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 PDFInfo
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- 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
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- 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
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- 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
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- 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
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- 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"
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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
- 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
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- 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
Description
仪器类型 | 本仪器 | QX200数字PCR | qPCR数字PCR |
R 2 | 0.9993 | 0.998 | 0.9923 |
Claims (59)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
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