CN110702588A - Digital PCR analysis device and PCR analysis method - Google Patents
Digital PCR analysis device and PCR analysis method Download PDFInfo
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Abstract
The application discloses a digital PCR analysis device and a PCR analysis method. The digital PCR analysis apparatus includes: droplet generation module, PCR temperature control module and optical detection module, wherein: the liquid drop generating module is used for generating a liquid drop reaction unit on the microfluidic chip; the PCR temperature control module is used for controlling the temperature of the liquid drop reaction unit generated on the microfluidic chip; and the optical detection module is used for determining the image of the droplet reaction unit on the microfluidic chip. Because the digital PCR analysis device integrates a plurality of processes such as generation of a liquid drop reaction unit, PCR amplification reaction, optical detection and the like into one instrument to be automatically carried out, the efficiency of PCR analysis can be improved, and the problems of pollution, damage and the like caused by transferring liquid drops in the prior art are solved.
Description
Technical Field
The present application relates to the field of biotechnology, and in particular, to a digital PCR analysis apparatus and a PCR analysis method.
Background
Polymerase Chain Reaction (PCR) can realize in-vitro amplification of DNA sequences, is a main method for nucleic acid detection at present, and is also an important support technology in the fields of life science research and clinical molecular diagnosis.
The basic principle of current digital PCR analysis is to distribute a sample into a large number of droplets, each of which contains one or more samples to be tested, then perform PCR amplification on the samples to be tested, and analyze the droplets after PCR amplification. Since the digital PCR analysis process involves multiple processes, which generally results in low PCR analysis efficiency, and the droplet transfer process may be contaminated or cause droplet breakage to affect the analysis quality, a new digital PCR analysis apparatus is needed to improve the PCR analysis efficiency and quality.
Disclosure of Invention
The embodiment of the application provides a digital PCR analysis device and a PCR analysis method, which are used for solving the problems of low PCR analysis efficiency, pollution in the liquid drop transfer process and liquid drop breakage in the prior art.
The embodiment of the present application provides a digital PCR analysis apparatus, including: droplet generation module, PCR temperature control module and optical detection module, wherein:
the liquid drop generating module is used for generating a liquid drop reaction unit on the microfluidic chip;
the PCR temperature control module is used for controlling the temperature of the liquid drop reaction unit generated on the microfluidic chip;
the optical detection module is used for determining the image of the droplet reaction unit on the microfluidic chip.
Preferably, the PCR temperature control module specifically comprises: and the sealed cavity submodule is used for hermetically accommodating the microfluidic chip which generates the droplet reaction unit.
Preferably, the PCR temperature control module further comprises: heating refrigeration submodule piece and heat dissipation submodule piece, wherein:
the heating and refrigerating submodule is used for heating and refrigerating the sealed cavity submodule;
and the heat dissipation submodule is used for dissipating heat of the sealed cavity submodule.
Preferably, the digital PCR analysis apparatus further comprises: and the scanning motion mechanism is used for driving the PCR temperature control module to perform two-dimensional motion along a preset optical scanning imaging path.
Preferably, the optical detection module is configured to capture images of a droplet reaction unit of a microfluidic chip on the PCR temperature control module in a plurality of positions of the preset optical scanning imaging path.
Preferably, the digital PCR analysis apparatus further comprises: image concatenation module and analysis module, wherein:
the image splicing module is used for generating an integral image of each liquid drop reaction unit on the microfluidic chip according to each image and the corresponding shooting position coordinate;
and the analysis module is used for determining an analysis result according to the whole image.
Preferably, the droplet generation module specifically includes: the backup pad, be used for supporting the backup pad is the stand that the slope was placed, set up in horizontal motion submodule piece in the backup pad and set up in vertical motion submodule piece in the backup pad, wherein:
the horizontal movement submodule is used for adjusting the horizontal position of the microfluidic chip;
and the vertical movement submodule is used for sealing the microfluidic chip by adjusting the vertical position of a sealing pressure head in the vertical movement submodule.
Preferably, the droplet generation module specifically includes: and the oiling needle is connected with the oil storage bottle and used for guiding sealing oil from the oil storage bottle to perform oiling sealing on the microfluidic chip.
Preferably, the digital PCR analysis apparatus further includes: the device comprises a device shell, a hardware control part, operating software and device peripheral equipment.
The embodiment of the present application further provides a PCR analysis method, and the digital PCR analysis apparatus includes: a droplet generation module, a PCR temperature control module and an optical detection module, the method comprising:
generating a liquid drop reaction unit on the microfluidic chip through the liquid drop generation module, and oiling and sealing a sample adding port, an oil filling port and an overflow port of the microfluidic chip;
placing the sealed microfluidic chip in a PCR temperature control module, and introducing an air source to maintain a certain positive air pressure;
controlling the temperature of a liquid drop reaction unit generated on the microfluidic chip through the PCR temperature control module, and performing PCR amplification reaction on a sample to be detected in the liquid drop reaction unit;
and after the PCR amplification reaction is finished, determining an image of a droplet reaction unit on the microfluidic chip through the optical detection module, and determining the concentration of the sample to be detected through the identification of the image.
The embodiment of the application adopts at least one technical scheme which can achieve the following beneficial effects:
by adopting the digital PCR analysis device provided by the embodiment of the application, the droplet reaction unit is generated on the microfluidic chip A through the droplet generation module, the temperature of the droplet reaction unit is controlled through the PCR temperature control module, so that PCR amplification reaction is carried out, and then the image of the droplet reaction unit is determined through the optical detection module. Because the digital PCR analysis device integrates a plurality of processes such as generation of a liquid drop reaction unit, PCR amplification reaction and the like into one instrument to be automatically carried out, the efficiency of PCR analysis can be improved, and the problems of pollution or liquid drop damage and the like during liquid drop transfer in the prior art are solved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
fig. 1 is a schematic structural diagram of a digital PCR analysis apparatus provided in an embodiment of the present application;
fig. 2 is a schematic structural diagram of a droplet generation module in the digital PCR analysis apparatus according to an embodiment of the present disclosure;
fig. 3 is a schematic structural diagram of a microfluidic chip provided in an embodiment of the present application;
fig. 4 is a schematic structural diagram of a flow channel layer of a microfluidic chip provided in an embodiment of the present application;
FIG. 5 is a schematic diagram of a specific structure of a PCR temperature control module and an optical detection module in the digital PCR analysis apparatus according to the embodiment of the present disclosure;
FIG. 6 is a diagram of an optical scanning imaging path provided by an embodiment of the present application;
FIG. 7-1 is a diagram illustrating the droplet recognition effect of the digital PCR analysis apparatus provided in the embodiments of the present application;
FIG. 7-2 is a diagram of the droplet recognition effect of the digital PCR analysis apparatus provided in the embodiments of the present application;
FIG. 8 is a schematic front view of a digital PCR analysis apparatus according to an embodiment of the present disclosure;
FIG. 9 is a schematic diagram of a back side of a digital PCR analysis apparatus according to an embodiment of the present application;
fig. 10 is a flowchart illustrating specific steps of a digital PCR analysis method according to an embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The technical solutions provided by the embodiments of the present application are described in detail below with reference to the accompanying drawings.
As mentioned above, since the digital PCR analysis process involves multiple processes such as generating reaction units, amplifying samples to be tested, etc., the PCR analysis efficiency is generally low and the droplets are contaminated and damaged during the transfer process. Based on this, the embodiments of the present application provide a digital PCR analysis apparatus, which can solve the problem.
As shown in fig. 1, a schematic structural diagram of the digital PCR analysis apparatus 100 is shown, the digital PCR analysis apparatus 100 includes: a liquid drop generating module 1, a PCR temperature control module 2 and an optical detection module 3. The liquid drop generating module 1 can be used for generating liquid drops on the microfluidic chip A, and the liquid drops are used as reaction units of subsequent PCR amplification reaction and are subsequently called liquid drop reaction units; the PCR temperature control module 2 can be used for controlling the temperature of the liquid drop reaction unit generated on the microfluidic chip A, so that the PCR amplification reaction of a sample to be detected is facilitated, and the optical detection module 3 can be used for determining the image of the liquid drop reaction unit on the microfluidic chip.
Therefore, with the digital PCR analysis apparatus 100 provided in this embodiment of the present application, the droplet generation module 1 generates the droplet reaction unit on the microfluidic chip a, the PCR temperature control module 2 controls the temperature of the droplet reaction unit, so as to perform PCR amplification reaction, and then the optical detection module 3 determines the image of the droplet reaction unit. Since the digital PCR analysis apparatus 100 integrates a plurality of processes such as generation of a droplet reaction unit, PCR amplification reaction, and the like into one instrument, it is possible to improve the efficiency of digital PCR analysis and solve the problems of contamination and damage caused by transferring droplets in the prior art.
In practical application, the droplet generating module 1 is mainly composed of a horizontal moving unit 11, a vertical moving unit 12, a vertical column 13 and a supporting plate 14. The upright columns 13 are used for supporting the supporting plate 14 to be obliquely placed, the horizontal movement submodule 11 and the vertical movement submodule 12 are arranged on the supporting plate 14, and the horizontal movement submodule 11 drives the microfluidic chip A to move in the horizontal direction to adjust the horizontal position of the microfluidic chip A. For example, the horizontal movement submodule 11 can drive the movement of the microfluidic chip a from the initial position, the sealing position and the refueling position.
In addition, the digital PCR analysis apparatus 100 further includes a scanning movement mechanism 4, an oil storage bottle 5, a precision pressure control module 6, a positive pressure gas source 7, and the like, wherein the positive pressure gas source 7 may be a nitrogen gas bottle or a compressor. The oil storage bottle 5, the precise pressure control module 6 and the positive pressure air source 7 are all connected with the vertical movement unit 12.
Fig. 2 is a schematic diagram of a specific structure of the droplet generation module 1. Wherein the horizontal movement unit 11 of the droplet generation module 1 comprises: a horizontal moving screw motor 111, a horizontal position sensor 112, a horizontal linear slide rail 113, a chip bracket 114 and a pressing block 115.
The chip carrier 114 is used for carrying the microfluidic chip a, and the pressing block 115 is used for fixing the microfluidic chip a carried on the chip carrier 114. And a horizontal position sensor 112 for sensing the specific position of the microfluidic chip a carried on the chip carrier 114 in the horizontal direction. The horizontal movement lead screw motor 111 can drive the chip carrier 114 to slide along the horizontal linear slide 113 in the horizontal direction, for example, the horizontal movement lead screw motor 111 drives the chip carrier 114 to move along the horizontal linear slide 113 from the initial position to the droplet generation position and then to the oiling sealing position.
The vertical movement unit 12 includes: a vertically moving screw motor 121, a vertical position sensor 122, a vertical linear guide 123, a sealing ram 124, and a refueling needle 125. The vertically moving screw motor 121 can drive the sealing pressure head 124 to slide in the vertical direction along the vertical linear guide rail 123, so that the sealing pressure head 124 can tightly seal an oil filling port, a sample adding port and the like on the microfluidic chip a. The vertical position sensor 122 is capable of sensing a particular position of the seal ram 124 in a vertical direction. In addition, the vertically moving screw motor 121 can also drive the oiling needle 125 to move up and down, so that sealing oil is extracted from the oil storage bottle 5 by matching with the oiling peristaltic pump 21, and oil sealing of an oiling port of the microfluidic chip A is performed. With reference to fig. 1 and 2, the oil filling needle 125 is connected to the oil storage bottle 5 for filling oil to the microfluidic chip a and sealing the microfluidic chip a, and the sealing oil in the oil storage bottle 5 is driven by the oil filling peristaltic pump 51 to flow to the oil filling needle 125 for filling oil to the microfluidic chip a and sealing the microfluidic chip a.
In addition, the oiling peristaltic pump 51 can also be used for controlling and outputting quantitative sealing oil, so that oiling and sealing of the microfluidic chip a are realized, and evaporation of liquid drops in a liquid drop reaction unit during PCR amplification reaction is avoided.
In practical applications, when the sealing ram 124 moves downward to be able to fasten the sealed microfluidic chip a, the pressure sensor on the sealing ram 124 can detect the pressure value thereof, and if the pressure value is greater than or equal to the preset value, the sealing ram 124 stops moving downward by vertically moving the lead screw motor 121. Then, a 2-way positive pressure air source is introduced into the sealing pressure head 124 through the positive force supply device 7, and the pressure value of the positive pressure air source can be 15-35 kPa.
After the droplet reaction unit is generated, the vertical movement unit 12 moves to an initial position, then the horizontal movement unit 11 moves the microfluidic chip a to an oil adding level, the vertical movement unit 12 moves downwards, the oil adding needle 125 is sequentially inserted into the oil filling port, the oil adding port and the overflow port, and 50-100uL of sealing oil is respectively added into the oil filling port, the oil adding port and the overflow port, so that the microfluidic chip a is sealed.
The precise pressure control module 6 mainly converts the pressure (such as nitrogen pressure) provided by the positive pressure gas source 7 through the precise pressure control module 6 into two paths of high-stability positive pressure gas sources, so as to provide the positive pressure gas sources to the microfluidic chip a and drive the generation of the droplet reaction units on the microfluidic chip, and meanwhile, the interruption or the introduction of the pressure provided by the positive pressure gas sources 7 can be controlled by at least one two-position two-way electromagnetic valve 71 shown in fig. 1.
In addition, the positive pressure gas source 7 can also be connected with the PCR temperature control module 2, and the connecting pipes include a plurality of pressure regulating valves 72 and a plurality of (for example, 2) two-position two-way solenoid valves 71 therebetween.
Fig. 3 shows a specific structure diagram of the microfluidic chip a. The micro-fluidic chip is composed of three layers of structures, namely an upper cover layer A1, a flow channel layer A2 and a lower cover layer A3. In the three-layer structure, the lower cover layer A3 is accurately positioned through the positioning column A31, and the three-layer structure is bonded together through the flow channel layer A2 in a double-sided adhesive-backed mode.
The upper cover layer A1 comprises three types of ports, namely an oil filling port A11, an adding sample port A12 and an overflow port A13. The oil filling port A11 is used for adding liquid drop generating oil, and the volume of the oil filling port A11 is not less than 70 uL; the sample adding port A12 is used for adding a sample to be detected, and the volume of the sample adding port A12 is not less than 30 uL; the overflow port a13 is used to overflow the seal oil out of the buffer area, and its volume is not less than 120 uL. The structure of the upper cover layer A1 can be formed by injection molding, and the material can be high-temperature resistant hard polymer, such as: cyclic Olefin Copolymer (COC), cyclic olefin polymer COP, etc., the thickness of the cap layer a1 may be less than 1 mm.
The lower cover layer A3 is usually a flat plate with 4 positioning posts a31 on the periphery, and the structure of the lower cover layer A3 can also be injection molded, and the material can be high temperature resistant hard polymer, such as: COC or COP, etc. In addition, to increase the thermal conductivity of the lower cap layer A3, its thickness is typically less than 0.5 mm.
The flow channel layer a2 is typically a COC or COP film with a thickness of 100-120mm, which is double-sided adhesive backed. The micro flow channel structure can be formed by etching, printing and other processes. As shown in fig. 4, the flow channel layer a2 is divided into a droplet generation area a21 and a droplet tiling area a 22. The droplet tiling area a22 generally adopts a double-V structure, which can accelerate the tiling speed of the droplet reaction unit, and can realize the single-layer tiling of droplets by controlling the thickness of the channel layer a 2.
In addition, the flow channel layer a2 of the microfluidic chip a may include at least one channel, for example, 1 channel, 2 channels, etc., and the number of the channels is not limited herein.
Before the microfluidic chip A is used, sealing oil can be added into the oil filling port A11, a small positive pressure acting force is applied to the oil filling port A11 by a liquid transfer machine, the flow channel is infiltrated, and redundant bubbles in the chip are discharged. Then, a sample is added into a sample adding port A12 of the microfluidic chip A, droplet generation oil is added into an oil adding port A11, and then the microfluidic chip A is placed into the droplet generation module 1, so that generation of a droplet reaction unit and oil adding sealing are performed.
In practical applications, since the PCR amplification reaction generally requires strict control of the reaction temperature and prevention of droplet evaporation, in order to prevent the influence of the external environment on the temperature of the droplet reaction unit and droplet evaporation, the PCR temperature control module 2 may include: and the sealed cavity submodule 21 is used for hermetically accommodating the microfluidic chip on which the droplet reaction unit is generated and maintaining the positive air pressure of the sealed cavity.
Fig. 5 shows a PCR temperature control module 2 and an optical detection module 3 of the digital PCR analysis apparatus 100 in practical application, wherein the PCR temperature control module 2 includes a sealed cavity sub-module 21, a heating and cooling sub-module 22, and a heat dissipation sub-module 23.
And the heating and refrigerating sub-module 22 is used for heating and refrigerating the sealed cavity sub-module. In order to rapidly perform heating and cooling, the heating and cooling submodule 22 may use a semiconductor cooling technology, and detect the temperature of the hot plate in real time through a temperature sensor, and implement temperature cycle control in combination with PID control.
The heat-dissipating sub-module 23 may be used to dissipate heat from the sealed-cavity sub-module. In general, the heat dissipation submodule 23 can dissipate heat by using a heat pipe and a fan is used to dissipate heat quickly, when the heat plate cools, the fan starts to move, and when the heat plate heats, the fan stops moving.
The sealed cavity submodule 21 includes a cavity unit and a sealing cover plate, so as to perform pressure-maintaining sealing on the microfluidic chip with the droplet reaction unit. The cavity unit can be sealed by an O ring, and the sealing cover plate can be screwed by threads and tightly presses the sealing ring. Generally, the sealing cover plate is provided with a transparent glass window, and the glass can be anti-reflection glass with high light transmission performance, so that the definition of optical imaging of the glass is improved.
Before the microfluidic chip A is placed in the PCR temperature control module 2 for PCR amplification reaction, a pressure source of 100-. After the pressure of the pressure source is stabilized, the PCR amplification reaction is started by adjusting the reaction temperature, and the pressure is sealed and maintained by the sealing cover plate on the sealing cavity submodule 21.
The optical detection module 3 includes a CCD camera 31, a filter unit 32, a light source unit 33, an objective lens 34, and an annular light source 35. The filter unit 32 can be driven by a stepping motor, so as to realize automatic switching of different fluorescent channels; the light source unit 33 may be excited by multiple (e.g. 2) LED light sources, and is coupled to a light source via a dichroic mirror for excitation; the objective lens 34 may be selected from an objective lens of 2 magnification, 4 magnification, 10 magnification, or other magnifications. The annular light source 35 is used for providing a bright field light source to realize the outline imaging of the liquid drop reaction unit in a bright field environment; the CCD camera 31 is used to capture an image of the droplet reaction unit.
It should be further noted that, in practical applications, in order to increase the throughput of detection, after the PCR amplification reaction is finished, images of the droplet reaction units on the microfluidic chip a can be collected at a plurality of image collection points, so that the final result can be determined by analyzing the images. Therefore, with reference to fig. 1 and 5, the digital PCR analysis apparatus 100 can further include a scanning motion mechanism 4 for driving the PCR temperature control module 2 to perform two-dimensional motion along the predetermined optical scanning imaging path. The scanning movement mechanism 4 can be composed of two paths of zero-gap screw rod motors and supporting plates, and two-dimensional movement is executed by driving the temperature control module, so that two-dimensional image acquisition of the droplet reaction unit in a large area range is realized after the PCR amplification reaction of the droplet reaction unit of the microfluidic chip A is finished.
One way of presetting the optical scanning imaging path is to preset at least two of the following parameters for the scanning movement mechanism 4: the chip ejection position coordinates, the PCR amplification position coordinates, the scanning starting position coordinates, the scanning ending position coordinates, the X-axis one-time scanning micro-step number and the Y-axis one-time scanning micro-step number.
This process can be exemplified by setting in advance the coordinates of the chip ejection position (X1, Y1), the coordinates of the PCR amplification position (X2, Y2), the coordinates of the scan start position (X3, Y3), the coordinates of the scan end position (X4, Y4), the number of X-axis one-scan micro-steps n being 4, and the number of Y-axis one-scan micro-steps m being 3, as shown in fig. 6. The process is as follows: firstly, the chip pop-up position coordinates (X1 and Y1) are that the PCR temperature control module 2 automatically moves to the chip pop-up position, and the microfluidic chip is manually placed on the PCR temperature control module 2 and sealed; then controlling the PCR temperature control module 2 to move from the chip ejection position to the PCR amplification position according to the coordinates (X2 and Y2) of the PCR amplification position, wherein the movement path is S1, and carrying out PCR amplification reaction at the PCR amplification position; after the PCR amplification reaction is finished, controlling the PCR temperature control module 2 to move from the PCR amplification position to the scanning initial position by scanning initial position coordinates (X3 and Y3), wherein the movement path is S2; and then controlling the PCR temperature control module 2 to move along the moving paths S3-S10 in sequence according to the X-axis one-time scanning micro step number n and the Y-axis one-time scanning micro step number m, and carrying out image acquisition by the optical detection module 3 after the movement of each step is finished, so as to carry out multi-position image acquisition in the processes of S3-S10. At this time, the optical detection module 3 can shoot images of the droplet reaction unit of the microfluidic chip on the PCR temperature control module in a plurality of positions of the preset optical scanning imaging path; after the scanning is completed, the PCR temperature control module 2 is controlled to return to the scanning start position according to the scanning start position coordinates (X3, Y3).
In practical application, if there are multiple fluorescence channels in the optical detection module 3, the fluorescence of other channels is used to scan again according to the preset optical scanning imaging path. After the scanning of all paths of fluorescence is finished, the fluorescence moves to a PCR amplification position from a scanning initial position and then moves to a chip ejection position, and therefore the scanning work of the microfluidic chip is finished.
The scanning process may obtain images of multiple positions, which need to be integrated to generate an overall image that can reflect the concentration of the sample in all or most of the droplet reaction units on the microfluidic chip a.
At this time, the digital PCR analysis apparatus in this embodiment of the application may further include an image stitching module, configured to generate the whole image according to the acquired images and their corresponding shooting position coordinates.
According to the acquired shooting position coordinates of each picture, after the pictures are spliced into an integral image, the outline of all liquid drop reaction units can be found by using an image processing algorithm, the brightness density of the outline area of all liquid drop reaction units is calculated, the percentage of fluorescent bright liquid drops to the total liquid drops is counted by setting the threshold value of fluorescent light and dark liquid drops, and finally, a test result is calculated by using Poisson distribution.
For example, after the pictures are stitched into the overall image, the analysis result may be determined from the overall image by the analysis module of the digital PCR analysis apparatus 100. Before analysis, a background noise threshold value, a droplet profile confidence level and a droplet profile size range value need to be set.
Wherein, the background noise threshold is used for deducting the influence of image background noise, and the outline of the similar liquid drop reaction unit below the threshold range is excluded; the confidence of the droplet profile is used for defining the similarity probability of the circular profile shape by an algorithm, and if the probability value of the circular profile of the droplet is larger than the set value, a droplet is confirmed to be found. The drop profile size range values are used to determine a drop profile diameter range, allowing for some fluctuation in drop size relative to a target value.
In the process of analyzing the whole image, firstly, the contour of each liquid drop reaction unit is determined by using an image recognition algorithm, and the average brightness value in the contour range of each liquid drop is calculated (the average brightness value of each liquid drop is obtained by dividing the total brightness value of each liquid drop by the contour area of each liquid drop). And then carrying out statistical analysis on the average brightness value of each liquid drop, if the average brightness value is larger than a preset target threshold value, considering the liquid drop as a positive liquid drop, and counting the number of all the liquid drops and the number of the positive liquid drops. And finally, calculating a concentration value of the sample according to parameters such as the volume of the liquid drop, the dilution multiple and the like according to the Poisson distribution, thereby determining a final analysis result. Fig. 7-1 and 7-2 are diagrams illustrating the recognition effect of the digital PCR analysis apparatus 100 on the droplet reaction units according to the embodiment of the present application, wherein the boundaries of the droplet reaction units are clear, and the boundaries of the droplet reaction units are further marked by circular borders in fig. 7-2.
In practical applications, the digital PCR analysis apparatus 100 includes: the device comprises a liquid drop generation module 1, a PCR temperature control module 2, an optical detection module 3, a device shell, a hardware control part, operating software, device peripheral equipment and the like.
Fig. 8 is a schematic structural diagram of the front side of the digital PCR analysis apparatus 100 in practical use. The droplet generation module 1 is disposed at one side of the digital PCR analysis apparatus 100, and the status indicator light 8 of the digital PCR analysis apparatus 100 is located below the droplet generation module, and the status indicator light may further include a droplet generation status indicator light and a PCR amplification detection indicator light. When the droplet generation state indicator light is on, the digital PCR analysis device 100 is prompted to generate a droplet reaction unit and ask the user to wait; when the droplet generation status indicator lamp is turned off, the digital PCR analysis device 100 is prompted to complete generation of the droplet reaction unit, and the user can take out the microfluidic chip a. Similarly, when the PCR amplification detection indicator light is on, the PCR amplification reaction is prompted to be carried out; when the PCR amplification detection indicator lamp is turned off, the PCR amplification reaction is indicated to be completed.
The oil storage bottle 5 is disposed in a storage area above the droplet generating module 1 and is shielded with a cover plate to prevent dust and impurities from entering.
The PCR temperature control module 2 and the optical detection module 3 are disposed on one side of the digital PCR analysis apparatus 100 and covered by a cover plate. Which is located inside the digital PCR analyzer 100 during operation, and performs amplification reaction and optical detection at a predetermined position inside the digital PCR analyzer 100.
The pop-in and pop-out button 24 of the PCR temperature control module 2 is located at one side of the digital PCR analysis device 100. The cover plate 25 can be opened by pressing the pop-in and pop-out button 24, at this time, the PCR temperature control module 2 pops out automatically, then the sealing cover plate of the PCR temperature control module 2 is opened, the microfluidic chip a after the droplet reaction unit is generated is placed at a designated position, then the sealing cover plate is screwed tightly, then the pop-in and pop-out button 24 is pressed, and the PCR temperature control module 2 moves to the PCR amplification position inside the digital PCR analysis apparatus 100.
The up-down push-pull shield plate 9 is located right in front of the PCR analyzer 100, the up-down push-pull shield plate 9 can slide up and down, and when the up-down push-pull shield plate 9 is pushed to the top, it is firmly attracted by the two magnetic attraction devices to prevent falling. When the digital PCR analysis apparatus 100 is idle, the up-and-down push-pull shield 9 can be pulled down to prevent dust and impurities from entering the digital PCR analysis apparatus 100.
Fig. 9 is a schematic diagram showing a structure of the back side of the digital PCR analysis apparatus 100 in practical use. The control board card B1 and the power supply module B2 are disposed on the back of the digital PCR analysis apparatus 100, and are used to control electromechanical motions, temperature control and optical imaging of the droplet generation module 1, the PCR temperature control module 2 and the optical detection module 3. The precise pressure control module 6 is disposed on the right side of the back of the digital PCR analysis apparatus 100.
The back interface of the digital PCR analysis apparatus 100 further includes: positive pressure air source inlet B3, USB interface B4, main switch B5 and secondary switch B6. The positive pressure gas source inlet B3 can be connected with a rubber tube with the outer diameter of 4mm, 6mm, 8mm or other sizes and is used for connecting a positive pressure gas source 7 (such as a nitrogen cylinder, an air compressor and the like). The USB interface B4 is used to connect to a peripheral computer for mutual communication, so as to control the digital PCR analysis apparatus 100 by the peripheral computer. The main switch B5 is used to control the power on/off of the digital PCR analysis apparatus 100. The secondary switch B6 is located at one side of the digital PCR analysis device 100 and is used for controlling the power on-off of the control board B1. The air inlet B7 of the PCR temperature control module 2 is located at the rear lower part of the digital PCR analysis device 100, and the air outlet B8 is located at the left side of the digital PCR analysis device 100, so that heat dissipation is achieved through air inlet from the rear lower part and air outlet from the left side.
The above-mentioned is a schematic diagram of a specific structure of the digital PCR analysis apparatus provided in the embodiments of the present application, and a specific method for using the digital PCR analysis apparatus can be provided below, so as to perform PCR analysis. As shown in fig. 10, the PCR analysis includes the following steps:
step S91: and adding the droplet generation oil and the sample to be detected into the provided microfluidic chip.
Droplet-generating oil (about 50. mu.L) and a sample (about 20. mu.L) can be added to the inside of the provided microfluidic chip from the oil port thereof. When the microfluidic chip is provided with 2 testing channels, the droplet generation oil and the sample can be added simultaneously, and if one of the testing channels is not used, the droplet generation oil with the same volume is added into the sample adding port and the oil adding port respectively.
Step S92: the digital PCR analysis device is electrified, the upper and lower push-pull shielding plates are manually opened, the operating software in the digital PCR analysis device is started, and actions such as mechanical reset, optical reset detection, air pressure calibration and the like are executed to ensure that the digital PCR analysis device is in a test preparation state.
Step S93: and generating a liquid drop reaction unit on the microfluidic chip through a liquid drop generation module in the digital PCR analysis device, and oiling and sealing a sample adding port, an oil filling port and an overflow port of the microfluidic chip.
In practical application, the droplet generating module 1 is mainly composed of a horizontal moving unit 11, a vertical moving unit 12, a vertical column 13 and a supporting plate 14. The upright column 13 is used for supporting the supporting plate 13 to be obliquely placed, the horizontal movement submodule 11 and the vertical movement submodule 12 are arranged on the supporting plate 14, and the horizontal movement submodule 11 drives the microfluidic chip A to move in the horizontal direction to adjust the horizontal position of the microfluidic chip A. For example, the horizontal movement submodule 11 can drive the movement of the microfluidic chip a from the initial position, the sealing position and the refueling position.
The microfluidic chip can be placed in the liquid drop generation area, the microfluidic chip is driven to move to the liquid drop generation position, the sealing pressure head compresses and seals the microfluidic chip, and 2 paths of positive pressure sources are introduced, so that the liquid drop generation and the flat laying process of the liquid drop reaction unit are carried out. Then the digital PCR analysis device drives the micro-fluidic chip to move to the position of the oil filling port, and oil filling sealing of three ports (the oil filling port, the oil filling port and the overflow port) of the micro-fluidic chip is executed.
Step S94: and placing the sealed microfluidic chip in a PCR temperature control module in a digital PCR analysis device, screwing a sealing cover plate, introducing positive air pressure and maintaining the air pressure to be stable. And then controlling the temperature of a liquid drop reaction unit generated on the microfluidic chip through the PCR temperature control module, and performing PCR amplification reaction on a sample to be detected in the liquid drop reaction unit.
After the oil-adding sealing is completed in step S93, the digital PCR analysis apparatus drives the microfluidic chip to move to the initial position, and then the microfluidic chip is taken out. And then pressing a PCR temperature control module to pop in a pop-up button, moving the temperature control module to a chip pop-up position, opening a sealing cover plate of the PCR temperature control module, placing the generated and tiled micro-fluidic chip with the droplet reaction unit in a temperature control heating area in the PCR temperature control module, and screwing the cover plate for sealing. Then pressing a pop-in and pop-out button of the PCR temperature control module, moving the temperature control module to a PCR amplification position, starting temperature control, and executing PCR amplification reaction.
Step S95: and after the PCR amplification reaction is finished, determining an image of a droplet reaction unit on the microfluidic chip through an optical detection module in the digital PCR analysis device, and determining the concentration of the sample to be detected through the identification of the image.
And after the amplification reaction is finished, turning on a light source lamp of the optical detection module, starting optical scanning and image recognition, and calculating the number of liquid drops to obtain a concentration result of the sample to be detected. And after all tests are finished, manually pressing a temperature control module ejection button, moving a chip ejection position by the temperature control module, and manually taking out the microfluidic chip to finish the tests.
With the method provided by the embodiment of the present application, since the method is based on the digital PCR analysis apparatus provided by the embodiment of the present application, the problems in the prior art can also be solved, and the description thereof is omitted here.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (10)
1. A digital PCR analysis apparatus, comprising: droplet generation module, PCR temperature control module and optical detection module, wherein:
the liquid drop generating module is used for generating a liquid drop reaction unit on the microfluidic chip;
the PCR temperature control module is used for controlling the PCR amplification reaction temperature of the liquid drop reaction unit generated on the microfluidic chip;
the optical detection module is used for determining the image of the droplet reaction unit on the microfluidic chip.
2. The digital PCR analysis device of claim 1, wherein the PCR temperature control module specifically comprises: and the sealed cavity submodule is used for hermetically accommodating the microfluidic chip which generates the droplet reaction unit.
3. The digital PCR analysis device of claim 1, wherein the PCR temperature control module further comprises: heating refrigeration submodule piece and heat dissipation submodule piece, wherein:
the heating and refrigerating submodule is used for heating and refrigerating the sealed cavity submodule;
and the heat dissipation submodule is used for dissipating heat of the sealed cavity submodule.
4. The digital PCR analysis apparatus according to any one of claims 1 to 3, further comprising: and the scanning motion mechanism is used for driving the PCR temperature control module to perform two-dimensional motion along a preset optical scanning imaging path.
5. The digital PCR analysis device according to claim 4, wherein the optical detection module is configured to capture images of the droplet reaction unit of the microfluidic chip on the PCR temperature control module at a plurality of positions of the predetermined optical scanning imaging path.
6. The digital PCR analysis device of claim 5, further comprising: image concatenation module and analysis module, wherein:
the image splicing module is used for generating an integral image of each liquid drop reaction unit on the microfluidic chip according to each image and the corresponding shooting position coordinate;
and the analysis module is used for determining an analysis result according to the whole image.
7. The digital PCR analysis device according to claim 1, wherein the droplet generation module specifically comprises: the backup pad, be used for supporting the backup pad is the stand that the slope was placed, set up in horizontal motion submodule piece in the backup pad and set up in vertical motion submodule piece in the backup pad, wherein:
the horizontal movement submodule is used for adjusting the horizontal position of the microfluidic chip;
and the vertical movement submodule is used for sealing the microfluidic chip by adjusting the vertical position of a sealing pressure head in the vertical movement submodule.
8. The digital PCR analysis device of claim 7, wherein the droplet generation module further comprises: and the oiling needle is connected with the oil storage bottle and used for guiding sealing oil from the oil storage bottle to perform oiling sealing on the microfluidic chip.
9. The digital PCR analysis device according to claim 1, further comprising: the device comprises a device shell, a hardware control part, operating software and device peripheral equipment.
10. A PCR analysis method, characterized in that a digital PCR analysis apparatus comprises: a droplet generation module, a PCR temperature control module and an optical detection module, the method comprising:
generating a liquid drop reaction unit on the microfluidic chip through the liquid drop generation module, and sealing a sample adding port, an oil filling port and an overflow port of the microfluidic chip;
placing the sealed microfluidic chip in a PCR temperature control module;
controlling the temperature of a liquid drop reaction unit generated on the microfluidic chip through the PCR temperature control module, and performing PCR amplification reaction on a sample to be detected in the liquid drop reaction unit;
and after the PCR amplification reaction is finished, determining an image of a droplet reaction unit on the microfluidic chip through the optical detection module, and determining the concentration of the sample to be detected through the identification of the image.
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