所述第二滤光片333被上方的所述物镜332收集，进入所述相机331，所述相机331采集所述多个微液滴的荧光图片。
所述激发光源340给所述多个微液滴提供蒸发、原子化或激发的能量。所述激发光源340具有光谱带宽窄、频谱纯度高、波长稳定性好、效率高、寿命长、可靠性好、光束质量好等特点，从而保证了检测结果的准确性与稳定性。
在一个实施例中，为了防止连续照射造成的荧光漂白，采用计算机来同步所述LED光源341的开启与所述相机331的采集。非采集状态下所述LED光源341保持关闭状态。
通过所述第三控制器310可以控制所述多个不同颜色的LED光源341之间的切换，从而构成不同的荧光检测通道。所述多个不同颜色的LED光源341可以轮流工作，不需要单独设置转轮。每个所述LED光源341前端依次设有准直镜342和第一滤光片343。
所述准直镜342分为反射式准直镜和透射式准直镜。反射式准直镜和透射式准直镜被用在光束传递系统中，以维持激光谐振腔和聚焦光学元件之间的光束的准直性。反射式准直镜一般使用的是铜制全反镜，而透射式准直镜则使用硒化锌透镜。
所述第一滤光片343用来选取所需辐射波段的光学器件。滤光片是塑料或玻璃片再加入特种染料做成的，红色滤光片只能让红光通过，如此类推。滤光片产品主要按光谱波段、光谱特性、膜层材料、应用特点等方式分类。
所述二向色镜344倾斜设置于所述第一滤光片343前端，用以将每个所述LED光源341发出的光折射成一条光路。所述二向色镜344的原理是在其内放置一无色方解石(冰洲石)，以将光线分解成两垂直振荡的光，而透过二向色镜分别观察这两光线的颜色。
所述复眼透镜345，用以提高经折射后的所述光路的均匀性。所述复眼透镜345是由一系列小透镜组合形成，将双排复眼透镜阵列应用于照明系统可以获得高的光能利用率和大面积的均匀照明。复眼透镜在微显示器及投影显示领域有广阔的应用前景。利用双排复眼透镜阵列可以实现均匀照明，提高了多个不同颜色的所述LED光源的均匀性和照明亮度，并且可以有效的计算自身与所观察物体的方位，距离，可以获取更加精确的荧光图片。所述复眼透镜阵列要实现均匀照明需两列复眼透镜阵列平行排列，第一列复眼透镜阵列中的各个小单元透镜的焦点与第二列的复眼透镜阵列中对应的小单元透镜的中心重合，两列复眼透镜的光轴互相平行，在第二列复眼透镜后放置聚光镜，聚光镜的焦平面放照明屏就形成了均匀照明系统。
所述复眼透镜阵列实现均匀照明的原理是：与光轴平行的光束通过第一块透镜后聚焦在第二块透镜的中心处，第一排复眼透镜交光源形成多个光源像进行照明，第二排复眼透镜的每个小透镜将第一排复眼透镜对就的小透镜重叠成像于照明面上。由于第一排复眼透镜将光源的整个宽光束分为多个细光束照明，且每个细光束范围内的垂泪不均匀性由于处于对称位置细光束的相互叠加，使细光束的垂泪不均匀性获得补偿，从而使整个孔径内的光能量得到有效均匀的利用。从第二排复眼透镜的出射的光斑通过聚光镜聚焦在照明屏 上，这样，照明屏上光斑的每一点均受到光源所有点发出的光线照射，同时，光源上每一点发出的光束又都交会重叠到照明光斑上的同一视场范围内，所以得到一个均匀的方形光斑。
所述聚焦透镜346设置于所述复眼透镜345的前端，用以聚焦成像。所述聚焦透镜346属于梯度折射率透镜。具有端面聚焦和成象的特性，以及其具有圆柱状的外形特点，因而可以应用于多种不同的微型光学系统中。
在一个实施例中，所述荧光探测组件330包括物镜332、相机331以及第二滤光片333，所述物镜332设置于所述相机331与所述第二滤光片333之间。
通过所述第三控制器310可以控制所述多个不同颜色的LED光源341之间的切换。所述第三控制器310可以同步开启所述多个不同颜色的LED光源341和所述相机331。所述多个微液滴的荧光图像的生成主要是通过所述相机331完成。所述相机331能够把光学影像转化为数字信号。所述相机331中排列有许多整齐的电容，能感应光线，并将影像转变成数字信号。经由外部电路的控制，每个小电容能将其所带的电荷转给它相邻的电容。采用所述相机331完成对所述多个微液滴的荧光采集，能够提供直观可视化的荧光图像，提高了荧光检测的速度，使得检测结果更加准确。
所述第二滤光片333采用多带通滤光片。所述多带通滤光片同时可以通过多个波段的光，每一个波段对应一种染料。在生物医学荧光检验分析系统中分离和选择物质的激发光与发射荧光的特征波段光谱。分子在吸收带吸收激发光谱，然后在发射带发射长波的辐射光谱，即形成了荧光光谱。
请参见图6，在一个实施例中，所述激发光源340包括5个不同颜色的LED光源341、5个所述准直镜342、5个所述第一滤光片343、4个所述二向色镜344、1个所述复眼透镜345以及1个聚焦透镜346。所述5个不同颜色的LED光源341可以生成不同颜色的光，照射至所述多个微液滴。通过对所述5个不同颜色的LED光源341进行选择，可以获得不同荧光颜色的照射，所述5个不同颜色的LED光源341可以轮流工作。每个LED光源发射的光路正前方依次设置有所述准直镜、所述第一滤光片343以及二向色镜344。所述准直镜342与所述第一滤光片343与光路呈垂直角度设置(90°角度设置)。所述二向色镜344与光路角度呈0°～45°设置。通过所述二向色镜344形成的一条光路，所述光路正前方依次设置有所述复眼透镜345以及所述聚焦透镜346。所述复眼透镜345与所述聚焦透镜346与光路呈垂直角度设置(90°角度设置)。
通过所述聚焦透镜346的光路倾斜照射于所述多个微液滴，使得所述微液滴容器中含有荧光物质的微液滴产生荧光。通过所述荧光探测组件330对所述含有荧光物质的微液滴进行荧光信息采集，并将所述含有荧光物质的微液滴进行荧光信息以荧光图像的形式传输至计算机，用以进行定量分析。
在一个实施例中，所述激发光源340中所述LED光源341、所述准直镜342、所述第一滤光片343、所述二向色镜344、所述复眼透镜345以及所述聚焦透镜346的个数不受限制。
所述激发光源340倾斜照射至所述微液滴容器60，用以照射所述多个微液滴。通过所述激发光源340形成的斜射光路可以有效降低激发光散射背景。同时，降低所述微液滴容器60的所述微液滴容器60侧壁的高度，有利于排除激发光从侧面照射时造成的的阴影，使得所述相机331能够获取所有微液滴的荧光信息，提高了所述荧光信号检测装置30的的灵敏度。
在一个实施例中，所述定量分析装置40为计算机。通过所述荧光信号检测装置30可以获得所述多个微液滴的荧光信息照片。所述计算机设置有分析软件，如matlab、microsoft office、origin以及Microsoft Office visual.c++等分析软件，用以实现对获得的所述多个微液滴的荧光信息进行定量分析。
在一个实施例中，所述控制器50分别与所述第一控制器170、所述第二控制器210以及所述第三控制器310连接，用以控制所述微液滴生成装置10、所述温控装置20、荧光信号检测装置30以及定量分析装置40工作。
所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化，形成多个微液滴。然后，通过所述温控装置20对所述多个微液滴进行加热的过程中，采用所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图像。通过所述定量分析装置40对所述多个微液滴的荧光变化图像进行分析，获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始核酸的浓度进行定量分析。
所述数字PCR检测仪1将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，使得所述操作人员可以通过一体式数字PCR检测机1实现自动化操作，提高了所述数字PCR检测仪1的工作效率。
所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化，形成多个微液滴。然后，通过所述温控装置20对所述多个微液滴进行核酸扩增。同时，采用所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图片。通过所述多个微液滴的荧光变化图片，获取所述多个微液滴的荧光变化曲线。根据所述荧光变化曲线，可以获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始DNA的浓度进行定量分析。其中，Ct值是指每个微液滴的荧光信号达到设定的阈值时所经历的循环数。
所述微液滴生成装置20生成的为均一大小微液滴，通过温控装置30对所述多个微液滴进行核酸扩增反应，并采集产物信号，如荧光、紫外吸收、浊度等信号。利用所述多个扩增与非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定量分析。通过实时监测所述多个微液滴的荧光变化图片，测序结果具有直接性，可以解决所述多个微液滴中的假阳性和假阴性的问题。
所述数字PCR检测仪将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，使得所述操作人员可以通过一体式数字PCR检测机1实现自动化操作，提高了工作效率，反应快速、重复性好、灵敏度高、特异性强、结果清晰。
在一个实施例中，所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化， 形成多个微液滴。其中，所述微液滴生成装置10生成的为均一大小的微液滴。然后，通过所述温控装置20对所述多个微液滴进行核酸扩增。同时，采用所述荧光信号检测装置30实时拍测所述多个微液滴的荧光变化图像。通过所述多个微液滴的荧光变化图像，获取所述多个微液滴的荧光变化曲线。根据所述荧光变化曲线，可以获取所述多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始核酸的浓度进行定量分析。
其中，Ct值中C代表Cycle，t代表threshold，Ct值的含义是：每个所述微液滴内的荧光信号到达设定的域值时所经历的循环数。在实时荧光PCR中，Ct值是指每个所述微液滴内的荧光信号达到设定的阈值时所经历的循环数。也就是说Ct值的含义是：每个微液滴的荧光信号到达设定的域值时所经历的循环数。
PCR循环在到达Ct值所在的循环数时，刚刚进入真正的指数扩增期(对数期)，此时微小误差尚未放大，因此Ct值的重现性极好，即同一核酸模板不同时间扩增或同一时间不同微液滴容器内扩增，得到的Ct值是恒定的。当所述微液滴对应的荧光曲线为扩增曲线时，此时表明所述微液滴中含有目标基因成分。当所述微液滴对应的荧光曲线为一条直线时，此时表明所述微液滴中不含目标基因成分。从获取的实时荧光曲线，可以获得Ct值，每个微液滴的Ct值在获取时，对所述实时荧光曲线进行求导，所述实时荧光曲线的斜率固定的荧光曲线的起始循环次数即为所需要的Ct值。
所述微液滴生成装置10生成的为均一大小微液滴，通过温控装置20对所述多个微液滴进行核酸扩增反应，并采集产物信号，如荧光、紫外吸收、浊度等信号。所述数字PCR检测仪1将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，使得所述操作人员可以通过一体式数字PCR检测机1实现自动化操作，提高了工作效率，反应快速、重复性好、灵敏度高、特异性强、结果清晰。
所述数字PCR检测仪1的检测过程主要包括5个环节：制备待测核酸扩增反应液、待测核酸扩增反应液微滴化、核酸扩增、荧光信息的采集以及定量分析。请参见图12，在一个实施例中，一种数字PCR检测仪的分析方法，包括以下步骤：S10，制备待测核酸扩增反应液；S20，将所述待测核酸扩增反应液微滴化，形成多个微液滴；S30，将所述多个微液滴进行核酸扩增，并实时获取所述多个微液滴的荧光信息；S40，根据所述多个微液滴的荧光信息，对所述多个微液滴进行定量分析。在一个实施例中，所述步骤S10包括：制备需要检测的核酸扩增反应液。所述核酸扩增反应液中包含待检测的核酸模板、反应缓冲水溶液、脱氧核糖核苷三磷酸、引物、聚合酶和产物标记物质等。
其中，核酸扩增反应液，可以是以脱氧核糖核酸(DNA)为模板的核酸扩增反应液(可称为DNA扩增反应液)，也可以是以核糖核酸(RNA)为模板的逆转录核酸扩增反应液(可称为RNA反转录反应液)，还可以是其它核酸扩增反应液，如环介导等温扩增(LAMP)反应液。其中，所述DNA扩增反应液的特点是含有DNA扩增所需要的dNTP、缓冲液、无机盐离子、聚合酶、引物、待检测的DNA模板以及荧光染料或荧光探针等。反应液中的荧光染料或荧光探针能指示核酸扩增，可以是SYBR Green等与DNA结合的荧光染料，也可以是同时含有荧光基团和淬灭基团的寡糖核苷酸探针，如TaqMan荧光探针等。
在一个实施例中，准备专供数字PCR使用的成套试剂和溶液，用来减少或避免外源DNA对模板DNA样本的潜在污染。所使用的所有仪器和耗材应该进行高温灭菌，高温干燥处理。所述待检测核酸扩增反应液成分包括：待扩增的模板DNA、用来扩增模板的特异性寡核苷酸引物、耐热DNA聚合酶、4种三磷酸脱氧核糖核苷酸底物、二价金属阳离子Mg2+、Taqman探针或荧光染料及PCR缓冲液等。
在一个实施例中，制备所述待测核酸扩增反应液时，采用Taqman探针对所述待测核酸扩增反应液进行标记。在一个实施例中，制备所述待测核酸扩增反应液时，采用SYBR荧光染料对所述待测核酸扩增反应液进行标记。在一个实施例中，所述步骤S20将所述待测核酸扩增反应液微滴化，用以形成多个微液滴包括两种微液滴生成方法：瞬时加速的微液滴生成方法以及变速周期的微液滴生成方法。通过所述微液滴生成装置10将所述待测核酸扩增反应液进行微滴化处理，可以获取大批量的微液滴，用于所述数字PCR检测仪1的检测。其中，所述驱动液体为一种与所述待测核酸扩增反应液互不相容且互不影响的液体。所述第一液体190为所述待测核酸扩增反应液，所述第二液体699为油相混合物。
将制备好的核酸扩增反应液通过所述微液滴生成装置，可以制备出大批量的微液滴。在制备所述多个微液滴的过程中，将所述多个微液滴放置于所述微液滴容器内，用以方便对所述多个微液滴进行检测。在一个实施例中，通过所述微液滴生成装置10在所述第二液体中生成大量的微液滴，可以保持所述多个微液滴之间不融合。
在一个实施例中，所述步骤S30包括S310：将所述多个微液滴平铺于所述微液滴容器中；S320：将平铺后的所述多个微液滴进行核酸扩增；S330：在所述多个微液滴进行核酸扩增时，实时对所述多个微液滴进行拍照检测。
请参见图13，在一个实施例中，所述步骤S310包括一种微液滴平铺方法。所述微液滴平铺方法包括：S311，提供一微液滴容器60，所述微液滴容器60具有开口631，且所述微液滴容器60内盛有第二液体699；S312，提供第一液体190，所述第一液体190的密度大于所述第二液体699并与所述第二液体699不互溶，并将所述第一液体190生成多个微液滴层叠堆积于所述微液滴容器底板610；S313，对所述多个微液滴进行高低温循环，直至所述多个微液滴平铺于所述容器底板610。
在所述微液滴容器60中生成多个微液滴，并将落至所述微液滴容器60的所述容器底板610，不规则的堆积在一起。当大量的微液滴降落至所述容器底板610时，会在所述容器底板610形成多层的微液滴。并且通过微液滴生成装置制备的多个微液滴在向下沉降过程当中，集中集合在微液滴容器的中间部位，聚集在一起，不利于观察。
请参见图14，在所述步骤S10中，将多个微液滴滴落至微液滴容器60中，所述多个微液滴堆积在所述微液滴容器底板610，亦即所述多个微液滴在所述微液滴容器底板610上形成多层微液滴。在荧光信号检测过程中，对所述多个微液滴进行拍照时，造成多层之间的相互影响，影响所述多个微液滴的拍照检测。因此，将容纳有所述多个微液滴的所述微液滴容器60进行高低温循环。将所述多个微液滴进行高低温循环多次，直至所述多个微液滴平铺于所述微液滴容器底板610，使得大批量的所述微液滴平铺于所述反应单元612 内，便于海量液滴大规模平行观测。
通过所述温控装置20进行高低温循环，利用热胀冷缩的原理，进行平铺。当物体温度升高时，分子的动能增加，分子的平均自由程增加，所以表现为热胀。同理，当物体温降低时，分子的动能减小，分子的平均自由程减少，所以表现为冷缩。随着温度的变化，当温度升高时，样本液滴的粘稠度变低、体积收缩。同时，温度越高粘度越低，当温度在60℃左右时，样本液滴形状最软，此时形状大概呈现为六边形，然而在其他的温度情况下，样本液滴形状的可变性较差，不容易实现在液滴容器中平铺。
在一个实施例中，通过所述温控装置20进行高低温循环的步骤如下：首先，将所述多个微液滴进行升温加热至90℃～95℃，并加热5min～10min；然后，将所述多个微液滴降温至40℃～60℃，并退火延伸30s～60s；最后，依次循环多次，降温至0℃～10℃，对所述多个微液滴保存。
在一个实施例中，通过所述温控装置20进行高低温循环的步骤还包括：首先，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min；将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动。然后，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；其次，所述多个微液变性之后，降温至55℃，并退火延伸45s，对所述多个微液滴进行拍照，并进行45次循环；最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
所述待测核酸扩增反应液通过所述微液滴生成装置10生成多个微液滴，用以进行检测。通过所述微液滴生成装置10制备的所述多个微液滴在向下沉降过程当中，集中集合在所述微液滴容器60的中间部位，聚集在一起，不利于观察。所以，为了更加精确的获取所述多个微液滴的核酸扩增反应的相关信息，需要将所述多个微液滴平铺在所述微液滴容器中。通过将所述多个微液滴平铺在所述微液滴容器中，形成一层，从而避免了多层微液滴之间的相互影响，使得所述荧光信号检测装置30拍照检测，获取更加精确地荧光信息，以便于定量分析。
PCR反应条件为温度、时间和循环次数。
温度与时间的设置：基于PCR原理三步骤而设置变性-退火-延伸三个温度点。在标准反应中采用三温度点法，双链DNA在90～95℃变性，再迅速冷却至40～60℃，引物退火并结合到靶序列上，然后快速升温至70～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸。对于较短靶基因(长度为100～300bp时)可采用二温度点法，除变性温度外、退火与延伸温度可合二为一，一般采用94℃变性，65℃左右退火与延伸(此温度Taq DNA酶仍有较高的催化活性)。
变性温度与时间：变性温度低，解链不完全是导致PCR失败的最主要原因。一般情况下，93℃～94℃min足以使模板DNA变性，若低于93℃则需延长时间，但温度不能过高，因为高温环境对酶的活性有影响。此步若不能使靶基因模板或PCR产物完全变性，就会导致PCR失败。
退火(复性)温度与时间：退火温度是影响PCR特异性的较重要因素。变性后温度快 速冷却至40℃～60℃，可使引物和模板发生结合。由于模板DNA比引物复杂得多，引物和模板之间的碰撞结合机会远远高于模板互补链之间的碰撞。退火温度与时间，取决于引物的长度、碱基组成及其浓度，还有靶基序列的长度。对于20个核苷酸，G+C含量约50％的引物，55℃为选择最适退火温度的起点较为理想。
在允许范围内，选择较高的复性温度可大大减少引物和模板间的非特异性结合，提高PCR反应的特异性。复性时间一般为30sec～60sec，足以使引物与模板之间完全结合。延伸温度与时间：Taq DNA聚合酶的生物学活性：70～80℃150核苷酸/S/酶分子；70℃60核苷酸/S/酶分子；55℃24核苷酸/S/酶分子；高于90℃时，DNA合成几乎不能进行。
所以，PCR反应的延伸温度一般选择在70～75℃之间，常用温度为72℃，过高的延伸温度不利于引物和模板的结合。PCR延伸反应的时间，可根据待扩增片段的长度而定，一般1Kb以内的DNA片段，延伸时间1min是足够的。3～4kb的靶序列需3min～4min；扩增10Kb需延伸至15min。延伸时间过长会导致非特异性扩增带的出现。对低浓度模板的扩增，延伸时间要稍长些。
在一个实施例中，所述步骤S320将所述多个微液滴进行核酸扩增的步骤如下：首先，将所述微液滴容器60放置于所述温控装置20的所述加热基板240上；然后，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min；将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动。其次，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；再次，所述多个微液变性之后，降温至55℃，并退火延伸45s，并进行45次循环；最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
所述温控装置20采用所述柔性电路板220和所述导热增强层250，使所述微液滴容器60温度分布均匀，加快了所述半导体致冷器的导热性能。当所述多个微液滴在不同温度范围内进行核酸扩增时，设置于所述导热增强层250表面的的温度传感器260与所述第二控制器210连接，可以检测所述微液滴容器60的实时温度，进而将温度信息反馈给所述第二控制器210，从而实现对所述多个微液滴加热温度的控制。可以实现迅速在几秒时间内进行切换。所述温控装置20可以实现瞬时升温降温，进而升温降温的过程缩短了，从而实现了高低温的循环，将所述数字PCR检测仪1的检测时间缩短了，提高了检测效率。
通过所述温控装置20可以使得所述多个微液滴进行核酸扩增。基于PCR原理三步骤而设置变性-退火-延伸的三个温度点。在标准反应中采用三温度点法，双链DNA在90～95℃变性，再迅速冷却至40～60℃，引物退火并结合到靶序列上，然后快速升温至70～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸，在适合的温度范围内对核酸进行扩增。同时，在核酸扩增过程中，所述微液滴容器底板610与所述温控装置20的所述导热增强层250紧密帖合，两者之间不会有间隙，加热均匀，提高了所述数字PCR检测仪1的准确性。
在一个实施例中，在所述多个微液滴进行核酸扩增的过程中，通过所述荧光信号检测装置30对所述多个微液滴进行拍照检测。
通过所述荧光信号检测装置30，对所述多个微液滴进行拍照。其中，所述激发光源340给所述多个微液滴提供蒸发、原子化或激发的能量，从所述微液滴容器60上方采取倾斜角度照射在所述微液滴容器60上。采用所述荧光信号检测装置30实现对所述多个微液滴进行周期性的二维扫描，并实时进行拍照。所述微液滴容器60内的所述多个微液滴的内部荧光被激发，通过所述第二滤光片333被上方的所述物镜332收集，进入所述相机331，所述相机331采集所述多个微液滴的荧光图像。所述第三控制器310可以同步开启所述多个不同颜色的LED光源341和所述相机331。
通过所述荧光信号检测装置30可以使得所述多个微液滴荧光成像，一次拍摄一定数量的所述多个微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。
在一个实施例中，通过所述荧光信号检测装置30拍照步骤，所述微液滴容器60中的每个微液滴可以获得45张荧光图片，用以进行定量分析。
在一个实施例中，所述步骤S330在所述多个微液滴进行核酸扩增时，实时对所述多个微液滴进行拍照检测的步骤如下：
首先，将所述多个微液滴进行升温加热，将温度加热至95℃，并加热10min；
将所述多个微液加热至95℃，并加热10min，用以将所述多个微液滴中的酶进行热启动；
然后，所述多个微液滴完成酶热启动之后，对所述多个微液滴进行变性30s；
其次，所述多个微液变性之后，降温至55℃，并退火延伸45s，通过所述荧光信号检测装置对所述多个微微液进行拍照，并进行45次循环，获得45张所述多个微液滴的荧光图像；
最后，循环45次之后，降温至4℃，对所述多个微液进行长时间保存。
通过所述聚焦透镜346的光路倾斜照射于所述多个微液滴，使得所述微液滴容器60中含有荧光物质的微液滴产生荧光。通过所述荧光探测组件330对所述含有荧光物质的微液滴进行荧光信息采集，并将所述含有荧光物质的微液滴进行荧光信息以荧光图像的形式传输至计算机，用以进行定量分析。
采用所述荧光成像的检测方法一次拍摄一定数量的所述微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。由于所述荧光成像的检测方法的成像范围大，因此，在检测的时候对所述微液滴所处的检测环境的要求较低。
在一个实施例中，所述核酸待测样本为包含有DNA的待测样本。所述微液滴生成装置10生成的均一大小微液滴，通过温控装置20对所述多个微液滴进行核酸扩增反应，并采集产物信号，如荧光、紫外吸收、浊度等信号。利用所述多个扩增与非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定量分析。在对微液滴进行加热的过程中，实时拍测所述多个微液滴的荧光变化图片，获取多个微液滴的Ct值，并通过Ct值与起始拷贝数的关系对初始DNA的浓度进行定量分析。
若含有目标DNA的微液滴扩增后，其荧光信号的强度达到一定水平，显示为阳性；若DNA含量为零的微液滴几乎检测不到荧光信号，视为阴性。
假设：数字PCR中的微液滴含有的起始DNA拷贝数为x，根据数理统计理论，x＝k(k＝0，1，2，3.....)的概率分布函数P符合泊松概率模型，其中λ为微液滴中含有的平均分子拷贝数。
Figure PCTCN2019072974-appb-000003
因此，通过所述泊松分布模型期望值μ与方差σ 2，可知期望值μ为λ，方差σ 2为λ。因此，可知数字PCR中每个微液滴中包含目标DNA分子的拷贝数为λ，所以求得的λ值就能够实现核酸的定量检测。
假设所述待测核酸扩增反应液的总体积为V(每个微液滴的体积为v)，则所述待测核酸扩增反应液浓度c(copy/μL)为：
Figure PCTCN2019072974-appb-000004
所以，求得λ的值，就能实现DNA的定量检测。
其中，实时荧光定量PCR无需内标是建立在Ct值的重现性与Ct值与起始DNA浓度的线性关系基础之上的。PCR循环在到达Ct值所在的循环数时，刚刚进入真正的指数扩增期(对数期)，此时微小误差尚未放大，因此Ct值的重现性极好，即同一DNA模板不同时间扩增或同一时间不同微液滴容器内扩增，得到的Ct值是恒定的。
当所述微液滴对应的荧光曲线为扩增曲线时，此时表明所述微液滴中含有目标基因成分。当所述微液滴对应的荧光曲线为一条直线时，此时表明所述微液滴中不含目标基因成分。
从获取的实时荧光曲线，可以获得Ct值，每个微液滴的Ct值在获取时，对所述实时荧光曲线进行求导，所述实时荧光曲线的斜率固定的荧光曲线的起始循环次数即为所需要的Ct值。
在一个实施例中，通过微液滴生成装置10可以生成多个体积大小均一的微液滴。每个所述微液滴大小在微米级。在所述多个微液滴体积均一的情况下，根据所述多个微液滴的荧光信息，对所述多个微液滴进行定量分析。
请参见图15，所述步骤S40包括一种数字PCR定量检测方法。所述数字PCR定量检测方法包括以下步骤：
S4110，获取所有微液滴的多个实时荧光图像，根据所述多个实时荧光图像获得进行核酸扩增的微液滴的实时荧光曲线；
S4120，根据所述实时荧光曲线，获得所有进行核酸扩增的微液滴的Ct值；
S4130，根据所述Ct值与进行核酸扩增的微液滴的核酸起始拷贝数的关系，获得所有进行核酸扩增的微液滴的核酸起始拷贝数；
S4140，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，获得所述核酸起始拷贝数的频数分布；
S4150，根据所述核酸起始拷贝数的频数分布，计算泊松分布的参数λ。
通过所述数字PCR定量检测方法解决了结果的假阳性和假阴性。测序平台的样本高通量性，可同时进行上百例样本的检测。同时能利用不同种类的荧光，进行多个位点的检测，加速检测的速度，降低了实验成本。采用数字PCR检测仪通过微滴化处理，使得稀有检测片段从大量的复杂背景中分离出来，大大简化操作步骤，有效节约了准备时间和检测时间，且结果判读直观可靠，具有可以稳定实施的特点，检测灵敏度及精确性都达到精准定量的要求，提高了检测的灵敏度和精确性。
在一个实施例中，所述S4110包括：
S4111，根据每个实时荧光图像，获得每个进行核酸扩增的微液滴的荧光强度值；
S4113，根据所述每个进行核酸扩增的微液滴的荧光强度值，获得每个进行核酸扩增的微液滴的实时荧光曲线；
S4115，根据所述每个进行核酸扩增的微液滴的荧光曲线，获得所述所有进行核酸扩增的微液滴的实时荧光曲线。
在一个实施例中，所述步骤采集所述多个微液滴的荧光图像，并进行图像追踪。在获取每个微液滴的实时荧光曲线时，需要对每张图像中的每个微液滴分别进行定位，获取每个微液滴的荧光强度。在所述数字PCR检测仪中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少根据之前。
在一个实施例中，对每个微液滴进行追踪时，还可以通过在每个温度循环过程中拍摄的照片，进行NCAST图像差分及聚类运算，识别到每个微液滴的位置，进而获取所述多个微液滴的荧光强度。
在一个实施例中，如果在一个温度循环过程中造成的微液滴移动距离不大于一个微液滴的直径，可以采用以下方法尽心微液滴追踪。其中，对每个微液滴进行追踪时，对每个微液滴进行图像追踪步骤如下：
首先，识别每次温度循环过程中拍摄的照片，获得每个微液滴的圆心位置；
然后，将当前识别的每个微液滴的圆心位置与前一次循环过程中的每个微液滴的圆心位置对比；
最后，若当前识别的每个微液滴的圆心位置与前一次循环过程中的每个微液滴的圆心位置的圆心间距小于一个微液滴直径，则标记为同一个微液滴。
在一个实施例中，根据每个温度循环过程中每个微液滴的荧光强度值，获取每个微液滴的荧光曲线。通过对每次温度循环过程中每个微液滴的各个部位的荧光强度值求和，作为每个微液滴的某一特定时刻的荧光强度值。
在一个实施例中，为了防止相邻的每个微液滴的边缘部位的互相影响，每个微液滴的 某一特定时刻的荧光强度值采用部分求和方式。通过每次温度循环过程中所述多个微液滴的荧光强度值，可以获得所述多个微液滴在全部循环过程中的变化情况，获取每个微液滴的荧光曲线。在一个实施例中，每个微液滴进行了45次循环，共获得45张荧光图片。通过对45张荧光图片中每个微液滴进行定位，并获取每个微液滴的45个荧光强度值，从而获取每个微液滴的荧光曲线。
在一个实施例中，所述S4120包括：
S4121，对每个进行核酸扩增的微液滴的实时荧光曲线求导，获取所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率；
S4123，根据所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率，获取所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率中斜率固定不变的数值；
S4125，根据所述斜率固定不变的数值，获取其对应的起始循环数，所述起始循环数为每个进行核酸扩增的微液滴的Ct值；
S4127，根据所述每个进行核酸扩增的微液滴的Ct值，获得所述所有进行核酸扩增的微液滴的Ct值。
在一个实施例中，所述S4120还包括：
S4122，根据每个进行核酸扩增的微液滴的实时荧光曲线，获得每个进行核酸扩增的微液滴的荧光域值的缺损值；
S4124，根据每个进行核酸扩增的微液滴的荧光域值的缺损值，获得其对应的循环数，所述循环数为每个进行核酸扩增的微液滴的Ct值；
S4126，根据所述每个进行核酸扩增的微液滴的Ct值，获得所述所有进行核酸扩增的微液滴的Ct值。
其中，Ct值中C代表Cycle，t代表threshold，Ct值的含义是：每个反应管内的荧光信号到达设定的域值时所经历的循环数。在实时荧光PCR中，Ct值是指每个反应管内的荧光信号达到设定的阈值时所经历的循环数。PCR反应的前15个循环的荧光信号作为荧光本底信号，荧光域值的缺损设置是3-15个循环的荧光信号的标准偏差的10倍，即：threshold＝10×SD cycle 3-15。
在一个实施例中，PCR反应的前15个循环的荧光信号作为荧光本底信号，荧光域值的缺损设置是3-15个循环的荧光信号的标准偏差的10倍，即：threshold＝10×SD cycle 3-15。根据所述荧光域值的缺损值threshold，获取相对应的循环数，所述循环数为所述Ct值。
在一个实施例中，所述S4130中所述每个微液滴的Ct值与所述每个微液滴的DNA起始拷贝数的对数存在线性关系。
在一个实施例中，每个模板(DNA)的Ct值与该模板(DNA)的起始拷贝数的对数存在线性关系。所述线性关系表示为：
Figure PCTCN2019072974-appb-000005
x 0为初始模板(DNA)量，E x为扩增效率，N为荧光扩增信号达到阈值强度时扩增产 物的量。
起始拷贝数越多，Ct值越小。利用已知起始拷贝数的标准品可作出标准曲线，其中横坐标代表起始拷贝数的对数，纵坐标代Ct值。因此，只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的起始拷贝数。
lg(x 0)＝lgN-Ctlg(1+E x),E x＜1
其中x 0为模板(DNA)的起始拷贝数。
Ct值与起始DNA浓度的关系为：每个DNA模板的Ct值与DNA模板的起始拷贝数的对数存在线性关系。起始拷贝数越多，Ct值越小。
在一个实施例中，所述S4140包括：
S4141，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，获得所述所有进行核酸扩增的微液滴的核酸起始拷贝数中的最大值和最小值；
S4143，根据所述最大值与所述最小值，选取组距和组数，获得所述核酸起始拷贝数的频数分布。
其中，频数是指落在不同区间中的数据个数。不同区间的频数之和等于这组数据的总数。
在一个实施例中，所述S4150中计算泊松分布的参数λ时，采用最大似然估计方法。
其中，对于液滴式PCR，单个液滴中所包含的起始拷贝数满足Poisson分布。
Figure PCTCN2019072974-appb-000006
其中λ为所述微液滴中平均包含的起始DNA拷贝数。每个液滴中包含的平均起始拷贝数用CPD(copies per droplet)来表示。
在一个实施例中，根据Ct值能够分别获得起始具有k(k＝0,1,2,3…)个DNA起始拷贝数的液滴数量n k，采用最大似然估计方法，获得：
Figure PCTCN2019072974-appb-000007
其中，所述微液滴的DNA起始拷贝数对应的频数值为n k，亦即：k＝0个DNA起始拷贝数时，对应出现的所述微液滴个数为n 0个；k＝1个DNA起始拷贝数时，对应出现的所述微液滴个数为n 1个；k＝2个DNA起始拷贝数时，对应出现的所述微液滴个数为n 2个；k＝3个DNA起始拷贝数时，对应出现的所述微液滴个数为n 3个，依次类推。采用此方法无需保证阴性暗液滴的数量。另外使用完整的频数分布数据对整体进行最优参数估计的精度要比单独采用一个频点进行估计的精度和稳定性高得多。
请参见图16，在一个实施例中，一种数字PCR定量检测方法，包括以下步骤：
S4210，获取所有微液滴的多个实时荧光图像，根据所述多个实时荧光图像获得进行核酸扩增的微液滴的实时荧光曲线；
S4220，根据所述实时荧光曲线，获得所有进行核酸扩增的微液滴的Ct值；
S4230，根据所述Ct值与进行核酸扩增的微液滴的核酸起始拷贝数的关系，获得所有进行核酸扩增的微液滴的核酸起始拷贝数；
S4240，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，选取部分核酸起始拷贝数；
S4250，根据所述部分核酸起始拷贝数，获取所述部分核酸起始拷贝数得频数分布；
S4260，根据所述部分核酸起始拷贝数的频数分布，对泊松分布进行点估计，获取泊松分布的参数λ。
其中，在一个实施例中，根据所有多个微液滴起始DNA浓度的不完全抽样，采用最小二乘法对泊松分布进行点估计。
根据所有多个微液滴起始DNA浓度的不完全抽样，还可以采用最大期望算法(Expectation Maximization Algorithm，EM)、蒙特卡罗(Markov chain Monte Carlo，MCMC)方法对泊松分布进行点估计。所述蒙特卡罗(Markov chain Monte Carlo，MCMC)方法为贝叶斯方法的一种。
在一个实施例中，所述S4260包括在一个区间[λ min,λ max]内搜索λ，使得所述部分核酸起始拷贝数的频数值误差平方和err最小。
在一个实施例中，当所述起始DNA浓度较小时，含有大于4个拷贝的液滴数量很少(或者可以忽略)。Biorad系统在20000个液滴体系的情况下，一般建议样本DNA浓度不大于6CPD。在实际实验过程中，k>4时，Ct值的区分变小，很难根据Ct值区分一个液滴的起始拷贝数为4或者5，因此只使用x 0，x 1，x 2，x 3不完整抽样来对Poisson分布进行点估计。根据不完全抽样对Poisson进行点估计的算法有很多，介绍一种可操作性的算法——最小二乘法。
在一个实施例中，给定一个区间[λ min,λ max]，λ在[λ min,λ max]区间中搜索，计算误差的平方和，选择合适的λ使得误差平方和最小。
Figure PCTCN2019072974-appb-000008
其中，每个微液滴中含有的DNA起始拷贝数为随机变量x，部分微液滴的DNA起始拷贝数对应的频数值为n k，N为所述多个微液滴的总数。
在一个实施例中，所述S4260中对泊松分布进行点估计的方法还包括矩估计法、顺序统计量法或最大似然法。
其中，点估计的方法还包括：
矩估计法：所述矩估计法是利用样本矩来估计总体中相应的参数。
首先，推导涉及感兴趣的参数的总体矩(即所考虑的随机变量的幂的期望值)的方程。然后，取出一个样本并从这个样本估计总体矩。接着使用样本矩取代(未知的)总体矩，解出感兴趣的参数。从而得到那些参数的估计。
顺序统计量法：所述顺序统计量法是用样本中位数估计总体的数学期望的方法。顺序统计量估计法的优点是计算简便，且不易受个别异常数据的影响。如果一组样本值某一数 据异常(如过于小或过于大)，则这个异常数据可能是总体的随机性造成的，也可能是受外来干扰造成的(如工作人员粗心，记录错误)。当原因属于后者，用样本平均值估计E(x)显然受到影响，但用样本中位数估计E(x)时，由于一个(甚至几个)异常的数据不易改变中位数的取值，所以估计值不易受到影响。
最大似然法：最大似然法(Maximum Likelihood，ML)也称为最大概似估计，也叫极大似然估计，是一种具有理论性的点估计法。所述最大似然法的基本思想是当从模型总体随机抽取n组样本观测值后，最合理的参数估计量应该使得从模型中抽取该n组样本观测值的概率最大，而不是像最小二乘估计法旨在得到使得模型能最好地拟合样本数据的参数估计量。
在实际过程中，所述数字PCR定量检测方法，不依赖于标准曲线而能够高精度地测定所述多个微液滴的起始DNA浓度。在所述数字PCR检测仪1中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少。
通过数字PCR定量检测方法可以实现所述多个微液滴的动态跟踪，在所述多个微液滴进行温度循环过程中都可以找到每个微液滴对应的具体位置，可以实现核酸扩增的全部过程的监测。因此，通过所述数字PCR定量检测方法可以解决所述多个微液滴中存在假阳性的问题。同时，通过对所述多个微液滴荧光曲线进行处理，并进行不依赖于均匀性假设进行的统计修正，而取得真正的绝对定量。
不仅摆脱了对标准曲线的依赖，排除了由标准曲线引起的定量结果不确定的问题，并且解决了液滴式数字PCR终点检测方式的限制，打破了只采用了一个p(x＝0)的数据对待测样本整体进行参数估计的局限性。采用所述实时荧光定量PCR检测方法，提高了数字PCR定量检测的准确性。
采用所述数字PCR定量检测方法无需保证阴性空液滴的数量。同时，采用多维度的频数分布数据对整体进行最优参数估计的精度要比单独采用一个p(x＝0)的数据进行估计的精度和稳定性高得多。
每一个荧光曲线代表一个有用信息的曲线的变化过程，参加了液滴样本信息，以实现实时监测；以设定算法消除相邻液滴之间的相互影响。
所述数字PCR定量检测方法依赖抽象的数学模型，实现了重复性、高灵敏性，并且动态范围变大，可以利用少量液滴实现监测。用小量的数据覆盖更多的信息。同时，所述数字PCR定量检测方法避免了之前泊松分布概率模型的误差，实现绝对定量，更加直观。综合了所有数据，消除了随机误差。获取液滴样本荧光曲线，实时监测液滴样本的荧光亮度的变化，用以去除假阳性；消除相邻液滴之间的相互影响，为后续定量分析模型提供更精确的数据源。
请参见图17，根据所述部分核酸起始拷贝数分别为0，1，2，3情况下获得的泊松分布拟合。其中，横坐标为每个液滴中包含的平均起始拷贝数(copies per droplet，CPD)。 纵坐标为每个液滴中包含的平均起始拷贝数的标准偏差(standard deviation，Std Dev，STD)。每个液滴中包含的平均起始拷贝数用CPD(copies per droplet)来表示。可知，采用所述部分核酸起始拷贝数获得的每个液滴中包含的平均起始拷贝数用CPD的标准偏差比其他算法获得的平均起始拷贝数用CPD的标准偏差小。因此，本算法获得的每个液滴中包含的平均起始拷贝数用CPD的值更加精确。对20000个液滴进行1000次仿真的结果表明。仅采用单点的估计方法只能覆盖有限的浓度范围，并且估计精度随着样品浓度的升高而急剧恶化。而采用不完全泊松分布拟合算法(N＝0，1，2，3)，随着样品浓度的增加，估计精度没有明显的恶化，并且能够将待测核酸扩增反应液的浓度扩大两倍。对于液滴数量较少的情况下，不完全泊松分布拟合算法(部分抽样泊松分布拟合算法)仍然具有很好的可靠性。
通过仿真结果表明，采用一种数字PCR定量检测方法时，200个液滴的实验体系的精度就可以优于传统的单点估计算法(uCount algorithm)。在液滴数量相近的情况下，泊松拟合算法的稳定性，精度已经可用动态范围都要远优于传统单点估计算法。在实现相同测定精度的情况下，采用泊松拟合算法所需的液滴数量要比传统单点估计算法所需的液滴数量低两个数量级。因此，可以提高数字PCR检测仪1的检测精度，并且扩大了检测范围，可以以少数液滴检测多个不同种类的核酸，提高了数字PCR检测仪1的使用效率。
在一个实施例中，如果所述微液滴生成装置10生成的所述多个均一微液滴的体积有特殊情况下，改变了所述微液滴的体积时，就会存在体积不均一的情况。同时，通过所述微液滴生成装置10也可以生成多个体积不同的微液滴，用以进行医学临床检测。
无论是微孔式还是液滴式数字PCR技术，其反应单元的体积往往保持高度一致，可视为单一体积数字PCR技术。单一体积数字PCR的定量上限主要取决于反应单元的体积和数量，检测下限与样本总体积相关。单一体积数字PCR技术的分辨率和动态范围无法独立调节。同时，连续对待测样本进行稀释，虽然可以扩展其动态范围，但是无法提高检测灵敏度。并且连续稀释的方法增加了试剂的用量和交叉污染的风险，操作步骤繁琐。
多重体积数字PCR(multivolume digital PCR，MVdPCR)，能够在规避连续稀释弊端的同时，使研究人员独立调节动态范围和分辨率，
多重体积数字PCR的微液滴容器中含有一系列不同体积的反应单元，小体积的反应单元可以对高浓度样本进行定量，大体积的反应单元利用足够的容积实现了高灵敏检测。多重体积数字PCR不需要大量的反应单元却能够达到单一体积数字PCR的动态范围，因此可以再所述微液滴容器中完成更多的样本分析，同时其试剂耗量有效降低。
在所述数字PCR检测仪中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少。
在一个实施例中，所述样本溶液为核酸扩增反应液，应用于数字PCR检测仪的定量分析方法中。
请参见图18，针对以上情况，提出一种不同体积数字PCR的定量分析方法包括：
S4310：获取所有微液滴体积v 1，v 2，...v m，所述体积为v 1，v 2，...v m依次对应的微液滴的数目n 1，n 2，…，n m，以及所述体积为v 1，v 2，...v m依次对应的微液滴核酸扩增后的阴性微液滴数目b 1，b 2，…，b m；
S4320：根据所有微液滴核酸扩增后的相关参数v 1、v 2，...v m，n 1，n 2，…，n m，b 1，b 2，…，b m，构建关于核酸扩增反应液浓度c的联合二项分布函数f(c)；
S4330：根据联合二项分布函数f(c)，求使得所述联合二项分布函数f(c)取极值时c的值；
S4340：将所述联合二项分布函数f(c)转化为关于ln(c)的联合二项分布函数F(Λ)，获得关于ln(c)的标准差以及置信区间；
S4350：根据ln(c)的标准差以及置信区间，获取所述核酸扩增反应液浓度c的标准差以及置信区间。
若含有目标DNA的微液滴扩增后，其荧光信号的强度达到一定水平，显示为阳性；若DNA含量为零的微液滴几乎检测不到荧光信号，视为阴性。
假设：数字PCR中的微液滴含有的起始DNA拷贝数为x，根据数理统计理论，x＝k(k＝0，1，2，3.....)的概率分布函数P符合泊松概率模型，其中λ为微液滴中含有的平均分子拷贝数。
Figure PCTCN2019072974-appb-000009
因此，通过所述泊松分布模型期望值μ与方差σ 2，可知期望值μ为λ，方差σ 2为λ。因此，可知数字PCR中每个微液滴中包含目标DNA分子的拷贝数为λ，所以求得的λ值就能够实现核酸的定量检测。
假设所述待测核酸扩增反应液的总体积为V(每个微液滴的体积为v)，则所述待测核酸扩增反应液浓度c(copy/μL)为：
Figure PCTCN2019072974-appb-000010
所以，求得λ的值，就能实现DNA的定量检测。
其中，实时荧光定量PCR无需内标是建立在Ct值的重现性与Ct值与起始DNA浓度的线性关系基础之上的。PCR循环在到达Ct值所在的循环数时，刚刚进入真正的指数扩增期(对数期)，此时微小误差尚未放大，因此Ct值的重现性极好，即同一DNA模板不同时间扩增或同一时间不同微液滴容器内扩增，得到的Ct值是恒定的。
当所述微液滴对应的荧光曲线为扩增曲线时，此时表明所述微液滴中含有目标基因成分。当所述微液滴对应的荧光曲线为一条直线时，此时表明所述微液滴中不含目标基因成分。
从获取的实时荧光曲线，可以获得Ct值，每个微液滴的Ct值在获取时，对所述实时荧光曲线进行求导，所述实时荧光曲线的斜率固定的荧光曲线的起始循环次数即为所需要 的Ct值。
在一个实施例中，所述S4310包括：
S4311：将含有目标核酸的样品溶液微滴化，获得多个不同体积v 1，v 2，...v m的微液滴，所述微液滴体积为v 1，v 2，...v m依次对应的微液滴的数目n 1，n 2，…，n m；
S4313：将所有微液滴进行核酸扩增，并拍照检测，获得所述所有微液滴的荧光图像；
S4315：根据所述所有微液滴的荧光图像，获取所述所有微液滴的体积为v 1，v 2，...v m依次对应的核酸扩增后的阴性微液滴数目b 1，b 2，…，b m。
在一个实施例中，所述S4310还包括：
S4312：将含有目标核酸的样品溶液微滴化，形成多个微液滴；
S4314：将所述多个微液滴进行核酸扩增，并拍照检测，获得所有微液滴核酸扩增后的荧光图像；
S4316：根据所述荧光图像，获取所述所有微液滴核酸扩增后的体积，所述体积分别为v 1，v 2，...v m，所述体积分别为v 1，v 2，...v m依次对应的核酸扩增后的微液滴的数目n 1，n 2，…，n m，以及所述体积为v 1，v 2，...v m依次对应的核酸扩增后的阴性微液滴数目b 1，b 2，…，b m。
一个实施例中，所述步骤采集所述多个微液滴的荧光图像，并进行图像追踪。在获取每个微液滴的实时荧光曲线时，需要对每张图像中的每个微液滴分别进行定位，获取每个微液滴的荧光强度。在所述数字PCR检测仪中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少根据之前。
在一个实施例中，对每个微液滴进行追踪时，还可以通过在每个温度循环过程中拍摄的照片，进行NCAST图像差分及聚类运算，识别到每个微液滴的位置，进而获取所述多个微液滴的荧光强度。
在一个实施例中，根据每个温度循环过程中每个微液滴的荧光强度值，获取每个微液滴的荧光曲线。每一个荧光曲线代表一个有用信息的曲线的变化过程，参加了液滴样本信息，以实现实时监测；以设定算法消除相邻液滴之间的相互影响。通过对每次温度循环过程中每个微液滴的各个部位的荧光强度值求和，作为每个微液滴的某一特定时刻的荧光强度值。
在一个实施例中，为了防止相邻的每个微液滴的边缘部位的互相影响，每个微液滴的某一特定时刻的荧光强度值采用部分求和方式。通过每次温度循环过程中所述多个微液滴的荧光强度值，可以获得所述多个微液滴在全部循环过程中的变化情况，获取每个微液滴的荧光曲线。在一个实施例中，每个微液滴进行了45次循环，共获得45张荧光图片。通过对45张荧光图片中每个微液滴进行定位，并获取每个微液滴的45个荧光强度值，从而获取每个微液滴的荧光曲线。
在一个实施例中，所述S4320构建关于待测核酸扩增反应液浓度c的联合二项分布函 数f(c)为：
Figure PCTCN2019072974-appb-000011
假设，在单一体积数字PCR中，每个微液滴的体积为v，待测核酸扩增反应液的核酸浓度为c，则每个微液滴含有的平均DNA数目为vc，假设每个微液滴含有的分子数为k，则可以通过泊松分布概率模型推导出k的概率分布P：
Figure PCTCN2019072974-appb-000012
对于k＝0即不含目标DNA分子的阴性微液滴，上式可改写为
p (k＝0)＝e -cv
在单一体积数字PCR分析中，可以通过微液滴总数n和阴性微液滴b估计出阴性微液滴的概率。
因此，推导出
Figure PCTCN2019072974-appb-000013
对于特定的实验结果，阴性微液滴数目b和总数n是已知的。因此，构建二项方程式：
Figure PCTCN2019072974-appb-000014
根据单一体积数字PCR分析过程，假设每个微液滴的体积分别为v 1、v 2，...v m，每个微液滴的体积分别对应的数目依次为n 1，n 2，…，n m。构建关于c的联合二项分布函数：
Figure PCTCN2019072974-appb-000015
在一个实施例中，所述S4330包括：
S4331：将所述联合二项分布函数f(c)求导，获取所述联合二项分布函数f(c)的导数
S4332：将所述联合二项分布函数f(c)的导数为0，获取所述联合二项分布函数f(c)取极值时的核酸扩增反应液浓度c的值。
通常函数导数为0时，函数值取极大或极小值。由于二项分布只有一个极大值，因此使函数的导函数为0时的解即为最可能的浓度值。通过使得所述联合二项分布函数f(c)取最大值时，获取相应的c的最大可能数。
在一个实施例中，所述步骤S4340中所述关于ln(c)的联合二项分布函数F(Λ)为：
Figure PCTCN2019072974-appb-000016
将ln(c)代替c，并令θ＝e -λ，Λ＝ln(c)，将所述二项分布函数f(c)转化为：
Figure PCTCN2019072974-appb-000017
P函数关于ln(c)比c更具有对称性，因此ln(c)的标准差σ更具有统计学意义。通过强化浓度为正值的约束条件，在低浓度样本分析时有较好的准确度。为简化计算，在计算ln(c)相应的标准差σ时，需要替换相应变量。
在一个实施例中，所述S4340包括：
S4341：将所述函数F(Λ)取对数，获取函数L(Λ)；
S4342：对所述函数L(Λ)求一阶导数，并将函数L(Λ)的一阶导数为0；
S4343：获取ln(c)相应的标准差σ；
S4344：根据ln(c)相应的标准差σ，获取ln(c)的置信区间。
将所述函数F(Λ)取对数，转化为：
Figure PCTCN2019072974-appb-000018
通过对所述函数F(Λ)取自然对数，可以使得相应的乘法关系变为独立的加法关系，使得对应的导函数更容易处理。
对所述L(Λ)求一阶导数，获得：
Figure PCTCN2019072974-appb-000019
令-v i代替ln(θ i)，并用t i表示第i重体积中阳性微液滴的数量，b i＝n i-t i，将步骤4中的式子转化为：
Figure PCTCN2019072974-appb-000020
将所述
Figure PCTCN2019072974-appb-000021
的式子为0，求解
Figure PCTCN2019072974-appb-000022
在一个实施例中，所述S4343中根据ln(c)的Fisher信息量I(Λ)获取标准差σ。
在一个实施例中，所述S4343中ln(c)的Fisher信息量I(Λ)为：
Figure PCTCN2019072974-appb-000023
对于标准差σ，可以结合ln(c)的Fisher信息量I(X)获取，相应的Fisher信息量可用下公式表示，其中E[]表示相应变量的期望值。
Figure PCTCN2019072974-appb-000024
在一个实施例中，所述ln(c)相应的标准差σ以及置信区间分别为：
Figure PCTCN2019072974-appb-000025
CI＝ln(c)±Zσ。
根据
Figure PCTCN2019072974-appb-000026
的的公式，求解：
Figure PCTCN2019072974-appb-000027
将
Figure PCTCN2019072974-appb-000028
的式子带入到步骤6中，获得：
Figure PCTCN2019072974-appb-000029
将公式
Figure PCTCN2019072974-appb-000030
带入步骤8中的式子，可以获得:
Figure PCTCN2019072974-appb-000031
从而可以从上式中可以获得ln(c)相应的标准差σ，以及置信区间：
Figure PCTCN2019072974-appb-000032
CI＝ln(c)±Zσ
其中，Z为标准正态分布的上临界值。
从获得的ln(c)相应的标准差σ，以及置信区间可以得到待测核酸扩增反应液的核酸浓度为c。通过标准正态分布表可以获知相应的数值，从而可以获知ln(c)的的置信区间，进而可以获知待测核酸扩增反应液的核酸浓度为c，从而可以获知所述待测核酸扩增反应液中含有的DNA起始拷贝数目。
其中，置信区间是指由样本统计量所构造的总体参数的估计区间。在统计学中，一个概率样本的置信区间(Confidence interval，CI)是对这个样本的某个总体参数的区间估计。置信区间展现的是这个参数的真实值有一定概率落在测量结果的周围的程度。置信区间给出的是被测量参数的测量值的可信程度，即前面所要求的"一个概率"。
数字PCR的定量结果通常需要结合置信区间和置信水平表示。在数字PCR中，置信区间展现的是样本真实浓度以一定概率落在测量结果λ的周围区间的程度，这个概率被称为置信水平。置信区间的两端被称为置信极限。
相比单一体积数字PCR，不同体积数字PCR可以利用少于200个微液滴实现5个数量级的检测动态范围，其性能可以和拥有12000个微液滴的单一体积数字PCR相媲美，节省了仪器的成本，耗材成本降低。同时，也可以对所述微液滴均一体积存在的特殊情况进行修正，使得所述数字PCR检测仪1检测精度提高。
通过所述不同体积数字PCR定量分析方法解决了结果的假阳性和假阴性。测序平台的样本高通量性，可同时进行上百例样本的检测。同时能利用不同种类的荧光，进行多个位点的检测，加速检测的速度，降低了实验成本。采用数字PCR检测仪通过微滴化处理，使得稀有检测片段从大量的复杂背景中分离出来，大大简化操作步骤，有效节约了准备时间和检测时间，且结果判读直观可靠，具有可以稳定实施的特点，检测灵敏度及精确性都达到精准定量的要求，提高了检测的灵敏度和精确性。
在实际过程中，所述不同体积数字PCR定量分析方法，不依赖于标准曲线而能够高精度地测定所述多个微液滴的起始DNA浓度。在所述数字PCR检测仪1中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少。
通过数字PCR定量检测方法可以实现所述多个微液滴的动态跟踪，在所述多个微液滴进行温度循环过程中都可以找到每个微液滴对应的具体位置，可以实现核酸扩增的全部过程的监测。因此，通过所述数字PCR定量检测方法可以解决所述多个微液滴中存在假阳性的问题。同时，通过对所述多个微液滴荧光曲线进行处理，并进行不依赖于均匀性假设进行的统计修正，而取得真正的绝对定量。
不仅摆脱了对标准曲线的依赖，排除了由标准曲线引起的定量结果不确定的问题，并且解决了液滴式数字PCR终点检测方式的限制，打破了只采用了一个p(x＝0)的数据对待测样本整体进行参数估计的局限性。采用所述实时荧光定量PCR检测方法，提高了数字PCR定量检测的准确性。
所述数字PCR定量检测方法依赖抽象的数学模型，实现了重复性、高灵敏性，并且动态范围变大，可以利用少量液滴实现监测。用小量的数据覆盖更多的信息。同时，所述数字PCR定量检测方法避免了之前泊松分布概率模型的误差，实现绝对定量，更加直观。综合了所有数据，消除了随机误差。获取液滴样本荧光曲线，实时监测液滴样本的荧光亮度的变化，用以去除假阳性；消除相邻液滴之间的相互影响，为后续定量分析模型提供更精确的数据源。
在一个实施例中，检测DNA为：人巨细胞病毒DNA。
以待测样本DNA为模板，加入待测样本DNA对应的检测引物及其探针；
所述实时荧光定量PCR检测方法采用的是Taqman荧光探针。
获取人巨细胞病毒核酸定量检测试剂盒，所述检测试剂盒包括人巨细胞病毒DNA实时荧光定量检测引物及其探针。
把试剂盒中10^6copies/mL浓度的阳性质控品(非标准浓度)比例稀释，所得浓度：10^6copies/mL、10^5copies/mL、10^4copies/mL、0.5×10^4copies/mL。同时，共制作5个样本浓度，分别为2×10^6copies/mL、10^6copies/mL、10^5copies/mL、10^4copies/mL以及0.5×10^4copies/mL。其中，所述待测样本的试剂配置比例为：1ul样本(2×10^6copies/mL加入2ul样本)、1ul DNA聚合酶、20ul Buffer，一共为22ul。
将所述样本浓度分别为2×10^6copies/mL、10^6copies/mL、10^5copies/mL、10^4copies/mL以及0.5×10^4copies/mL的样本，分别通过本申请提供的一种数字PCR检测仪、一种QX200数字PCR检测仪以及一种qPCR数字PCR检测仪进行检测，获得表1中通过各个仪器的5个核酸扩增反应液起始拷贝数的检测值与真实值相关系数的对比表。
5个核酸扩增反应液起始拷贝数的检测值与真实值相关系数的对比表
仪器类型 本仪器 QX200数字PCR qPCR数字PCR
R 2 0.9993 0.998 0.9923
相关系数R是用以反映变量之间相关关系密切程度的统计指标，用来度量两个变量间的线性关系。从表1中可以看出通过本申请提供的一种数字PCR检测仪检测的5个核酸扩增反应液的起始拷贝数的检测值与真实值的相关系数最大，最接近于1。因此，通过本申请提供的一种数字PCR检测仪检测的5个核酸扩增反应液的起始拷贝数的检测值与真实值的相关系数最大，最接近于1。所以，本申请提供的一种数字PCR检测仪1的检测精度更高，准确度更高。
所述数字PCR检测仪1将所述微液滴生成装置10、所述温控装置20、所述荧光信号检测装置30以及所述定量分析装置40集成化，通过控制器50使得所述操作人员可以通过一体式数字PCR检测机1实现自动化操作，提高了工作效率，反应快速、重复性好、灵敏度高、特异性强、结果清晰。
针对传统的数字PCR检测方法工作量繁重、耗费时间效率低的问题，提供一种简便、效率高的数字PCR检测方法。
请参见图19，本申请提供一种数字PCR检测方法，包括：
S10，制备待测核酸扩增反应液；
S20，将所述待测核酸扩增反应液微滴化，形成微液滴阵列；
S30，将所述微液滴阵列进行聚合酶链式反应，并获取所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线；以及
S40，根据所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线，对所述微液滴阵列进行分析，以获得所述待测核酸信息。
在制备待测核酸扩增反应液时，可以使用一种饱和荧光染料即可实现对不同类型变异的分类，具有高分辨率与灵敏度，降低了数字PCR检测仪检测的成本。并且，通过所述数字PCR检测方法，可以使得所述微液滴阵列在同一个高度集成化的数字PCR检测仪上完成聚合酶链式反应(Polymerase Chain Reaction，PCR)，并在所述微液滴阵列PCR扩增后对PCR产物进行熔解曲线分析。在所述微液滴阵列进行聚合酶链式反应过程中，获取每个所述微液滴的荧光曲线。当所述微液滴阵列完成PCR扩增之后，再进行一次高低温循环，获取此次循环过程中的每个所述微液滴的熔解曲线。通过所述数字PCR检测方法，可以获得所述微液滴阵列的荧光曲线与所述微液滴阵列的熔解曲线，完全可以实现PCR扩增整个过程的实时监测与PCR产物的熔解曲线分析的无痕连接。从而，通过所述微液滴阵列的荧光曲线与熔解曲线，实现对所述微液滴阵列的定性以及定量分析，更加全面、简便、高效的完成数字PCR的检测。
在一个实施例中，在所述步骤S10中所述核酸扩增反应液中包含待检测的核酸模板、反应缓冲溶液、脱氧核糖核苷三磷酸、引物、聚合酶和产物标记物质等。耐热DNA聚合酶可以为FastStart Taq DNA聚合酶、Ex Taq、Z-Taq、AccuPrime Taq DNA聚合酶和HS Taq DNA聚合酶等。
其中，核酸扩增反应液，可以是以脱氧核糖核酸(DNA)为模板的核酸扩增反应液(可称为DNA扩增反应液)，也可以是互补脱氧核糖核酸(cDNA)即以核糖核酸(RNA)为模板的逆转录核酸扩增反应液(可称为RNA反转录反应液)，还可以是其它核酸扩增反应液，如环介导等温扩增(LAMP)反应液。其中，所述DNA扩增反应液的特点是含有DNA扩增所需要的dNTP、缓冲液、无机盐离子、聚合酶、引物、待检测的DNA模板以及染料等。反应液中的染料能指示核酸扩增，可以是SYBR Green等与DNA结合的荧光染料。
在一个实施例中，准备专供数字PCR使用的成套试剂和溶液，用来减少或避免外源DNA对模板DNA样本的潜在污染。所使用的所有仪器和耗材应该进行高温灭菌，高温干燥处理。
在一个实施例中，制备所述待测核酸扩增反应液时，采用SYBR Green荧光染料对所述待测核酸扩增反应液进行标记。由于SYBR Green荧光染料因能与所有的双链DNA相结合，对模板没有选择性，并且价格便宜，适用于多种目的产物的检测。采用熔解曲线分析时一般是用SYBR Green作为荧光染料的时候使用。因为SYBR Green染料是非特异的染料，只要有扩增，染料就与双链DNA结合，荧光大大增强。通过荧光信号的改变与温度作图，形成熔解曲线是为了观察扩增发出荧光的片段是不是数字PCR检测时需要检测的目 的基因。如果熔解曲线做出来只有单峰，出峰位置是退火温度，且为窄峰，那么产物是PCR扩增的特异性产物，如果峰位置不对或为宽峰，则可能是产物不是特异性或无设计对应产物，若在熔解曲线峰值之前存在小峰值，可能是引物二聚体，需要考虑重新设计引物等。如果高分辨率溶解曲线，有轻微的温度或曲线形状变化，说明有但核苷酸的变化，通过大量重复的微液滴阵列的溶解曲线分析，通过密集的曲线分析匹配程序，可以自动对核酸序列分型等。
请参见图20，在一个实施例中，在所述步骤S20中将所述待测核酸扩增反应液微滴化，形成微液滴阵列。所述微液滴阵列包括大量的多个微液滴。
在一个实施例中，一种数字PCR检测仪1，所述数字PCR检测仪1包括微液滴生成装置10、温控装置20、荧光信号检测装置30、定量分析装置40以及控制器50。
所述微液滴生成装置10用以将核酸扩增反应液微滴化，形成所述微液滴阵列，进而在微液滴容器中形成微液滴阵列，可以同时检测出多种目标序列。所述温控装置20用以进行温度循环，实现核酸扩增。所述荧光信号检测装置30与所述温控装置20相对设置，用以对核酸扩增后的所述微液滴阵列进行信号采集。所述定量分析装置40与所述荧光信号检测装置30通过数据线连接，用以实现所述微液滴阵列荧光信息的传输，进行定量分析。所述控制器50分别与所述微液滴生成装置10、所述温控装置20、荧光信号检测装置30以及定量分析装置40连接，用以控制所述微液滴生成装置10、所述温控装置20、荧光信号检测装置30以及定量分析装置40。
所述数字PCR检测仪在工作时，所述微液滴生成装置10可以将所述待测核酸扩增反应液进行微滴化，从而形成所述微液滴阵列。所述微液滴阵列包括多个大量的微液滴。所述温控装置20可以对所述微液滴阵列进行核酸扩增。所述荧光信号检测装置30实时采集所述微液滴阵列的荧光变化图像。所述温控装置20对所述微液滴阵列进行核酸扩增反应，并通过所述荧光信号检测装置30采集核酸扩增反应后的所述微液滴阵列的产物信号，如荧光、紫外吸收、浊度等信号。利用所述多个扩增与非扩增微液滴在组成上的差异，对获得目标序列扩增的液滴数量进行分析，最终实现对核酸分子的定性以及定量分析。通过实时监测所述微液滴阵列的信号的变化，检测结果具有直接性，并可以解决所述微液滴阵列中的假阳性和假阴性的问题。
通过所述微液滴生成装置10在微液滴容器中生成所述微液滴阵列，并将落至微液滴容器的底部，不规则的堆积在一起。通过所述微液滴生成装置10制备的所述微液滴阵列在向下沉降过程当中，集中集合在微液滴容器的中间部位，聚集在一起，此时需要在对所述微液滴阵列进行信号采集之前，将多个所述微液滴阵列平铺在微液滴容器的底部。
在一个实施例中，在对多个所述微液滴阵列进行信号采集之前，通过所述温控装置20进行高低温，将多个所述微液滴阵列平铺在微液滴容器的底部。首先，将所述微液滴阵列升温。其次，将所述微液滴阵列降温。再次，将所述微液滴阵列进行高低温循环，直至所述微液滴阵列平铺于所述微液滴容器底板。最后，将所述微液滴阵列平铺于微液滴容器中，并将平铺后的所述微液滴阵列进行PCR扩增并进行拍照检测。
在一个实施例中，通过微液滴生成装置10可以生成多个体积大小均一的微液滴。每个所述微液滴大小在微米级。在多个所述微液滴体积均一的情况下，根据多个所述微液滴的荧光信息，对多个所述微液滴进行定量分析。
在一个实施例中，所述步骤S30包括：
S310，设置聚合酶链式反应的温度参数、时间参数以及循环次数；
S320，根据所述温度参数以及所述时间参数对所述微液滴阵列进行聚合酶链式反应，依次完成所述循环次数，并获取每次循环过程的每个所述微液滴的荧光曲线；以及
S330，将完成聚合酶链式反应扩增后的所述微液滴阵列降温，并以特定的温度间隔进行升温，获取每个所述微液滴的熔解曲线。
PCR由变性--退火(复性)--延伸三个基本反应步骤组成构成。模板DNA的变性是指模板DNA经加热至90℃～95℃一定时间后，使模板DNA双链或经PCR扩增形成的双链DNA解离，使之成为单链，以便它与引物结合，为下轮反应作准备。模板DNA与引物的退火(复性)是指模板DNA经加热变性成单链后，温度降至50～60℃，引物与模板DNA单链的互补序列配对结合。引物的延伸是指DNA模板--引物结合物在DNA聚合酶的作用下，于70～75℃，以dNTP为反应原料，靶序列为模板，按碱基配对与半保留复制原理，合成一条新的与模板DNA链互补的半保留复制链重复循环变性--退火--延伸三过程，就可获得更多的“半保留复制链”，而且这种新链又可成为下次循环的模板。每完成一个循环需2～4分钟，2～3小时就能将待扩目的基因扩增放大几百万倍。
其中，退火温度是影响PCR特异性的较重要因素。变性后温度快速冷却至40℃～60℃，可使引物和模板发生结合。由于模板DNA比引物复杂得多，引物和模板之间的碰撞结合机会远远高于模板互补链之间的碰撞。退火温度与时间，取决于引物的长度、碱基组成及其浓度，还有靶基序列的长度。
在所述步骤S310中，所述微液滴阵列在引物、待检测DNA样本和耐热DNA聚合酶存在下，通过使变性、退火和延伸步骤的循环重复大约30次～50次来进行PCR。所述循环次数一般设置为30次～50次的变性、退火和延伸三步过程循环。所述温度参数一般设置为40℃～95℃，所述时间参数根据每一个具体过程而定。
在一个实施例中，所述步骤S320包括：
S321，根据所述温度参数以及所述时间参数对所述微液滴阵列进行聚合酶链式反应，获取所述微液滴阵列的荧光图像；
S322，根据所述循环次数依次循环，获取所述微液滴阵列在聚合酶链式反应过程中的全部荧光图像；
S323，根据所述微液滴阵列的全部荧光图像，获取每次循环过程的每个所述微液滴的荧光信息；以及
S324，根据每次循环过程的每个所述微液滴的荧光信息，获取每个所述微液滴的荧光曲线，从而获得所述微液滴阵列的荧光曲线。
在所述步骤S321中，所述步骤S321包括：
首先，将所述微液滴阵列加热至95℃，并加热4min，用以将所述微液滴阵列中的酶进行热启动，所述微液滴阵列完成酶热启动之后，对所述微液滴阵列进行变性1min；
其次，所述微液滴阵列变性之后，降温至55℃，引物与DNA模板结合，形成局部双链，退火(复性)1min，此时通过所述荧光信号检测装置30对所述微液滴阵列进行拍照，获得第一次循环的所述微液滴阵列的荧光图像；
再次，将所述微液滴阵列进行升温至70℃，延伸7min；
最后，根据所述循环次数依次按照上述变性-退火(复性)-延伸三步骤进行循环45次，循环45次之后，降温至4℃，对所述多个微液进行保存。
在一个实施例中，通过所述温控装置20对所述微液滴阵列进行升温降温，所述循环次数设置为45次，进而每个微液滴可以获取45次循环过程中的45张荧光图像。每个所述微液滴进行了45次循环，共获得45张荧光图像。通过对45张荧光图像中每个所述微液滴进行定位，并获取每个所述微液滴的45个荧光强度值，从而获取每个所述微液滴的荧光曲线。
在一个实施例中，在所述步骤S323中，首先，根据每个荧光图像，获得每个进行PCR扩增的所述微液滴的荧光强度值。然后，根据所述每个进行PCR扩增的微液滴的荧光强度值，获得每个进行PCR扩增的微液滴的荧光曲线。最后，根据所述每个进行PCR扩增的微液滴的荧光曲线，获得所述所有进行PCR扩增的微液滴的荧光曲线，也就是所述微液滴阵列的荧光曲线。
请参见图21，在一个实施例中，采集所述微液滴阵列的荧光图像，并进行图像追踪。在获取每个所述微液滴阵列的荧光曲线时，需要对每张图像中的每个所述微液滴分别进行定位，获取每个所述微液滴的荧光强度。在所述数字PCR检测仪中，会在成像系统中标定，所述荧光图像的每一个像素对应的实际的比例是多少。根据所述荧光图像，提取出所述微液滴的直径对应的是多少个像素，从而得到直径对应多少个微米，也就可以获得所述微液滴的直径是多少。
在一个实施例中，对每个所述微液滴进行追踪时，还可以通过在每个温度循环过程中采集的图像，进行NCAST图像差分及聚类运算，识别到每个所述微液滴的位置，进而获取所述微液滴阵列的荧光强度。
在一个实施例中，如果在一个温度循环过程中造成的微液滴移动距离不大于一个微液滴的直径，可以采用以下方法进行微液滴追踪。其中，对每个所述微液滴进行追踪时，对每个所述微液滴进行图像追踪步骤如下：
首先，识别每次温度循环过程中拍摄的照片，获得每个微液滴的圆心位置；
然后，将当前识别的每个微液滴的圆心位置与前一次循环过程中的每个微液滴的圆心位置对比；
最后，若当前识别的每个微液滴的圆心位置与前一次循环过程中的每个微液滴的圆心位置的圆心间距小于一个微液滴直径，则标记为同一个微液滴。
请参见图22，在一个实施例中，根据每个温度循环过程中每个所述微液滴的荧光强度 值，获取所述每个微液滴的荧光曲线。通过对每次温度循环过程中每个所述微液滴的各个部位的荧光强度值求和，作为每个所述微液滴的某一特定时刻的荧光强度值。
在一个实施例中，为了防止相邻的每个所述微液滴的边缘部位的互相影响，每个所述微液滴的某一特定时刻的荧光强度值采用部分求和方式。通过每次温度循环过程中所述微液滴阵列的荧光强度值，可以获得所述微液滴阵列在全部循环过程中的变化情况，获取每个所述微液滴的荧光曲线。
在一个实施例中，所述步骤S330包括：
S331，将完成聚合酶链式反应扩增后的所述微液滴阵列降温至40摄氏度以下；
S332，将降温至40摄氏度以下的所述微液滴阵列以特定的温度间隔进行升温，获取所述温度间隔对应的所述微液滴阵列的荧光图像；
S333，根据所述温度间隔对应的所述微液滴阵列的荧光图像，获取所述温度间隔对应的每个所述微液滴的荧光信息；以及
S334，根据所述温度间隔对应的每个所述微液滴的荧光信息，获取每个所述微液滴的熔解曲线，从而获得所述微液滴阵列的熔解曲线。
PCR扩增反应完成后，通过逐渐增加温度同时监测每一步的荧光信号来产生熔解曲线。由于不同的DNA有不同的温度熔解线，随着反应中双链DNA变性，荧光染料又恢复到游离状态导致荧光信号降低，用荧光信号改变与温度作图。在扩增产物染料法需要做熔解曲线，因为熔解曲线是由于染料法的特异性差，所以通过熔解曲线考察扩增产物是否是目标产物。熔解温度上有一特征峰，用这个特征峰就可以将特异产物与其它产物如引物二聚体区或非特异性产物分开。
在所述步骤S332中，将PCR扩增后对PCR产物进行熔解曲线分析，使得PCR扩增与PCR产物的熔解曲线分析无痕连接。在所述步骤S332中，通过所述温控装置20将温度降至40摄氏度以下，并以所述温度间隔为0.1℃进行依次升温至95℃，每间隔0.1℃通过所述荧光信号检测装置30进行拍照一次直至升温至95℃。然后，拍照结束后，将所述微液滴阵列降温至4℃，对所述微液滴阵列进行保存。
在一个实施例中，获取每个所述微液滴的荧光曲线时处理荧光图像的方法与获得每个所述微液滴的熔解曲线时处理荧光图像的方法相同。
在所述步骤S333中，根据每间隔0.1℃采集获得的荧光图像，获取每个所述微液滴对应的荧光强度，绘制关于温度与荧光强度的曲线，并一次微分获得含有波峰的峰性图。
熔解曲线(Dissociation curve)是指随温度升高DNA的双螺旋结构降解程度的曲线。熔解曲线分析可以用来确定不同的反应产物，包括非特异性产物。总的DNA双螺旋结构降解一半的温度称为熔解温度(Tm)，不同序列的DNA，Tm值不同。也就是说一个DNA的熔解曲线是一个DNA的指纹，对应特定的一个DNA。根据熔解曲线图，其波峰所在的温度代表双链DNA分子的Tm值(熔点温度)。根据扩增产物的Tm值即可判断其基因型。DNA片段的Tm依赖于它的长度、G+C组成、序列、链互补性、浓度和缓冲区成分，诸如盐、染料和PCR增强剂。
每一条熔解曲线代表着每个所述微液滴里产物的单一情况，若一条熔解曲线为单峰且在一定合理的温度范围内(一般为80～90℃)则认为正常，如果熔解曲线为双峰则可能有非特异性扩增。由此可以判断产物是否为目的基因且是否单一。内参有内参的线，同一条熔解曲线不会出现两个基因的峰。
对于所述微液滴阵列的每一条高分辨率熔解曲线分析，可以检测单核苷酸多态性及扫描突变。
在一个实施例中，在数字PCR检测过程中，通过所述荧光信号检测装置30对所述微液滴阵列进行图像采集。
通过所述荧光信号检测装置30，对所述微液滴阵列进行图像采集。其中，从微液滴容器上方采取倾斜角度照射在微液滴容器上。采用所述荧光信号检测装置30实现对所述微液滴阵列进行周期性的二维扫描，并实时进行图像采集。微液滴容器内的所述微液滴阵列的内部荧光被激发，通过所述荧光信号检测装置30的物镜收集，进入相机，相机采集所述微液滴阵列的荧光图像。
通过所述荧光信号检测装置30可以使得所述微液滴阵列荧光成像，一次拍摄一定数量的所述微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。
通过所述荧光信号检测装置的荧光探测组件对含有荧光物质的所述微液滴阵列进行荧光信息采集，并将检测到的荧光信息以荧光图像的形式传输至计算机，用以进行定量分析。采用荧光成像的检测方法一次拍摄一定数量的所述微液滴的荧光图像，然后利用图像处理技术，将图像中的液滴荧光进行自动识别，从而得到液滴的荧光信息。由于所述荧光成像的检测方法的成像范围大，因此，在检测的时候对所述微液滴阵列所处的检测环境的要求较低。
在一个实施例中，所述步骤S40包括：
S410，根据所述微液滴阵列的荧光曲线，获取所述微液滴阵列的核酸起始拷贝数；以及
S420，根据所述微液滴阵列的熔解曲线，获取所述微液滴阵列的核酸信息。
在一个实施例中，所述步骤S410包括：
S411，根据所述微液滴阵列的荧光曲线，获取每个所述微液滴的所述荧光曲线对应的Ct值；
S412，根据每个所述微液滴的所述荧光曲线的Ct值进行聚类，并依次由大到小进行排序，获得x1，x2，…，xn个类别；
S413，根据所述x1，x2，….xn个类别，获得每个类别对应的所述微液滴的数量y1、y2、….yn；
S414，根据每个类别对应的所述微液滴的数量y1、y2、….yn，获得所述x1，x2，…，xn个类别对应的所述微液滴的数量y1、y2、….yn频数分布；
S415，根据所述频数分布，计算所述微液滴阵列的核酸起始拷贝数。
在所述步骤S411中，PCR循环在到达Ct值所在的循环数时，刚刚进入真正的指数扩增期(对数期)，此时微小误差尚未放大，因此Ct值的重现性极好，即同一DNA模板不同时间扩增或同一时间不同微液滴容器内扩增，得到的Ct值是恒定的。当所述微液滴对应的荧光曲线为扩增曲线时，此时表明所述微液滴中含有目标基因成分。当所述微液滴对应的荧光曲线为一条直线时，此时表明所述微液滴中不含目标基因成分。
从获取的实时荧光曲线，可以获得Ct值，每个所述微液滴的Ct值在获取时，对所述荧光曲线进行求导，所述荧光曲线的斜率固定的荧光曲线的起始循环次数即为所需要的Ct值。
在一个实施例中，在所述步骤S411中，首先，对每个进行PCR扩增的微液滴的荧光曲线求导，获取所述每个进行PCR扩增的微液滴的荧光曲线的斜率。其次，根据所述每个进行PCR扩增的微液滴的荧光曲线的斜率，获取所述每个进行PCR扩增的微液滴的荧光曲线的斜率中斜率固定不变的数值。再次，根据所述斜率固定不变的数值，获取其对应的起始循环数，所述起始循环数为每个进行PCR扩增的微液滴的Ct值。最后，根据所述每个进行PCR扩增的微液滴的Ct值，获得所述所有进行PCR扩增的微液滴的Ct值。
在一个实施例中，在所述步骤S411中，首先，根据每个进行PCR扩增的微液滴的荧光曲线，获得每个进行PCR扩增的微液滴的荧光域值的缺损值。其次，根据每个进行PCR扩增的微液滴的荧光域值的缺损值，获得其对应的循环数，所述循环数为每个进行PCR扩增的微液滴的Ct值。再次，根据所述每个进行PCR扩增的微液滴的Ct值，获得所述所有进行PCR扩增的微液滴的Ct值。
其中，Ct值中C代表Cycle，t代表threshold，Ct值的含义是：每个反应管内的荧光信号到达设定的域值时所经历的循环数。在实时荧光PCR中，Ct值是指每个反应管内的荧光信号达到设定的阈值时所经历的循环数。PCR反应的前15个循环的荧光信号作为荧光本底信号，荧光域值的缺损设置是3-15个循环的荧光信号的标准偏差的10倍，即：threshold＝10×SDcycle 3-15。
在一个实施例中，PCR反应的前15个循环的荧光信号作为荧光本底信号，荧光域值的缺损设置是3-15个循环的荧光信号的标准偏差的10倍，即：threshold＝10×SDcycle3-15。根据所述荧光域值的缺损值threshold，获取相对应的循环数，所述循环数为所述Ct值。Ct值与起始DNA浓度的关系为：起始拷贝数越多，Ct值越小。
在所述步骤S412中根据每个所述微液滴的所述荧光曲线的Ct值进行聚类，并依次由大到小进行排序，获得，x 2，…，x n个类别。其中，所述微液滴阵列中暗液滴对应的类别为x 1，也就是说所述微液滴阵列中不含核酸起始拷贝数的液滴对应的类别为x 1类别。由于核酸起始拷贝数越多，Ct值越小，所以此时暗液滴(阴性液滴)对应的Ct值为无穷大，也就是所述x 1类别对应的Ct值为无穷大。以此类推，所述x 2类别对应的核酸起始拷贝数为1，所述x 3类别对应的核酸起始拷贝数为2，所述x 4类别对应的核酸起始拷贝数为3，所述x 5类别对应的核酸起始拷贝数为4等。
在一个实施例中，在所述步骤S415中，当所述x 1类别的所述微液滴的数量y 1大于或 等于特征值m时，根据所述频数分布进行泊松分布拟合，获得泊松分布的参数λ，从而获得所述微液滴阵列的核酸起始拷贝数。
其中，所述特征值m范围为所述微液滴阵列中所述微液滴总数的0.5％-10％。
在一个实施例中，所述特征值m可以为所述微液滴总数的5％。
当所述x 1类别的所述微液滴的数量y 1大于或等于特征值m时，此时所述微液滴阵列中暗液滴的数量y 1大于或等于特征值m。此时，所述微液滴阵列中暗液滴的数量对整体计算所述微液滴阵列的核酸起始拷贝数起到一定的作用，从而对所述微液滴的数量y 1、y 2、….y n频数分布进行泊松分布拟合，获得对应的泊松分布的参数λ。
假设：数字PCR中的微液滴含有的起始DNA拷贝数为x，根据数理统计理论，x＝k(k＝0，1，2，3.....)的概率分布函数P符合泊松概率模型，其中λ为微液滴中含有的平均分子拷贝数。
Figure PCTCN2019072974-appb-000033
因此，通过所述泊松分布模型期望值μ与方差σ 2，可知期望值μ为λ，方差σ 2为λ。因此，可知数字PCR中每个微液滴中包含目标DNA分子的拷贝数为λ，所以求得的λ值就能够实现核酸的定量检测。
其中，对于液滴式PCR，单个液滴中所包含的起始拷贝数满足Poisson分布。
Figure PCTCN2019072974-appb-000034
其中λ为所述微液滴中平均包含的起始DNA拷贝数。每个液滴中包含的平均起始拷贝数用CPD(copies per droplet)来表示。
此时，所述微液滴阵列的核酸起始拷贝数为λ乘以所述微液滴阵列的所述微液滴的数量。
假设所述待测核酸扩增反应液的总体积为V(每个微液滴的体积为v)，则所述待测核酸扩增反应液浓度c(copy/μL)为：
Figure PCTCN2019072974-appb-000035
所以，求得λ的值，也就能实现DNA的定量检测。
在一个实施例中，在所述步骤S415中，当所述x 1类别的所述微液滴的数量y 1小于特征值m时，包括：
S4151，根据所述x 2，…，x n个类别对应的所述微液滴的数量y 2、…，y n的部分频数分布，依次假设所述x 2类别对应的所述核酸起始拷贝数，并进行泊松分布拟合，获取每个泊松分布对应的的参数λ j(j＝0，1，2….)；
S4152，在一个区间[λ min,λ max]内搜索λ j，使得频数值误差平方和err最小，获得最优 λ optimal；
S4153，根据所述最优λ optimal，计算所述微液滴阵列的核酸起始拷贝数。
当所述x 1类别的所述微液滴的数量y 1小于特征值m时，此时所述微液滴阵列中暗液滴的数量对整体计算所述微液滴阵列的核酸起始拷贝数没有作用，可以忽略。
Biorad系统在20000个液滴体系的情况下，一般建议样本DNA浓度不大于6CPD。在实际实验过程中，k>4时，Ct值的区分变小，很难根据Ct值区分一个液滴的起始拷贝数为4或者5，因此可以只使用所述x 2，…，x n个类别进行不完整抽样来对Poisson分布进行拟合。
在一个实施例中，给定一个区间[λ min,λ max]，在[λ min,λ max]区间中搜索，计算误差的平方和err最小，选择最优λ optimal使得误差平方和最小。
在一个实施例中，获得泊松分布的参数λ时，采用最大似然估计方法对参数λ进行估计。
在一个实施例中，对参数λ进行估计的方法还可以为矩估计法、顺序统计量法或最大似然法。
采用此方法无需保证阴性暗液滴的数量，且比单独采用一个频点进行估计的精度和稳定性高得多。
在所述步骤S4153中，所述微液滴阵列的核酸起始拷贝数为所述最优λ optimal乘以所述微液滴阵列的所述微液滴的数量。
在一个实施例中，根据不完全抽样，采用最小二乘法对泊松分布的参数λ进行点估计。
在实际过程中，所述数字PCR定量检测方法，不依赖于标准曲线而能够高精度地测定所述微液滴阵列的核酸起始拷贝数。
同时，通过实时荧光曲线可以解决所述微液滴阵列中存在假阳性的问题。通过对所述微液滴阵列的荧光曲线进行处理，并进行不依赖于均匀性假设进行的统计修正，而取得真正的绝对定量。
采用所述数字PCR定量检测方法不仅摆脱了对标准荧光曲线的依赖，排除了由标准荧光曲线引起的定量结果不确定的问题，并且解决了液滴式数字PCR终点检测方式的限制，打破了只采用了一个p(x＝0)的数据对待测样本整体进行参数估计的局限性，提高了数字PCR定量检测的准确性。
采用所述数字PCR定量检测方法无需保证阴性空液滴的数量。同时，采用多维度的频数分布数据对整体进行最优参数估计的精度要比单独采用一个p(x＝0)的数据进行估计的精度和稳定性高得多。
每一个荧光曲线代表一个有用信息的曲线的变化过程，参加了液滴样本信息，以实现实时监测，以设定算法消除相邻液滴之间的相互影响。
所述数字PCR检测方法依赖抽象的数学模型，实现了重复性、高灵敏性，并且动态范围变大，可以利用少量液滴实现监测。用小量的数据覆盖更多的信息。同时，所述数字PCR定量检测方法避免了之前泊松分布概率模型的误差，实现绝对定量，更加直观。并且，通 过所述数字PCR定量检测方法可以结合所有数据，从而可以避免随机误差的产生。获取液滴样本荧光曲线，实时监测液滴样本的荧光亮度的变化，用以去除假阳性，消除相邻液滴之间的相互影响，为后续定量分析模型提供更精确的数据源。
请参见图23，根据部分核酸起始拷贝数分别为0，1，2，3情况下获得的泊松分布拟合。其中，横坐标为每个所述微液滴中包含的平均起始拷贝数(copies per droplet，CPD)。纵坐标为每个所述微液滴中包含的平均起始拷贝数的标准偏差(standard deviation，Std Dev，STD)。每个所述微液滴中包含的平均起始拷贝数用CPD(copies per droplet)来表示。可知，采用部分所述核酸起始拷贝数获得的每个所述微液滴中包含的平均起始拷贝数用CPD的标准偏差比其他算法获得的平均起始拷贝数用CPD的标准偏差小。因此，所述数字PCR检测方法获得的每个所述微液滴中包含的平均起始拷贝数用CPD的值更加精确。对20000个液滴进行1000次仿真的结果表明。仅采用单点的估计方法只能覆盖有限的浓度范围，并且估计精度随着样品浓度的升高而急剧恶化。而采用不完全泊松分布拟合算法，随着样品浓度的增加，估计精度没有明显的恶化，并且能够将待测核酸扩增反应液的浓度扩大两倍。对于液滴数量较少的情况下，不完全泊松分布拟合算法(部分抽样泊松分布拟合算法)仍然具有很好的可靠性。
通过所述数字PCR定量检测方法解决了结果的假阳性和假阴性。测序平台的样本高通量性，可同时进行上百例样本的检测。同时能利用不同种类的荧光，进行多个位点的检测，加速检测的速度，降低了实验成本。采用数字PCR检测仪通过微滴化处理，使得稀有检测片段从大量的复杂背景中分离出来，大大简化操作步骤，有效节约了准备时间和检测时间，且结果判读直观可靠，具有可以稳定实施的特点，检测灵敏度及精确性都达到精准定量的要求，提高了检测的灵敏度和精确性。
在一个实施例中，所述步骤S420包括：
S421，根据所述微液滴阵列的熔解曲线，获取每个所述微液滴的所述熔解曲线对应的熔解温度；以及
S422，根据所述熔解温度，对所述微液滴阵列进行分类，获取所述微液滴阵列的核酸信息，进而获得所述待测核酸的核酸信息。
不同序列的DNA，Tm值不同。也就是说一个DNA的熔解曲线是一个DNA的指纹，对应特定的一个DNA。根据熔解曲线图，其波峰所在的温度代表双链DNA分子的Tm值(熔点温度)。根据扩增产物的Tm值即可判断其基因型。通过将相同的熔解曲线进行归类划分，不同形状熔解曲线的基因分型或归类，并与目标基因的熔解曲线进行对比，可以去除非特异性假阳性，排除与目标基因不同的序列。
在一个实施例中，通过熔解曲线对所述微液滴阵列进行分类时，可以采用决策树、贝叶斯、人工神经网络、K-近邻、支持向量机和基于关联规则的分类、Bagging以及Boosting等算法。
在一个实施例中，所述数字PCR检测方法还包括：
S50，获取所述微液滴阵列的高分辨率熔解曲线，对所述微液滴阵列进行分类，并获 得所述微液滴阵列的基因分型、突变检测等核酸信息。
根据所述熔解曲线，可以获取所述微液滴阵列的核酸扩增的特异性，以及核酸扩层过程中是否有引物二聚体现象等。
根据所述微液滴阵列的实时检测dsDNA熔解过程中荧光信号值的变化，获取所述微液滴阵列的高分辨率熔解曲线。高分辨率熔解曲线是一种基于单核苷酸熔解温度不同而形成不同形态熔解曲线的基因分析新技术，具有极高的敏感性，可以检测出单个碱基的差异，并且成本低、通量高、速度快、结果准确、不受检测位点的局限，实现了真正的闭管操作。在突变扫描、单核苷酸多态性分析、甲基化研究、基因分型、序列匹配等方面HRM分析技术发挥着重要作用。双链核苷酸的热稳定性受其长度和碱基组成的影响，序列变化会导致升温过程中dsDNA解链行为的改变。因为所用的荧光染料只能嵌入并结合到dsDNA上，因此利用实时PCR技术，通过实时检测dsDNA熔解过程中荧光信号值的变化，就能以生成不同形状熔解曲线的方式将PCR产物中存在的差异直观地展示出来。同时，借助于专业性的分析软件就可以对测试群体实现基于不同形状熔解曲线的基因分型或归类。
在一个实施例中，在所述步骤S20中形成所述微液滴阵列时，可以采用瞬时加速的微液滴生成方法或变速周期的微液滴生成方法。
在一个实施例中，在所述步骤S10中所述待测核酸扩增反应液中采用饱和染料对聚合酶链式反应产物进行分析。
在一个实施例中，对多个所述微液滴进行定性分类分析时，也可以采用高分辨率熔解曲线分析(high resolution melting analysis，HRM)无需使用序列特异性探针，而是利用一种饱和染料对PCR反应产物进行分析。双链核苷酸(double strand DNA，dsDNA)的热稳定性受其长度和碱基组成的影响，序列变化会导致升温过程中dsDNA解链行为的改变。因为所用的荧光染料只能嵌入并结合到dsDNA上，因此利用实时PCR技术，通过实时检测dsDNA熔解过程中荧光信号值的变化，就能以生成不同形状熔解曲线的方式将PCR产物中存在的差异直观地展示出来。同时，借助于专业性的分析软件就可以对测试群体实现基于不同形状熔解曲线的基因分型或归类。
在一个实施例中，引物设计有三条基本原则。首先，引物与模板的序列要紧密互补。其次，引物与引物之间避免形成稳定的二聚体或发夹结构。再次，引物不能在模板的非目的位点引发DNA聚合反应(即错配)。具体实现这三条基本原则需要考虑到诸多因素，如引物长度、产物长度、序列Tm值、引物与模板形成双链的内部稳定性、形成引物二聚体以及发夹结构的能值、在错配位点的引发效率，引物及产物的GC含量等等。同时，针对特殊的检测也可以对引物进行修饰，如增加限制性内切酶位点、引进突变等。
引物所对应模板位置序列的Tm值在72℃左右可使复性条件最佳。Tm值的计算有多种方法，如按公式Tm＝4(G+C)+2(A+T)，在Oligo软件中使用的是最邻近法。
请参见图24，在一个实施例中，通过所述数字PCR检测方法获得的所述微液滴阵列的熔解曲线图，用荧光信号改变的负的一次导数与温度作图，其波峰所在的温度代表双链DNA分子的Tm值(熔点温度)。根据扩增产物的Tm值即可判断其基因型。由此可以看 出，存在两个熔解温度Tm1以及Tm2。熔解温度Tm1与熔解温度Tm2分别对应着两种不同类别的DNA由此可以将所述微液滴阵列中的目标DNA区分出来。
针对传统PCR检测技术不断反复的多次检测、工作量大且耗费时间的问题，提供一种可以实现高通量、高灵敏度、检测时间短的用于高通量核酸检测分析的核酸检测微球、制备方法、试剂盒及高通量核酸检测方法。
请参见图25-27，本申请提供一种核酸检测微球700，用于高通量核酸检测分析。所述核酸检测微球700包括核体730以及包覆层710。所述核体730具有荧光编码信息。所述包覆层710包裹所述核体730，所述包覆层710包括基体711以及分散于所述基体711的引物712，且所述引物712与所述核体730唯一对应。
其中，所述包裹层710将所述核体730包裹，形成所述所述核酸检测微球700。所述基体711为在疏水性油中形成的含水聚合物凝胶，没有流动性，且形状和体积不易改变。含水聚合物凝胶在室温下为凝胶状态，并在高于室温的温度下熔融，且不影响酶、反应液等的扩散及活性。同时，分散于所述基体711的所述引物712可以对目标检测核酸进行定性分析识别。所述核体730为耐高温材料，具有荧光编码信息。所述荧光编码信息通过所述核体730的荧光编码信号来显示，从而通过荧光编码信号来实现特殊标记功能。并且每个所述核体730对应着一种所述引物712，且唯一对应，从而可以通过所述核体730来标记所述核酸检测微球700，以便可以进行追踪检测。
在进行PCR检测时，将多个且多种类的所述核酸检测微球700与需要检测的核酸扩增反应液混合，可以获得核酸检测液。将所述核酸检测液微滴化可以形成多个微液滴，并将多个微液滴进行PCR反应。在PCR反应过程中，双链DNA在90℃～95℃变性，再迅速冷却至50℃～60℃，引物退火并结合到靶序列上，然后快速升温至70℃～75℃，在Taq DNA聚合酶的作用下，使引物链沿模板延伸，在适合的温度范围内对核酸进行扩增。在多个微液滴进行PCR温控过程中，所述包裹层710熔融分解，将所述包裹层710中携带的所述引物712释放到所对应的微液滴中，并与微液滴中含有的目标核酸分子反应，最终可以对所述核体730进行定位追踪识别，并通过所述核体730对应的所述引物712来获知目标核酸分子，以此来实现PCR高通量检测。
在实际应用过程中，可以批量制备多个多种所述核酸检测微球700。并将多个多种所述核酸检测微球700按照实际检测目标核酸的需要按照一定比例混合，并与需要检测的核酸扩增反应液混合，可以获得核酸检测液，以此来对目标核酸进行检测，一次即可检测多种目标核酸分子，不需要反复的多次检测，并且工作量小、检测时间短、灵敏度高。
请参见图28-29，在其中一个实施例中，所述包覆层710还包括探针713，且所述探针713与所述引物712分散于所述基体711，并与所述核体730唯一对应。
其中，所述探针713可以为荧光探针能指示核酸扩增，可以为同时含有荧光基团和淬灭基团的寡糖核苷酸探针，如TaqMan荧光探针等。所述探针713分散于所述基体711中可以对目标检测核酸分子进行定性分析识别。在多个微液滴进行PCR温控过程中，所述包裹层710熔融分解，将所述包裹层710中携带的所述引物712与所述探针713释放到所对 应的微液滴中，并与微液滴中含有的目标检测核酸分子反应，最终可以对所述核体730进行定位追踪识别，并通过所述核体730对应的所述引物712与所述探针713来获知目标核酸分子，以此来实现PCR高通量检测。
在一个实施例中，所述引物712可以设置为不同类型的引物，当进行批量检测时，可以将多种所述引物712设置为不同类型的引物，可以用于检测不同类型的目标核酸分子。同时，一种所述引物712对应着一种所述核体730，也就相当于每个所述引物712有其相应的代表编号即所述核体730，从而可以通过对所述核体730的检测来进行识别。
在一个实施例中，以100个所述核酸检测微球700为例子，100个所述核酸检测微球700对应着100种不同的所述引物712，也就是100个所述核酸检测微球700对应着100种不同的所述核体730。亦即100种不同的所述引物712对应着100种不同的所述核体730。将含有100个所述核酸检测微球700的多个微液滴进行PCR升温过程中，每个所述核酸检测微球700中的所述包裹层710熔融分解将所述包裹层710中携带的所述引物712释放到所对应的微液滴中进行PCR扩增。此时，部分所述微液滴中含有1个所述核体730，通过所述核体730可以获知所述微液滴中含有的所述引物712，进而通过所述引物712获知所述微液滴中的目标核酸分子，实现PCR检测。
在其中一个实施例中，所述基体为琼脂糖凝胶。琼脂糖凝胶是以琼脂糖为支持介质制备的凝胶，琼脂糖的熔点在62℃～65℃之间，融化后在37℃下可维持液态数小时，30℃时凝固成胶。在多个微液滴进行PCR温控过程中，所述包裹层710熔融分解，将所述包裹层710中携带的所述引物712、所述探针713释放到所对应的微液滴中，从而实现所述引物712与微液滴中含有的目标核酸分子进行PCR反应，并通过荧光染料或所述探针713来指示核酸是否进行扩增。
在其中一个实施例中，所述核体730为含有荧光染料的实体球。
其中，所述核体730的材料可以为耐高温材料，可以为聚酰亚胺、聚四氟乙烯、聚苯硫醚或聚酰胺等。同时，所述核体730中含有荧光染料，可以发射出荧光信号。通过不同种类的荧光染料以及荧光的强度大小，可以对所述核体730进行编码，获得大量的不同类型的所述核体730，使得所述核体730具有荧光编码信号，从而实现对多个所述核酸检测微球700进行编码。
在一个实施例中，本申请采用两种不同类型的荧光染料，每种荧光染料采用10个不同级别的荧光信号强度，进而可以获得10×10＝100中不同标记的所述核体730，使得所述核体730具有荧光编码信息，从而获得10×10＝100中不同标记的所述核酸检测微球700。其中，每一种所述核酸检测微球700对应着一种所述核体730，一种所述核体730对应着一种所述引物712，一种所述核体730对应着一种所述探针713。通过多个所述核酸检测微球700可以一次性实现对多个不同种类的目标核酸分子进行PCR检测，不需要反复的多次检测，具有工作量小、检测时间短、高通量以及灵敏度高的特点。
在其中一个实施例中，所述核体730的直径为10微米～100微米。所述包覆层厚度为10微米～100微米。
所述核酸检测微球700的直径大小一般为20微米～150微米，可以使得在图像采集时采集足够多的微液滴。其中，所述核体730的直径可以为10微米～100微米，所述包覆层厚度可以为10微米～100微米。所述核酸检测微球700不宜过大或过小，如果过小则不容易被识别，如果过大则在生成多个微液滴时容易堵住微液滴生成装置的出口端，阻碍多个微液滴的生成。通过对所述核酸检测微球700的直径大小的设置，可以使得再生成多个微液滴时既可以被荧光信号检测装置30识别，又可以最大可能的涵盖更多的微液滴以方便图像采集，并且也不容易阻碍微液滴生成装置的出口端生成多个微液滴。
在其中一个实施例中，一种核酸检测微球的制备方法，包括：
S110，提供多个核体730与引物溶液；
S120，提供凝胶粉末，将所述凝胶粉末加入到双蒸水中获得凝胶粉溶液，并将所述凝胶粉溶液加热至澄清，获得包覆层制备液；
S130，在凝胶熔融温度，将所述多个核体730、所述引物溶液与所述包覆层制备液混合，获得核酸检测微球制备溶液；
S140，在所述凝胶熔融温度，将所述核酸检测微球制备溶液微滴化，形成多个核酸检测微球液滴；
S150，将所述多个核酸检测微球液滴冷却，并通过流式分选获得多个核酸检测微球700。
在所述步骤S110中，多个所述核体730为同一种类的含有荧光染料的实体球，以此来制备所述核酸检测微球700，使得一种所述核酸检测微球700与一种所述核体730唯一对应，且一种所述引物712与一种所述核体730唯一对应。
并且，所述引物溶液包含所述引物712。采用灭过菌的超纯水稀释干粉状引物712时，稀释到引物浓度为100μM也就是100μmol/L。然后，将浓度为100μM的引物溶液100ul放入到所述包覆层制备液900ul中，配置为引物浓度为10μM(μmol/L)。
在所述步骤S120中，所述凝胶粉末可以为琼脂粉或乙二醇双丙烯酸酯等可以制作凝胶的物质。其中，所述包覆层制备液可以为琼脂粉溶液，提供质量比为1.5％～4.5％的琼脂粉与10ml的双蒸水，将所述琼脂粉加入到所述双蒸水中高温溶解至澄清，以获得所述包覆层制备液。其中，所述包覆层制备液也就是琼脂粉溶液。
在所述步骤S130中，所述引物溶液中含有的多个所述引物712为同一种类型的引物，多个所述核体730为同一种荧光类型的核体。所述凝胶熔融温度为凝胶转换为液体溶液的温度。其中，所述琼脂糖的熔点在62℃～65℃之间，30℃时凝固成胶。所以，在高温环境62℃～65℃下将多个所述引物712与多个所述核体730加入所述包覆层制备液，所述包覆层制备液也就是琼脂粉溶液，从而获得所述核酸检测微球制备溶液。
在一个实施例中，在所述步骤S130中，将所述引物712与所述核体730加入所述琼脂糖溶液中时，一般根据生成所述核酸检测微球700的大小来选定所述核体730的浓度。
请参见图30-31，在所述步骤S140中，在高温环境下通过微流控芯片、微流体发生器或微液滴生成装置在疏水性油中形成多个所述核酸检测微球液滴。
请参见图30，在其中一个实施例中，所述步骤S140包括：
S141，提供具有出口端的吐液枪头，吐液枪头内储存有所述核酸检测微球制备溶液，且提供储存有疏水性油的开口容器；
S142，在所述凝胶熔融温度，将所述吐液枪头的出口端插入所述疏水性油的液面下；
S143，所述吐液枪头的出口端在所述疏水性油的液面下做瞬时加速的运动或变速周期的运动，将所述核酸检测微球制备溶液由所述吐液枪头的出口端排出，在所述疏水性油的液面下形成所述多个核酸检测微球液滴。
本申请提供一种微液滴生成装置，所述微液滴生成装置包括吐液枪头、流体驱动机构以及运动控制机构。所述吐液枪头具有出口端及入口端，所述流体驱动机构驱动所述吐液枪头通过入口端将核酸检测微球制备溶液吸入所述吐液枪头中，并将所述吐液枪头的出口端插入储存有油性液体的容器中，使得所述吐液枪头的出口端进入油性液体的液面下。同时，通过所述运动控制机构在所述油性液体的液面下做瞬时加速的运动或变速周期的运动，使得所述核酸检测微球制备溶液从所述吐液枪头的出口端排出，在所述油性液体的液面下形成所述多个核酸检测微球液滴。其中，所述油性液体与所述核酸检测微球制备溶液为互不相溶或具有界面反应的两种液体，所述油性液体可以为矿物油(包括正十四烷等)、植物油、硅油和全氟烷烃油等。
在所述步骤S150中，将多个所述核酸检测微球液滴冷却至常温30℃左右，此时多个所述凝固成胶，并通过流式分选获得多个所述核酸检测微球700。所述流式分选以高能量激光照射高速流动状态下的多个所述核酸检测微球液滴。由于多个所述核酸检测微球液滴中含有0个、1个或多个所述核体730，且所述核体730为含有荧光染料的实体球，所以可以测量出产生的散射光和发射荧光的强度，以此来进行筛选，获取只含有单个所述核体730的核酸检测微球700。
此时，冷却后的多个所述核酸检测微球700为凝胶状态，可以方便在常温环境下进行储存和运输，有利于批量进行运输用于PCR检测。
在其中一个实施例中，一种核酸检测微球的制备方法，包括：
S210，提供引物溶液、探针溶液以及多个核体730；
S220，提供凝胶粉末，将所述凝胶粉末加入到双蒸水中获得凝胶粉溶液，并将所述凝胶粉溶液加热至澄清，获得包覆层制备液；
S230，在凝胶熔融温度，将所述多个核体730、所述引物溶液和所述探针溶液与所述包覆层制备液混合，获得核酸检测微球制备溶液；
S240，在所述凝胶熔融温度，将所述核酸检测微球制备溶液微滴化，形成多个核酸检测微球液滴；
S250，将所述多个核酸检测微球液滴冷却，并通过流式分选获得多个核酸检测微球。
在所述步骤S210中，所述探针溶液包含所述探针713，用以检测核酸是否发生扩增，可以为同时含有荧光基团和淬灭基团的寡糖核苷酸探针，如TaqMan荧光探针等。在多个微液滴进行PCR温控过程中，所述包裹层710熔融分解，将所述包裹层710中携带的所述 引物712与所述探针713释放到所对应的微液滴中，并与微液滴中含有的目标检测核酸分子反应，最终可以对所述核体730进行定位追踪识别，并通过所述核体730对应的所述引物712与所述探针713来获知目标核酸分子，以此来实现PCR高通量检测。
其中，多个所述核体730为同一种类的含有荧光染料的实体球，以此来制备所述核酸检测微球700，使得一种所述核酸检测微球700与一种所述核体730唯一对应，且一种所述引物712与一种所述核体730唯一对应，一种所述探针713与一种所述引物712唯一对应。
在所述步骤S220中，所述包覆层制备液与所述步骤120中的制备方法可以相同。
在所述步骤S240中，形成多个所述核酸检测微球液滴的制备方法与所述步骤S140方法可以相同。
在所述步骤S250中，获得多个所述多个核酸检测微球700的方法与所述步骤150方法可以相同。
在其中一个实施例中，在所述步骤S250中，所述核酸检测微球为含有单个所述核体的核酸检测微球。
在其中一个实施例中，在所述步骤S220中，所述凝胶粉末为琼脂粉或聚乙二醇双丙烯酸酯等。
在其中一个实施例中，一种试剂盒，用于高通量核酸检测分析。所述试剂盒包括如上述任一实施例中所述的核酸检测微球与核酸反应液。
其中，所述核酸反应液含有PCR扩增所需的酶、dNTP、荧光染料以及离子等。如若所述核酸检测微球700中包含所述探针713，则所述核酸反应液中可以不包括荧光染料。
所述试剂盒可以用于存储、运载多个多种不同的所述核酸检测微球700。其中，所述核酸检测微球700可以保存在甘油中。
在一个实施例中，准备专供数字PCR使用的成套试剂和溶液，用来减少或避免外源DNA对模板DNA样本的潜在污染。所使用的所有仪器和耗材应该进行高温灭菌并进行高温干燥处理。
请参见图32，在其中一个实施例中，一种高通量核酸检测方法，包括：
S310，提供核酸扩增反应液与多种不同类型的核酸检测微球700，且所述核酸检测微球700包括核体730与包覆层710，所述核体730具有编码信息，所述包覆层710包裹所述核体730，所述包覆层710包括基体711以及分散于所述基体711的引物712，且所述引物712与所述核体730唯一对应，所述核体730为含有荧光染料的实体球；
S320，将所述多种不同类型的核酸检测微球700与所述核酸扩增反应液混合，获得核酸检测液；
S330，将所述核酸检测液微滴化，形成多个微液滴800；
S340，将所述多个微液滴800进行核酸扩增，获得扩增完成后的所述多个微液滴800；
S350，根据扩增完成后的所述多个微液滴800，检测每个所述微液滴800中的所述核体730，筛选出仅含有一个所述核体730的所述微液滴800，获得第一有效微液滴810；
S360，根据所述第一有效微液滴810，检测所述第一有效微液滴810中的所述核体730的荧光信号，获得所述核体730对应的所述引物712，并读取核酸扩增反应之后的报告荧光信号，获取所述有第一效微液滴810中是否有对应的目标核酸分子。
在所述步骤S310中，所述核酸扩增反应液为以脱氧核糖核酸为模板的核酸扩增反应液、以核糖核酸为模板的逆转录核酸扩增反应液或环介导等温扩增反应液，且所述核酸扩增反应液中包含荧光染料。所述核酸扩增反应液包括核酸模板、反应缓冲液、脱氧核糖核苷三磷酸、聚合酶以及二价金属阳离子等，且若所述包覆层710中无所述探针713时，所述反应缓冲液中包含荧光染料。
其中，所述核酸扩增反应液，可以是以脱氧核糖核酸(DNA)为模板的核酸扩增反应液(可称为DNA扩增反应液)，也可以是以核糖核酸(RNA)为模板的逆转录核酸扩增反应液(可称为RNA反转录反应液)，还可以是其它核酸扩增反应液，如环介导等温扩增(LAMP)反应液。其中，所述DNA扩增反应液的特点是含有DNA扩增所需要的dNTP、反应缓冲液、无机盐离子、聚合酶、待检测的DNA模板以及荧光染料。所述荧光染料可以为SYBR Green等与DNA结合的荧光染料。
在PCR反应体系中，在游离状态下，加入SYBR Green荧光染料后发出微弱的荧光，但一旦与双链DNA结合后，荧光大大增强，发射荧光信号，从而可以保证荧光信号的增加与PCR产物的增加完全同步。此时，可以通过检测SYBR Green荧光染料发出的荧光信号，获得核酸扩增反应之后的报告荧光信号，从而获知所述有第一效微液滴810中是否有对应的目标核酸分子。
其中，所述多个多种不同类型的所述核酸检测微球700中所述核酸检测微球700的大小、形状以及含有的所述引物712可以相同可以不同。多个所述核酸检测微球700中可以为含有不同类型的所述引物712，用以检测不同种类的所述目标核酸分子。
在所述步骤S320中，将所述多种不同类型的核酸检测微球700与所述核酸扩增反应液混合混合形成所述核酸检测液时，可以通过调整所述核酸检测液中所述核酸检测微球700的浓度，使得生成所述多个微液滴800时单包裹数量最大，微球分布符合泊松分布理论模型。此时，通过计算每个所述微液滴800中含有一个所述核体730的概率为p(x＝1)＝λe -λ，p’(x＝1)＝e -λ-λe -λ＝0，此时λ＝1，即平均每个所述微液滴800中含有1个所述核体730时，所述核体730单个被包裹的概率最大。此时，每个所述微液滴800中含有1个所述核体730的概率为p(x＝1)＝e -1＝0.368。
请参见图1，在所述步骤S330中，在一个实施例中本申请提供一种核酸高通量检测仪，所述核酸高通量检测仪包括微液滴生成装置、温控装置、荧光信号检测装置、分析装置以及控制器。所述微液滴生成装置用以将所述核酸检测液微滴化，形成所述多个微液滴800。所述温控装置与所述微液滴生成装置通过轨道连接，用以将所述多个微液滴转移至所述温控装置，并通过所述温控装置进行温度循环，实现核酸扩增。待所述多个微液滴扩增完成后，通过所述荧光信号检测装置对核酸扩增完成后的所述多个微液滴进行荧光检测。所述控制器分别与所述微液滴生成装置、所述温控装置以及所述荧光信号检测装置连接，用以 控制所述微液滴生成装置、所述温控装置以及所述荧光信号检测装置。
其中，所述微液滴生成装置包括吐液枪头、流体驱动机构以及运动控制机构。所述吐液枪头具有出口端及入口端，所述流体驱动机构驱动所述吐液枪头通过入口端将所述核酸检测液吸入所述吐液枪头中，并将所述吐液枪头的出口端插入储存有油性液体的容器中，使得所述吐液枪头的出口端进入油性液体的液面下。同时，通过所述运动控制机构在所述油性液体的液面下做瞬时加速的运动或变速周期的运动，使得所述核酸检测液从所述吐液枪头的出口端排出，在所述油性液体的液面下形成所述多个微液滴800。其中，所述油性液体与所述核酸检测液为互不相溶或具有界面反应的两种液体，所述油性液体可以为矿物油(包括正十四烷等)、植物油、硅油和全氟烷烃油等。
请参见图33-34，在一个实施例中，在所述步骤S330中将所述核酸检测液微滴化，形成多个微液滴800时可以采用微流控芯片、微流体发生器或微液滴生成装置等。且制备所述多个微液滴800的方法不仅限于上述所述装置，也可以为其他装置，用以制备所述多个微液滴800。
其中，每个所述微液滴800中可能包括零个、一个或多个所述核酸检测微球700，且每个所述微液滴800中含有所述核酸扩增反应液，用以进行核酸扩增。
在一个实施例中，在所述步骤S330中将所述核酸检测液微滴化形成的所述多个微液滴800的大小可以相同也可以不同。
在一个实施例中，在所述步骤S330中，将所述核酸检测液微滴化时可以采用微流控芯片。
将所述核酸检测液微滴化时，每个所述微液滴800中可能包括零个、一个或多个所述核酸检测微球700。将所述多个微液滴800进行核酸扩增温度高于所述琼脂糖的熔点时，所述包裹层710熔融，释放出所述引物712，从而，使得所述引物712与所述微液滴800中的核酸分子同时进行PCR扩增，此时通过识别所述微液滴800中对应的所述核体730即可获知所述引物712的种类，从而获知目标核酸分子。
在一个实施例中，在所述步骤S350中，所述第一有效微液滴810包括一个所述核体730。对于含有零个或多于1个的所述微液滴800视为无效微液滴。其中，所述第一有效微液滴810中含有荧光染料以及所述引物712。如果在所述引物712与所述第一有效微液滴810中的核酸分子进行PCR扩增过程中，所述荧光染料与双链DNA结合后，荧光大大增强，可以发射出较强的荧光信号，使得所述第一有效微液滴810具有较强的荧光信号，从而可以根据所述核体730以及所述引物712来获得所述有第一效微液滴810中对应的目标核酸分子的种类。
在其中一个实施例中，所述步骤S360包括：
S361，提供荧光信号检测装置，所述荧光信号检测装置包括编码荧光通道与荧光染料检测通道，根据编码荧光通道识别所述有效微液滴中所述核体730的荧光信号；
S362，根据所述核体730的荧光信号，获取所述730对应的所述引物712；
S363，根据所述荧光染料检测通道检测所述第一有效微液滴810中核酸扩增反应之后 的报告荧光信号，获取所述第一有效微液滴810中是否有对应的目标核酸分子。
所述核体730为含有荧光染料的实体球，通过采用不同种类的荧光染料以及荧光的强度大小，可以对所述核体730进行标记。每一种荧光对应着一种所述核体730，每一种所述核体730对应着一种所述引物712也就相当于每种所述712有其相应的代表编号即所述核体730。
在所述步骤S360中，根据有无报告荧光信号检测出目标核酸分子的有无，实现定性检测。其中，在筛选过程中可以将含有同一种类别的所述核体730的所述第一有效微液滴810分为一组。同时，通过检测报告荧光有无，可以得到此类别所述核体730中所述第一有效微液滴810中没有报告荧光信号的微液滴数量占该类别微液滴总数的比例，从而可以根据泊松分布计算得到同一种类别的所述核体730对应的目标核酸分子的浓度。
请参见图35，在其中一个实施例中，一种高通量核酸检测方法，包括：
S410，提供核酸扩增反应液与多种不同类型的核酸检测微球700，且所述核酸检测微球700包括核体730与包覆层710，所述核体730具有编码信息，所述包覆层710包裹所述核体730，所述包覆层710包括基体711以及分散于所述基体711的引物712和探针713，且所述引物712和所述探针713与所述核体730唯一对应，所述核体730为具有荧光编码信息的实体球；
S420，将所述多种不同类型的核酸检测微球700与所述核酸扩增反应液混合，获得核酸检测液；
S430，将所述核酸检测液微滴化，形成多个微液滴800；
S440，将所述多个微液滴800进行核酸扩增，获得扩增完成后的所述多个微液滴800；
S450，根据扩增完成后的所述多个微液滴800，检测每个所述微液滴800中的所述核体730，筛选出仅含有一个所述核体730的所述微液滴800，获得第二有效微液滴820；
S460，根据所述第二有效微液滴820，检测所述第二有效微液滴820中的所述核体730的荧光信号，获得所述核体730对应的所述引物712与所述探针713，并读取核酸扩增反应之后的报告荧光信号，获取所述第二有效微液滴820中是否有对应的目标核酸分子。
将所述核酸检测液微滴化时，每个所述微液滴800中可能包括零个、一个或多个所述核酸检测微球700。将所述多个微液滴800进行核酸扩增温度高于所述琼脂糖的熔点时，所述包裹层710熔融，释放出所述引物712与所述探针713。从而，使得所述引物712与所述探针713与所述微液滴800中的核酸分子同时进行PCR扩增，此时通过识别所述微液滴800中对应的所述核体730即可获知所述引物712与所述探针713，进而即可获知所述引物740的种类，从而获知目标核酸分子。
在所述步骤S450中，所述第二有效微液滴820包括一个所述核体730。对于含有零个或多于1个所述核体730的所述微液滴800视为无效微液滴。同时，所述第二有效微液滴820中还包括含有所述探针713以及所述引物712。当所述第二有效微液滴820中含有所述探针713时，所述第二有效微液滴820中可以不含有荧光染料，此时所述探针713起到荧光标定的作用。在所述引物712与所述第二有效微液滴820中的核酸分子进行PCR扩增 过程中，所述探针713与双链DNA结合，以便可以识别出含有对应目标核酸分子的所述第二有效微液滴820。
假设在所述引物712与所述第二有效微液滴820中的核酸分子进行PCR扩增过程中，所述探针713与双链DNA结合后，可以通过识别所述探针713来判断所述第二有效微液滴820中是否含有对应的目标核酸分子。从而，可以根据所述核体730以及所述引物712来获得所述有第一效微液滴810中对应的目标核酸分子的种类。
在一个实施例中，在所述步骤S430中与在所述步骤S330中将所述核酸检测液微滴化的方法相同。
请参见图6，在一个实施例中，本申请提供一种荧光信号检测装置30。所述荧光信号检测装置30包括激发光源340、荧光探测组件330以及第三控制器310。所述激发光源340设置于所述多个微液滴800检测区域上方，并与所述多个微液滴800检测区域呈倾斜角度进行照射，形成斜射光路。所述荧光探测组件330设置于所述多个微液滴800检测区域正上方，用以采集所述多个微液滴800的荧光图像。所述第三控制器310分别与所述激发光源340和所述荧光探测组件330连接，用以控制所述激发光源340与所述荧光探测组件330。所述荧光信号检测装置30可以对微液滴进行多个荧光通道成像以及进行明场暗场成像。其中多个荧光通道成像用于微液滴反应信号的探测，明场暗场成像用于检测形成微液滴的尺寸信息以及在反应过程中监测液滴的状态。
所述激发光源340包括不同颜色的LED光源341、准直镜342、第一滤光片343、二向色镜344、复眼透镜345、聚焦透镜346。所述不同颜色的LED光源341可以生成不同颜色的光，照射至所述多个微液滴800。通过对所述不同颜色的LED光源341进行选择，可以获得不同荧光颜色的照射，所述不同颜色的LED光源341可以轮流工作。每个LED光源发射的光路正前方依次设置有所述准直镜342、所述第一滤光片343以及二向色镜344。所述准直镜342与所述第一滤光片343与光路呈垂直角度设置(90°角度设置)。所述二向色镜344与光路角度呈0°～45°设置。通过所述二向色镜344形成的一条光路，所述光路正前方依次设置有所述复眼透镜345以及所述聚焦透镜346。所述复眼透镜345与所述聚焦透镜346与光路呈垂直角度设置(90°角度设置)。所述多个微液滴800的内部荧光被激发，通过所述第二滤光片333被上方的所述物镜332收集，进入相机331，所述相机331采集所述多个微液滴的荧光图像。
通过所述激发光源340发射的光路倾斜照射于所述多个微液滴800，使得所述多个微液滴800中含有荧光物质的微液滴产生荧光。通过所述荧光探测组件330对所述含有荧光物质的微液滴进行荧光信息采集，并将所述含有荧光物质的微液滴进行荧光信息以荧光图像的形式传输至分析装置(计算机)，用以进行分析。
所述第二控制器310用于切换不同的滤光片，从而构成不同的荧光检测通道。所述荧光信号检测装置包括编码荧光通道、荧光染料检测通道、荧光探针检测通道、微液滴识别通道以及多个备用通道等。
其中，在生成多个所述微液滴800时，在所述核酸检测液中加入ROX内参染料。所 述ROX内参染料不参与PCR反应，可以用于识别出多个所述微液滴800的具体位置、轮廓与数量等信息。所述微液滴识别通道用于识别ROX内参染料的荧光，用以准确定位每个所述微液滴800。所述编码荧光通道用于识别所述核体730的荧光信号及荧光信号强度，用以获取含有一个所述核体730的所述第一有效微液滴810。所述荧光染料检测通道或荧光探针检测通道用于识别所述第一有效微液滴810中核酸扩增反应之后的报告荧光信号，从而根据所述报告荧光信号来判断所述引物712或所述引物712与所述探针713与目标核酸分子是否进行了PCR扩增。
在所述步骤S460中，根据有无报告荧光信号检测出目标核酸分子的有无，实现定性检测。其中，在筛选过程中可以将含有同一种类别的所述核体730的所述第二有效微液滴820分为一组。同时，通过检测报告荧光有无，可以得到此类别所述核体730中所述第二有效微液滴820中没有报告荧光信号的微液滴数量占该类别微液滴总数的比例，从而可以根据泊松分布计算得到同一种类别的所述核体730对应的目标核酸分子的浓度。
通过所述编码荧光通道检测多个所述微液滴800中的所述核体730的荧光信号，可以从多个所述微液滴800中筛选出所述第一有效微液滴810。其中，所述第一有效微液滴810含有一个所述核体730。从而，通过所述荧光染料检测通道对所述第一有效微液滴810进行检测，读取核酸扩增反应之后的报告荧光信号，判断所述第一有效微液滴810中是否含有对应的目标核酸分子。若所述第一有效微液滴810中含有对应的目标核酸分子，则可以根据所述第一有效微液滴810中的所述核体730对应的所述引物712，来获知对应的目标核酸分子的种类。
同理，通过所述编码荧光通道检测多个所述微液滴800中的所述核体730的荧光信号，可以从多个所述微液滴800中筛选出所述第二有效微液滴810。其中，所述第二有效微液滴820含有一个所述核体730。从而，通过所述荧光探针检测通道对所述第二有效微液滴820进行检测，读取核酸扩增反应之后的报告荧光信号，判断所述第二有效微液滴820中是否含有对应的目标核酸分子。若所述第二有效微液滴820中含有对应的目标核酸分子，则可以根据所述第二有效微液滴820中的所述核体730对应的所述引物712或所述探针713，来获知对应的目标核酸分子的种类。
在一个实施例中，所述荧光信号检测装置包括多个所述编码荧光通道，多个所述编码荧光通道可以用以识别多种不同荧光标记的所述核体730。具体地，第一编码荧光通道设置为荧光A，设置10个梯度的浓度，第二编码荧光通道设置为荧光B，同样设置10个梯度的浓度，则可以通过所述荧光信号检测装置识别10×10＝100种不同荧光通道及强度标记的所述核体730，也就是说可以通过100种荧光标记的所述核体730标记100种不同类型的所述引物712或所述引物712与所述探针713。以此类推，可以获得大量的不同种类的所述核体730，进而可以用以标记大量不同种类的所述核酸检测微球700。
所述核酸检测微球700由具有荧光编码信息的所述核体730、所述引物712、所述探针713的所述包覆层710组成。多种所述核酸检测微球700随机分布到所述核酸扩增反应液中，并进行混合获得所述核酸检测液，所述核酸检测液再微液滴化生成的所述多个微液滴 800。当温度升高到60摄氏度以上，所述包覆层710融化，将所述引物712、所述探针713释放到所述微液滴800中，从而组成完整的核酸扩增反应体系，所述核体730留在所述微液滴800中，作为标记所述微液滴800的荧光标签。待扩增完成后，若所述微液滴800中含有目标核酸分子，则在扩增过程中荧光染料或所述探针713会与双链DNA进行结合，荧光信号增强，会有报告荧光信号产生。
然后，通过检测多个所述微液滴800中的具有荧光编码信息的所述核体730，筛选出有且只有一个具有荧光编码信息的所述核体730的所述微液滴800作为有效微液滴进入后续的分析。根据获得的有效微液滴(有效微液滴为上述实施例中所述第一有效微液滴810或所述第二有效微液滴820)，检测有效微液滴中的所述核体730的荧光信号，获得对应的所述引物712、所述探针713的种类。然后获取有效微液滴中核酸扩增反应之后的报告荧光信号，根据所述报告荧光信号判断有效微液滴中是否有相应的目标核酸分子。因此，通过所述核酸检测微球700、制备方法、试剂盒及高通量核酸检测方法能够通过一次加入多种所述核酸检测微球700来实现一次检测多个目标核酸分子的有无，且可以根据泊松分布分获得每种检测目标核酸的浓度。
因此，通过将大量不同种类的所述核酸检测微球700与需要检测的核酸扩增反应液混合来检测目标核酸，一次即可检测多种目标核酸，不需要反复的多次检测，工作量小、节省时间、灵敏度高。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。
最后，还需要说明的是，本文中为部件所编序号本身，例如“第一”、“第二”等，仅用于区分所描述的对象，不具有任何顺序或技术含义。而本申请所说“连接”、“联接”，如无特别说明，均包括直接和间接连接(联接)。在本申请的描述中，需要理解的是，术语“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。
本文中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方”和“上面”可以是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度小于第二特 征。
在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。对所提供的实施例的上述说明，使本领域专业技术人员能够实现或使用本申请。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本申请的精神或范围的情况下，在其它实施例中实现。因此，本申请将不会被限制于本文所示的这些实施例，而是要符合与本文所提供的原理和新颖特点相一致的最宽的范围。

Claims (59)

  1. 一种数字PCR检测仪，包括：
    微液滴生成装置，设置于将核酸扩增反应液微滴化，形成多个微液滴；
    温控装置，所述温控装置与所述微液滴生成装置通过轨道连接，设置于将所述多个微液滴转移至所述温控装置，进行温度循环，实现核酸扩增；
    荧光信号检测装置，所述荧光信号检测装置与所述温控装置相对设置，设置于对核酸扩增后的所述多个微液滴进行拍照检测；所述荧光信号检测装置可以对微液滴进行多个荧光通道成像以及进行明场暗场成像；其中多个荧光通道成像设置于微液滴反应信号的探测，明场暗场成像设置于检测形成微液滴的尺寸信息以及在反应过程中监测液滴的状态；
    定量分析装置，所述定量分析装置与所述荧光信号检测装置通过数据线连接，设置于实现所述多个微液滴荧光信息的传输，进行定量分析；
    控制器，所述控制器分别与所述微液滴生成装置、所述温控装置、荧光信号检测装置以及定量分析装置连接，设置于控制所述微液滴生成装置、所述温控装置、所述荧光信号检测装置以及所述定量分析装置。
  2. 如权利要求1所述的数字PCR检测仪，其特征在于，所述微液滴生成装置包括：
    吐液枪头，所述吐液枪头具有出口端及入口端，所述吐液枪头用于储存第一液体；
    流体驱动机构，所述流体驱动机构与所述吐液枪头的入口端连接，用于将储存在所述吐液枪头内部的第一液体从所述吐液枪头的出口端排出；
    运动控制机构，所述运动控制机构用于控制所述吐液枪头的出口端与第二液体之间产生设定轨迹或设定速度或设定加速度的相对运动，以使排出所述吐液枪头的出口端的第一液体克服表面张力及附着力形成微液滴；
    第一控制器，所述第一控制器分别与所述流体驱动机构以及所述运动控制机构连接，用以控制所述流体驱动机构以及所述运动控制机构工作。
  3. 如权利要求2所述的数字PCR检测仪，其特征在于，所述流体驱动机构包括：
    变容积组件，包括注射筒及推杆，所述推杆与所述注射筒的内壁动配合，所述注射筒内能够储存驱动液体，所述注射筒具有进出液口，所述进出液口用于连通储存有第一液体的吐液枪头的入口端；
    动力组件，与所述推杆传动连接，用于驱动所述推杆沿所述注射筒的延伸方向滑动；
    储液罐，用于储存驱动液体；
    三通换向阀，具有第一接口、第二接口及第三接口，所述吐液枪头的入口端、所述进出液口及所述储液罐分别与所述第一接口、所述第二接口及所述第三接口连通。
  4. 如权利要求2所述的数字PCR检测仪，其特征在于，所述吐液枪头包括具有中空腔体的针梗及设置于所述针梗一端的出口端。
  5. 如权利要求2所述的数字PCR检测仪，其特征在于，所述运动控制机构包括：
    支撑架；
    连接件，用于与吐液枪头连接；
    驱动元件，固定于所述支撑架，所述驱动元件与所述连接件传动连接；
    在所述驱动元件的驱动下，吐液枪头的出口端相对第二液体做位移大小呈正弦变化或者速度大小呈方波变化的相对运动。
  6. 如权利要求1所述的数字PCR检测仪，其特征在于，所述温控装置包括：
    柔性电路板；
    与所述柔性电路板间隔设置的加热基板，所述加热基板包括相对设置的第一表面和第二表面；
    多个半导体电偶对，设置于所述柔性电路板与所述第一表面之间，所述多个半导体电偶对相互串联、并联或者混合连接。
  7. 如权利要求6所述的数字PCR检测仪，其特征在于，所述温控装置还包括：
    第二控制器，与所述多个半导体电偶对电连接，用于控制电流大小；
    温度传感器，设置于所述加热基板表面，所述温度传感器与所述第二控制器电连接，用于检测所述加热基板的温度并将该温度发送给所述第二控制器。
  8. 如权利要求1所述的数字PCR检测仪，其特征在于，所述荧光信号检测装置包括：
    激发光源，所述激发光源设置于所述微液滴容器检测区域上方，并与所述微液滴容器检测区域呈倾斜角度进行照射，形成斜射光路；
    荧光探测组件，所述荧光探测组件设置于所述微液滴容器检测区域正上方，用以采集所述多个微液滴的荧光图像及明场暗场图像；
    第三控制器，所述第三控制器分别与所述激发光源和所述荧光探测组件连接，用以控制所述激发光源与所述荧光探测组件。
  9. 如权利要求8所述的数字PCR检测仪，其特征在于，所述激发光源包括：
    多个不同颜色的LED光源，每个所述LED光源前端依次设有准直镜和第一滤光片；
    二向色镜，所述二向色镜倾斜设置于所述第一滤光片前端，用以将每个所述LED光源发出的光折射成一条光路；
    复眼透镜，用以提高经折射后的所述光路的均匀性；
    聚焦透镜，所述聚焦透镜设置于所述复眼透镜的前端，用以聚焦形成照明光斑。
  10. 如权利要求8所述的数字PCR检测仪，其特征在于，所述荧光探测组件包括物镜、相机以及第二滤光片，所述物镜设置于所述相机与所述第二滤光片之间。
  11. 如权利要求1所述的数字PCR检测仪，其特征在于，所述控制器分别与所述第一控制器、所述第二控制器以及所述第三控制器连接，用以控制所述微液滴生成装置、所述温控装置、所述荧光信号检测装置以及所述定量分析装置工作。
  12. 一种数字PCR检测仪的分析方法，包括以下步骤：
    S10，制备待测核酸扩增反应液；
    S20，将所述待测核酸扩增反应液微滴化，形成多个微液滴；
    S30，将所述多个微液滴进行核酸扩增，并实时获取所述多个微液滴的荧光信息；
    S40，根据所述多个微液滴的荧光信息，对所述多个微液滴进行定量分析。
  13. 一种数字PCR定量检测方法，包括以下步骤：
    S4110，获取所有微液滴的多个实时荧光图像，根据所述多个实时荧光图像获得进行核酸扩增的微液滴的实时荧光曲线；
    S4120，根据所述实时荧光曲线，获得所有进行核酸扩增的微液滴的Ct值；
    S4130，根据所述Ct值与进行核酸扩增的微液滴的核酸起始拷贝数的关系，获得所有进行核酸扩增的微液滴的核酸起始拷贝数；
    S4140，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，获得所述核酸起始拷贝数的频数分布；
    S4150，根据所述核酸起始拷贝数的频数分布，计算泊松分布的参数λ。
  14. 如权利要求13所述的数字PCR定量检测方法，其特征在于，所述S4110包括：
    S4111，根据每个实时荧光图像，获得每个进行核酸扩增的微液滴的荧光强度值；
    S4113，根据所述每个进行核酸扩增的微液滴的荧光强度值，获得每个进行核酸扩增的微液滴的实时荧光曲线；
    S4115，根据所述每个进行核酸扩增的微液滴的荧光曲线，获得所述所有进行核酸扩增的微液滴的实时荧光曲线。
  15. 如权利要求13所述的数字PCR定量检测方法，其特征在于，所述S4120包括：
    S4121，对每个进行核酸扩增的微液滴的实时荧光曲线求导，获取所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率；
    S4123，根据所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率，获取所述每个进行核酸扩增的微液滴的实时荧光曲线的斜率中斜率固定不变的数值；
    S4125，根据所述斜率固定不变的数值，获取其对应的起始循环数，所述起始循环数为每个进行核酸扩增的微液滴的Ct值；
    S4127，根据所述每个进行核酸扩增的微液滴的Ct值，获得所述所有进行核酸扩增的微液滴的Ct值。
  16. 如权利要求13所述的数字PCR定量检测方法，其特征在于，所述S4120还包括：
    S4122，根据每个进行核酸扩增的微液滴的实时荧光曲线，获得每个进行核酸扩增的微液滴的荧光域值的缺损值；
    S4124，根据每个进行核酸扩增的微液滴的荧光域值的缺损值，获得其对应的循环数，所述循环数为每个进行核酸扩增的微液滴的Ct值；
    S4126，根据所述每个进行核酸扩增的微液滴的Ct值，获得所述所有进行核酸扩增的微液滴的Ct值。
  17. 如权利要求13所述的数字PCR定量检测方法，其特征在于，所述S4140包括：
    S4141，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，获得所述所有进行核酸扩增的微液滴的核酸起始拷贝数中的最大值和最小值；
    S4143，根据所述最大值与所述最小值，选取组距和组数，获得所述核酸起始拷贝数的频数分布。
  18. 如权利要求13所述的数字PCR定量检测方法，其特征在于，所述S4150中计算泊松分布的参数λ时，采用最大似然估计方法。
  19. 一种数字PCR定量检测方法，包括以下步骤：
    S4210，获取所有微液滴的多个实时荧光图像，根据所述多个实时荧光图像获得进行核酸扩增的微液滴的实时荧光曲线；
    S4220，根据所述实时荧光曲线，获得所有进行核酸扩增的微液滴的Ct值；
    S4230，根据所述Ct值与进行核酸扩增的微液滴的核酸起始拷贝数的关系，获得所有进行核酸扩增的微液滴的核酸起始拷贝数；
    S4240，根据所述所有进行核酸扩增的微液滴的核酸起始拷贝数，选取部分核酸起始拷贝数；
    S4250，根据所述部分核酸起始拷贝数，获取所述部分核酸起始拷贝数得频数分布；
    S4260，根据所述部分核酸起始拷贝数的频数分布，对泊松分布进行点估计，获取泊松分布的参数λ。
  20. 如权利要求19所述的数字PCR定量检测方法，其特征在于，所述S4260包括在一个区间[λ min,λ max]内搜索λ，使得所述部分核酸起始拷贝数的频数值误差平方和err最小。
  21. 如权利要求19所述的数字PCR定量检测方法，其特征在于，所述S4260中对泊松分布进行点估计的方法还包括矩估计法、顺序统计量法或最大似然法。
  22. 如权利要求19所述的数字PCR定量检测方法，其特征在于，所述S4260中的所述误差平方和err为：
    Figure PCTCN2019072974-appb-100001
    其中，每个微液滴中含有的DNA起始拷贝数为随机变量x，部分微液滴的DNA起始拷贝数对应的频数值为n k，N为所述多个微液滴的总数。
  23. 一种不同体积数字PCR的定量分析方法，包括：
    S4310：获取所有微液滴体积v 1，v 2，...v m，所述体积为v 1，v 2，...v m依次对应的微液滴的数目n 1，n 2，…，n m，以及所述体积为v 1，v 2，...v m依次对应的微液滴核酸扩增后的阴性微液滴数目b 1，b 2，…，b m；
    S4320：根据所有微液滴核酸扩增后的相关参数v 1、v 2，...v m，n 1，n 2，…，n m，b 1，b 2，…，b m，构建关于待测核酸扩增反应液浓度c的联合二项分布函数f(c)；
    S4330：根据联合二项分布函数f(c)，求使得所述联合二项分布函数f(c)取极值时c的值；
    S4340：将所述联合二项分布函数f(c)转化为关于ln(c)的联合二项分布函数F(Λ)，获得关于ln(c)的标准差以及置信区间；
    S4350：根据ln(c)的标准差以及置信区间，获取所述待测核酸扩增反应液浓度c的标准差以及置信区间。
  24. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4310包括：
    S4311：将含有目标核酸的样品溶液微滴化，获得多个不同体积v 1，v 2，...v m的微液滴，所述微液滴体积为v 1，v 2，...v m依次对应的微液滴的数目n 1，n 2，…，n m；
    S4313：将所有微液滴进行核酸扩增，并拍照检测，获得所述所有微液滴的荧光图像；
    S4315：根据所述所有微液滴的荧光图像，获取所述所有微液滴的体积为v 1，v 2，...v m依次对应的核酸扩增后的阴性微液滴数目b 1，b 2，…，b m。
  25. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4310还包括：
    S4312：将含有目标核酸的样品溶液微滴化，形成多个微液滴；
    S4314：将所述多个微液滴进行核酸扩增，并拍照检测，获得所有微液滴核酸扩增后的荧光图像；
    S4316：根据所述荧光图像，获取所述所有微液滴核酸扩增后的体积，所述体积分别为v 1，v 2，...v m，所述体积分别为v 1，v 2，...v m依次对应的核酸扩增后的微液滴的数目n 1，n 2，…，n m，以及所述体积为v 1，v 2，...v m依次对应的核酸扩增后的阴性微液滴数目b 1，b 2，…，b m。
  26. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4320构建关于待测核酸扩增反应液浓度c的联合二项分布函数f(c)为：
    Figure PCTCN2019072974-appb-100002
  27. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4330包括：
    S4331：将所述联合二项分布函数f(c)求导，获取所述联合二项分布函数f(c)的导数；
    S4332：将所述联合二项分布函数f(c)的导数为0，获取所述联合二项分布函数f(c)取极值时的待测核酸扩增反应液浓度c的值。
  28. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述
    S4340中所述关于ln(c)的联合二项分布函数F(Λ)为：
    Figure PCTCN2019072974-appb-100003
  29. 如权利要求23所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4340包括：
    S4341：将所述函数F(Λ)取对数，获取函数L(Λ)；
    S4342：对所述函数L(Λ)求一阶导数，并将函数L(Λ)的一阶导数为0；
    S4343：获取ln(c)相应的标准差σ；
    S4344：根据ln(c)相应的标准差σ，获取ln(c)的置信区间。
  30. 如权利要求27所述的不同体积数字PCR的定量分析方法，其特征在于，所述S4343 中根据ln(c)的Fisher信息量I(Λ)获取标准差σ。
  31. 如权利要求30所述的不同体积数字PCR的定量分析方法，其特征在于，ln(c)的Fisher信息量I(Λ)为：
    Figure PCTCN2019072974-appb-100004
  32. 如权利要求29所述的不同体积数字PCR的定量分析方法，其特征在于，所述ln(c)相应的标准差σ以及置信区间分别为：
    Figure PCTCN2019072974-appb-100005
    CI＝ln(c)±Zσ。
  33. 一种数字PCR检测方法，包括：
    S10，制备待测核酸扩增反应液；
    S20，将所述待测核酸扩增反应液微滴化，形成微液滴阵列；
    S30，将所述微液滴阵列进行聚合酶链式反应，并获取所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线；以及
    S40，根据所述微液滴阵列中每个微液滴的荧光曲线与每个微液滴的熔解曲线，对所述微液滴阵列进行分析，以获得所述待测核酸信息。
  34. 如权利要求33所述的数字PCR检测方法，其特征在于，所述步骤S30包括：
    S310，设置聚合酶链式反应的温度参数、时间参数以及循环次数；
    S320，根据所述温度参数以及所述时间参数对所述微液滴阵列进行聚合酶链式反应，依次完成所述循环次数，并获取每次循环过程的每个所述微液滴的荧光曲线；以及
    S330，将完成聚合酶链式反应扩增后的所述微液滴阵列降温，并以特定的温度间隔进行升温，获取每个所述微液滴的熔解曲线。
  35. 如权利要求34所述的数字PCR检测方法，其特征在于，所述步骤S320包括：
    S321，根据所述温度参数以及所述时间参数对所述微液滴阵列进行聚合酶链式反应，获取所述微液滴阵列的荧光图像；
    S322，根据所述循环次数依次循环，获取所述微液滴阵列在聚合酶链式反应过程中的全部荧光图像；
    S323，根据所述微液滴阵列的全部荧光图像，获取每次循环过程的每个所述微液滴的荧光信息；以及
    S324，根据每次循环过程的每个所述微液滴的荧光信息，获取每个所述微液滴的荧光曲线，从而获得所述微液滴阵列的荧光曲线。
  36. 如权利要求34所述的数字PCR检测方法，其特征在于，所述步骤S330包括：
    S331，将完成聚合酶链式反应扩增后的所述微液滴阵列降温至40摄氏度以下；
    S332，将降温至40摄氏度以下的所述微液滴阵列以特定的温度间隔进行升温，获取所述温度间隔对应的所述微液滴阵列的荧光图像；
    S333，根据所述温度间隔对应的所述微液滴阵列的荧光图像，获取所述温度间隔对应的每个所述微液滴的荧光信息；以及
    S334，根据所述温度间隔对应的每个所述微液滴的荧光信息，获取每个所述微液滴的熔解曲线，从而获得所述微液滴阵列的熔解曲线。
  37. 如权利要求33所述的数字PCR检测方法，其特征在于，所述步骤S40包括：
    S410，根据所述微液滴阵列的荧光曲线，获取所述微液滴阵列的核酸起始拷贝数；以及S420，根据所述微液滴阵列的熔解曲线，获取所述微液滴阵列的核酸信息。
  38. 如权利要求37所述的数字PCR检测方法，其特征在于，所述步骤S410包括：
    S411，根据所述微液滴阵列的荧光曲线，获取每个所述微液滴的所述荧光曲线对应的Ct值；
    S412，根据每个所述微液滴的所述荧光曲线的Ct值进行聚类，并依次由大到小进行排序，获得x 1，x 2，····x n个类别；
    S413，根据所述x 1，x 2，····x n个类别，获得每个类别对应的所述微液滴的数量y 1、y 2、····y n；
    S414，根据每个类别对应的所述微液滴的数量y 1、y 2、····y n，获得所述x 1，x 2，····x n个类别对应的所述微液滴的数量y 1、y 2、····y n频数分布；
    S415，根据所述频数分布，计算所述微液滴阵列的核酸起始拷贝数。
  39. 如权利要求38所述的数字PCR检测方法，其特征在于，在所述步骤S415中，当所述x 1类别的所述微液滴的数量y 1大于或等于特征值m时，根据所述频数分布进行泊松分布拟合，获得泊松分布的参数λ，从而获得所述微液滴阵列的核酸起始拷贝数。
  40. 如权利要求38所述的数字PCR检测方法，其特征在于，在所述步骤S415中，当所述x 1类别的所述微液滴的数量y 1小于特征值m时，包括：
    S4151，根据所述x 2，····x n个类别对应的所述微液滴的数量y 2、····y n的部分频数分布，依次假设所述x 2类别对应的所述核酸起始拷贝数，并进行泊松分布拟合，获取每个泊松分布对应的的参数λ j(j＝0，1，2····)；
    S4152，在一个区间[λ min,λ max]内搜索λ j，使得频数值误差平方和err最小，获得最优λ optimal；
    S4153，根据所述最优λ optimal，计算所述微液滴阵列的核酸起始拷贝数。
  41. 如权利要求37所述的数字PCR检测方法，其特征在于，所述步骤S420包括：
    S421，根据所述微液滴阵列的熔解曲线，获取每个所述微液滴的所述熔解曲线对应的熔解温度；以及
    S422，根据所述熔解温度，对所述微液滴阵列进行分类，获取所述微液滴阵列的核酸信息，进而获得所述待测核酸的核酸信息。
  42. 如权利要求33所述的数字PCR检测方法，其特征在于，所述数字PCR检测方法还包括：
    S50，获取所述微液滴阵列的高分辨率熔解曲线，对所述微液滴阵列进行分类，并获得所述微液滴阵列的基因分型、突变检测等核酸信息。
  43. 一种核酸检测微球，包括：
    核体，所述核体具有荧光编码信息；
    包覆层，包裹所述核体，所述包覆层包括基体以及分散于所述基体的引物，且所述引物与所述核体唯一对应。
  44. 如权利要求43所述的核酸检测微球，其特征在于，所述包覆层还包括探针，且所述探针与所述引物分散于所述基体，并与所述核体唯一对应。
  45. 如权利要求43所述的核酸检测微球，其特征在于，所述基体为琼脂糖凝胶。
  46. 如权利要求43所述的核酸检测微球，其特征在于，所述核体为含有荧光染料的实体球。
  47. 如权利要求43所述的核酸检测微球，其特征在于，所述核体的直径为10微米～100微米。
  48. 如权利要求43所述的核酸检测微球，其特征在于，所述包覆层厚度为10微米～100微米。
  49. 一种核酸检测微球的制备方法，包括：
    S110，提供多个核体与引物溶液；
    S120，提供凝胶粉末，将所述凝胶粉末加入到双蒸水中获得凝胶粉溶液，并将所述凝胶粉溶液加热至澄清，获得包覆层制备液；
    S130，在凝胶熔融温度，将所述多个核体、所述引物溶液与所述包覆层制备液混合，获得核酸检测微球制备溶液；
    S140，在所述凝胶熔融温度，将所述核酸检测微球制备溶液微滴化，形成多个核酸检测微球液滴；
    S150，将所述多个核酸检测微球液滴冷却，并通过流式分选获得多个核酸检测微球。
  50. 如权利要求49所述的核酸检测微球的制备方法，其特征在于，在所述步骤S150中，所述核酸检测微球为含有单个所述核体的核酸检测微球。
  51. 如权利要求49所述的核酸检测微球的制备方法，其特征在于，在所述步骤S120中，所述凝胶粉末为琼脂粉、或聚乙二醇双丙烯酸酯等。
  52. 如权利要求49所述的核酸检测微球的制备方法，其特征在于，所述步骤S140包括：
    S141，提供具有出口端的吐液枪头，吐液枪头内储存有所述核酸检测微球制备溶液，且提供储存有疏水性油的开口容器；
    S142，在所述凝胶熔融温度，将所述吐液枪头的出口端插入所述疏水性油的液面下；
    S143，所述吐液枪头的出口端在所述疏水性油的液面下做瞬时加速的运动或变速周期 的运动，将所述核酸检测微球制备溶液由所述吐液枪头的出口端排出，在所述疏水性油的液面下形成所述多个核酸检测微球液滴。
  53. 一种核酸检测微球的制备方法，包括：
    S210，提供引物溶液、探针溶液以及多个核体；
    S220，提供凝胶粉末，将所述凝胶粉末加入到双蒸水中获得凝胶粉溶液，并将所述凝胶粉溶液加热至澄清，获得包覆层制备液；
    S230，在凝胶熔融温度，将所述多个核体、所述引物和探针溶液与所述包覆层制备液混合，获得核酸检测微球制备溶液；
    S240，在所述凝胶熔融温度，将所述核酸检测微球制备溶液微滴化，形成多个核酸检测微球液滴；
    S250，将所述多个核酸检测微球液滴冷却，并通过流式分选获得多个核酸检测微球。
  54. 如权利要求53所述的核酸检测微球的制备方法，其特征在于，在所述步骤S250中，所述核酸检测微球为含有单个所述核体的核酸检测微球。
  55. 一种试剂盒，用于高通量核酸检测分析，其特征在于，包括如权利要求43至48中任一项所述的核酸检测微球与核酸反应液。
  56. 一种高通量核酸检测方法，包括：
    S310，提供核酸扩增反应液与多种不同类型的核酸检测微球，且所述核酸检测微球包括核体与包覆层，所述核体具有编码信息，所述包覆层包裹所述核体，所述包覆层包括基体以及分散于所述基体的引物，且所述引物与所述核体唯一对应，所述核体为含有荧光染料的实体球；
    S320，将所述多种不同类型的核酸检测微球与所述核酸扩增反应液混合，获得核酸检测液；
    S330，将所述核酸检测液微滴化，形成多个微液滴；
    S340，将所述多个微液滴进行核酸扩增，获得扩增完成后的所述多个微液滴；
    S350，根据扩增完成后的所述多个微液滴，检测每个所述微液滴中的所述核体，筛选出仅含有一个所述核体的所述微液滴，获得第一有效微液滴；
    S360，根据所述第一有效微液滴，检测所述第一有效微液滴中的所述核体的荧光编码信号，获得所述核体对应的所述引物，并读取核酸扩增反应之后的报告荧光信号，获取所述第一有效微液滴中是否有对应的目标核酸分子。
  57. 如权利要求56所述的高通量核酸检测方法，其特征在于，在所述步骤S310中，所述核酸扩增反应液为以脱氧核糖核酸为模板的核酸扩增反应液、以核糖核酸为模板的逆转录核酸扩增反应液或环介导等温扩增反应液，且所述核酸扩增反应液中包含荧光染料。
  58. 如权利要求56所述的高通量核酸检测方法，其特征在于，所述步骤S360包括：
    S361，提供荧光信号检测装置，所述荧光信号检测装置包括编码荧光通道与荧光染料检测通道，根据编码荧光通道识别所述有效微液滴中所述核体的荧光编码信号；
    S362，根据所述核体的荧光编码信号，获取所述核体对应的所述引物；
    S363，根据所述荧光染料检测通道检测所述第一有效微液滴中核酸扩增反应之后的报告荧光信号，获取所述第一有效微液滴中是否有对应的目标核酸分子。
  59. 一种高通量核酸检测方法，包括：
    S410，提供核酸扩增反应液与多种不同类型的核酸检测微球，且所述核酸检测微球包括核体与包覆层，所述核体具有编码信息，所述包覆层包裹所述核体，所述包覆层包括基体以及分散于所述基体的引物和探针，且所述引物和所述探针与所述核体唯一对应，所述核体为具有荧光编码信息的实体球；
    S420，将所述多种不同类型的核酸检测微球与所述核酸扩增反应液混合，获得核酸检测液；
    S430，将所述核酸检测液微滴化，形成多个微液滴；
    S440，将所述多个微液滴进行核酸扩增，获得扩增完成后的所述多个微液滴；
    S450，根据扩增完成后的所述多个微液滴，检测每个所述微液滴中的所述核体，筛选出仅含有一个所述核体的所述微液滴，获得第二有效微液滴；
    S460，根据所述第二有效微液滴，检测所述第二有效微液滴中的所述核体的荧光编码信号，获得所述核体对应的所述引物与所述探针，并读取核酸扩增反应之后的报告荧光信号，获取所述第二有效微液滴中是否有对应的目标核酸分子。
PCT/CN2019/072974 2018-01-24 2019-01-24 数字pcr检测仪、数字pcr定量检测方法、不同体积数字pcr的定量分析方法、数字pcr检测方法、核酸检测微球、核酸检测微球制备方法、核酸检测微球试剂盒以及高通量核酸检测方法 WO2019144907A1 (zh)

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