Integrated digital PCR device and its control method

Abstract

The present invention provides an integrated digital PCR device and its control method. The integrated digital PCR device includes a chip loading module, a droplet generation module, a temperature cycle module, a fluorescence detection module, a chip disposal module, and a scheduling mechanism. The droplet generation module can generate microdroplets from the sample in the sample addition chamber during the microdroplet generation process, and then transfer them to the reaction chamber for storage. The temperature cycle module realizes the amplification of the microdroplets in the reaction chamber. The fluorescence detection module can transfer the microdroplets in the reaction chamber to the fluorescence detection area and then to the sample addition chamber during the microdroplet detection process, and complete the fluorescence detection of the microdroplets during this process. The scheduling mechanism is used to transfer the integrated microdroplet chip between the chip loading module, the droplet generation module, the temperature cycle module, the fluorescence detection module, and the chip disposal module under the control of the main control module, and the integrated microdroplet chip in the temperature cycle module can be flipped up and down 180° and then transferred to the fluorescence detection module. The present invention has a high degree of automation and integration, which can improve the efficiency of the detection analysis work and reduce the labor costs.

Description

[Technical Field]

[0001] The present invention relates to the technical field of digital PCR analysis devices, and more particularly to an integrated digital PCR device and a control method thereof. [Background technology]

[0002] Digital PCR is a cutting-edge quantitative technology that quantifies nucleic acids by counting them using single-molecule PCR methods, achieving absolute quantification. It primarily employs microfluidics or microdropletization, a popular analytical chemistry research method, to distribute large volumes of diluted nucleic acid solutions into microreactors or microdroplets on a biochip. Each reactor contains no more than one nucleic acid template. After PCR cycles, reactors containing one nucleic acid template emit a fluorescent signal, while reactors without templates emit no fluorescent signal. The relative ratio and reactor volume can be used to estimate the nucleic acid concentration of the original solution. Traditional digital PCR analyzers are single-function and require the manual operation of multiple devices to complete a single amplification and detection cycle. With the development of society, the medical industry and scientific research fields are placing increasing demands on high-throughput, automated, fast, and integrated PCR analysis. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide an integrated digital PCR device and a control method thereof to overcome the drawbacks of the prior art digital PCR devices, which only have one function, and require manual operation of multiple devices to complete one amplification and detection operation, resulting in low detection efficiency.

[0004] To solve the above problems, the present invention provides an integrated digital PCR device. This integrated digital PCR device includes a chip loading module, a droplet generation module, a temperature cycling module, a fluorescence detection module, a chip disposal module, and a scheduling mechanism. The chip loading module is used to load an integrated micro-droplet chip containing a sample. The integrated micro-droplet chip includes a chip body, which is configured with a sample addition chamber, a reaction chamber, and a fluorescence detection region. The droplet generation module is used to supply gas to the integrated micro-droplet chip. During the micro-droplet generation process, micro-droplets can be generated from the sample in the sample addition chamber and then transferred to the reaction chamber for storage. The temperature cycling module is used to create a heating / cooling cycle for the micro-droplets in the reaction chamber to amplify the micro-droplets in the reaction chamber. During the micro-droplet detection process, the fluorescence detection module transfers the micro-droplets in the reaction chamber to the fluorescence detection region and then to the sample addition chamber, and performs fluorescence detection on the micro-droplets located in the fluorescence detection region. The chip disposal module is used to store the integrated micro-droplet chip after detection. The scheduling mechanism is used to transfer the integrated micro-droplet chip between the chip loading module, droplet generation module, temperature cycling module, fluorescence detection module, and chip disposal module under the control of the main control module, and can transfer the integrated micro-droplet chip in the temperature cycling module into the fluorescence detection module after flipping it upside down 180°.

[0005] In some embodiments, the integrated micro-droplet chip includes a chip body, in which a micro-droplet generation structure, an oil-liquid port, and a gas-liquid port are configured. The gas-liquid port is in communication with the reaction chamber, the sample addition chamber is in communication with the micro-droplet generation structure, and the oil-liquid port is in communication with the micro-droplet generation structure. When the integrated micro-droplet chip is located within the droplet generation module, the droplet generation module can create a first pressure difference between the sample addition chamber and the gas-liquid port, and a second pressure difference between the oil-liquid port and the gas-liquid port. The first pressure difference and the second pressure difference drive the sample in the sample addition chamber and the generated oil in the oil-liquid port, respectively, to enter the micro-droplet generation structure. The generated micro-droplets enter the reaction chamber and are stored. When the integrated micro-droplet chip is located within the fluorescence detection module, the fluorescence detection module can drive the detection oil to enter the reaction chamber through the gas-liquid port, causing the micro-droplets in the reaction chamber to flow out from the reaction chamber to the micro-droplet generating structure. The droplet generating module can also drive the detection separation oil to enter the micro-droplet generating structure through the oil-liquid port. The detection separation oil separates the micro-droplets flowing out from the reaction chamber into the micro-droplet generating structure to form a train, which then enters the fluorescence detection region.

[0006] In some embodiments, the microdroplet generation structure includes an oil-liquid conduit and a communication conduit, the oil-liquid conduit and the communication conduit intersecting in a cross shape. The communication conduit includes a first conduit located on a first side of the cross intersection and communicating with the reaction chamber, and a second conduit located on a second side of the cross intersection and communicating with the sample addition chamber. The oil-liquid port communicates with the oil-liquid conduit.

[0007] In some embodiments, the first side of the chip body is positioned in a horizontal orientation, the reaction chamber and the sample addition chamber are positioned on the first side, and the connection port between the reaction chamber and the first side of the chip body extends upward and forms a trumpet-shaped mouth that is small at the bottom and large at the top.

[0008] In some embodiments, a gas-liquid conduit extending from bottom to top is configured within the reaction chamber, a lower opening of the gas-liquid conduit communicates with the gas-liquid port, and an upper opening of the gas-liquid conduit is higher than an upper opening of the connection port.

[0009] In some embodiments, a microdroplet observation region is located between the first conduit and the connection port.

[0010] In some embodiments, the fluorescence detection region is located in the second conduit.

[0011] In some embodiments, the sample addition chamber includes an open chamber and a sealed cover sealed to the opening of the open chamber, and / or the sample addition chamber is provided with a filtration membrane or a vent.

[0012] In some embodiments, a light oil is pre-disposed in the reaction chamber before the microdroplets enter the reaction chamber.

[0013] The present invention further provides a method for controlling an integrated digital PCR device, which includes a chip loading step, a droplet generation step, a temperature cycling step, a fluorescence detection step, and a chip discarding step. In the chip loading step, the integrated micro-droplet chip is placed in a chip loading module. In the droplet generation step, the integrated micro-droplet chip in the chip loading module is transferred into the droplet generation module, and the sample in the sample addition chamber of the integrated micro-droplet chip is controlled to generate micro-droplets, which are then stored in the reaction chamber. In the temperature cycle step, the integrated micro-droplet chip is turned upside down by 180°, and then a heating and cooling cycle is performed on the micro-droplets in the micro-droplets in the reaction chamber located at the bottom of the integrated micro-droplet chip. In the fluorescence detection step, after the amplification of the microdroplets in the reaction chamber is completed, the microdroplets in the reaction chamber are controlled to enter a fluorescence detection region, thereby completing the fluorescence detection. In the tip disposal step, the integrated micro-droplet chip after the completion of the fluorescence detection is transferred into a tip disposal module.

[0014] In the integrated digital PCR device and its control method provided by the present invention, the scheduling mechanism controls the main control module to coordinate the operation of each functional module of the integrated micro-droplet chip, realizing integrated operation of the entire process from chip loading, droplet generation, PCR amplification, fluorescence detection and analysis to chip disposal, with a high degree of automation and integration, improving the efficiency of detection and analysis work and reducing labor costs. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing the structure of an integrated digital PCR device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram of the three-dimensional structure of the integrated microdroplet chip of FIG. 1. [Figure 3] FIG. 3 is a structural schematic diagram of the microdroplet generating structure of FIG. 2. [Figure 4] FIG. 1 is a schematic diagram of a microdroplet generation process. [Figure 5] FIG. 1 is a schematic diagram of microdroplets being generated and then stored in a reaction chamber. [Figure 6] FIG. 2 is a schematic diagram of the state inside the reaction chamber after the integrated microdroplet chip of FIG. 1 is turned upside down by 180°. [Figure 7] FIG. 7 is a schematic diagram of the state after oil liquid is introduced into the reaction chamber in the state of FIG. 6 (arrows in the figure indicate the flow directions of the microdroplets and oil liquid). [Figure 8] FIG. 1 is a schematic diagram of the state of a microdroplet after it has been extruded from the reaction chamber. [Figure 9] FIG. 2 is a schematic diagram of the operating optical path principle of the fluorescence detection module of FIG. 1. [Figure 10]1 shows the detection results of a digital PCR device according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] 1 to 10, an integrated digital PCR device is provided according to an embodiment of the present invention. The integrated digital PCR device includes a chip loading module 200, a droplet generation module 300, a temperature cycle module 400, a fluorescence detection module 500, a chip disposal module 600, and a scheduling mechanism 700. The chip loading module 200 is used to place an integrated micro-droplet chip 100 to which a sample has been added. The integrated micro-droplet chip 100 includes a chip body 1, which is configured with a sample addition chamber 12, a reaction chamber 11, and a fluorescence detection region 33. The droplet generation module 300 is used to supply oil (i.e., oil or pressurized gas) to the integrated micro-droplet chip 100. During the micro-droplet generation process, micro-droplets 4 can be generated from the sample in the sample addition chamber 12 and then transferred to and stored in the reaction chamber 11. The temperature cycle module 400 is used to create a heating / cooling cycle for the microdroplets 4 in the reaction chamber 11 in order to amplify the microdroplets 4 in the reaction chamber 11. During the microdroplet detection process, the fluorescence detection module 500 transfers the microdroplets 4 in the reaction chamber 11 to the fluorescence detection area 33 and then to the sample addition chamber 12, and performs fluorescence detection on the microdroplets 4 located in the fluorescence detection area 33. The chip disposal module 600 is used to store the integrated microdroplet chip 100 after detection. The scheduling mechanism 700 is used to transfer the integrated microdroplet chip 100 between the chip loading module 200, the droplet generation module 300, the temperature cycle module 400, the fluorescence detection module 500, and the chip disposal module 600 under the control of the main control module 800. The integrated microdroplet chip 100 in the temperature cycle module 400 can be flipped upside down by 180° before being transferred to the fluorescence detection module 500.In this technical solution, under the control of the main control module 800, the scheduling mechanism 700 can coordinate the operations of each functional module of the integrated micro-droplet chip 100, realizing the integrated operation of the entire process from chip loading, droplet generation, PCR amplification, fluorescence detection and analysis, to chip disposal, with a high degree of automation and integration, improving the efficiency of detection and analysis work and reducing labor costs. More importantly, the scheduling mechanism 700 of the present invention can be configured to flip the integrated micro-droplet chip 100 up and down 180 degrees, so that the operation processes of droplet generation, PCR amplification, and fluorescence detection and analysis can be integrated into the same chip, i.e., the integrated micro-droplet chip 100, further improving the degree of integration and automation.

[0017] In some embodiments, the integrated micro-droplet chip 100 includes a chip body 1, in which a micro-droplet generating structure, an oil / liquid port 31, and an air / liquid port 32 are configured. The air / liquid port 32 communicates with the reaction chamber 11, the sample addition chamber 12 communicates with the micro-droplet generating structure, and the oil / liquid port 31 communicates with the micro-droplet generating structure. When the integrated micro-droplet chip 100 is positioned within the droplet generation module 300, the droplet generation module 300 can create a first pressure difference between the sample addition chamber 12 and the gas-liquid port 32 (i.e., the air path of the droplet generation module 300 communicates with the sample addition chamber 12 and the gas-liquid port 32), and a second pressure difference between the oil-liquid port 31 and the gas-liquid port 32 (i.e., the liquid path of the droplet generation module 300 communicates with the oil-liquid port 31 and the gas-liquid port 32). The first pressure difference and the second pressure difference drive the sample in the sample addition chamber 12 and the oil produced at the oil-liquid port 31, respectively, to enter the micro-droplet generation structure, and the generated micro-droplets 4 enter the reaction chamber 11 and are stored. When the integrated micro-droplet chip 100 is located within the fluorescence detection module 500, the droplet generation module 300 can drive the detection oil 5 to enter the reaction chamber 11 through the gas-liquid port 32, causing the micro-droplets 4 in the reaction chamber 11 to flow out from the reaction chamber 11 to the micro-droplet generation structure. The droplet generation module 300 can also drive the detection separation oil to enter the micro-droplet generation structure through the oil-liquid port 31. The detection separation oil separates the micro-droplets 4 flowing out from the reaction chamber 11 into the micro-droplet generation structure, forming a train, and causing them to enter the fluorescence detection region 33. By time-division multiplexing the micro-droplet generation structure (time-division boundaries are defined before and after the inversion of the integrated micro-droplet chip 100), droplet generation, amplification, and detection are all integrated into a single chip, realizing a fully integrated and sealed digital PCR process.It not only inherits the advantages of uniform droplet size, less droplet number limitation, and high fluorescence detection signal-to-noise ratio, but also overcomes the original difficulties of complex chip structure, generation and detection completed on different chips, low integration, and difficulty in automation, which marks an important technological breakthrough in the field of digital PCR.

[0018] In one specific embodiment of the microdroplet generation structure, the microdroplet generation structure includes an oil-liquid pipe line 21 and a connecting pipe line, the oil-liquid pipe line 21 and the connecting pipe line intersect in a cross shape, the connecting pipe line includes a first pipe line 22 located on a first side of the cross intersection and connecting with the reaction chamber 11, and a second pipe line 23 located on a second side of the cross intersection and connecting with the sample addition chamber 12, and the oil-liquid port 31 is connected with the oil-liquid pipe line 21.

[0019] In some embodiments, as shown in FIG. 2 , the first side of the chip body 1 is positioned horizontally, the reaction chamber 11 and the sample addition chamber 12 are positioned on the first side, and the connection port 111 between the reaction chamber 11 and the first side of the chip body 1 extends upward and forms a horn-shaped opening with a small bottom and a large top. The horn-shaped connection port 111 can facilitate the entry of the microdroplets 4 from the first conduit 22 into the reaction chamber 11 and can also facilitate the entry of the microdroplets 4 from the reaction chamber 11 into the first conduit 22, preventing the microdroplets 4 from stagnating. In this case, the reaction chamber 11 and the sample addition chamber 12 are both positioned on the first side (specifically, the top surface) of the chip body 1, and all the microdroplets 4 that enter the reaction chamber 11 are collected at the connection port 111. During PCR amplification, the entire chip body 1 needs to be turned upside down, i.e., flipped 180°, to position the microdroplets 4 in the reaction region of the reaction chamber 11.

[0020] In some embodiments, a gas-liquid conduit 112 extending from bottom to top is further configured in the reaction chamber 11, the lower opening of the gas-liquid conduit 112 communicates with the gas-liquid port 32, and the upper opening of the gas-liquid conduit 112 is higher than the upper opening of the connection port 111. Thus, when the reaction chamber 11 is under negative pressure, the microdroplets 4 generated by the microdroplet generating structure can be prevented from flowing out of the gas-liquid conduit 112 after entering the reaction chamber 11.

[0021] In some embodiments, a microdroplet observation region 34 is located between the first conduit 22 and the connection port 111. The flow area of ​​the microdroplet observation region 34 is much larger than the flow area of ​​the first conduit 22. That is, the microdroplet observation region 34 is an enlarged (wide) region on the first conduit 22, which reduces the flow rate of the microdroplets 4 entering this region, making it easier to image the microdroplets 4 with an external camera, record the droplet morphology, and determine whether the droplet generation process is normal.

[0022] In one embodiment, the fluorescence detection area 33 is located in the second pipeline 23, and in the second pipeline 23, the microdroplets 4 flowing out of the reaction chamber 11 can be separated into a series of droplets with appropriate spacing when passing through the cross-junction under the action of the detection oil in the oil-liquid pipeline 21, thereby completing fluorescence detection under the action of an external system.

[0023] In a specific embodiment, the sample addition chamber 12 includes an open chamber 121 and a sealed cover 122 sealed to the opening of the open chamber 121, making it easy for an operator to add sample to the sample addition chamber 12. Furthermore, the sample addition chamber 12 is provided with a filter membrane or an exhaust hole. When the sample addition chamber 12 becomes a waste liquid reservoir (i.e., when the droplet chip is turned upside down), a certain amount of air is exhausted to prevent pressure buildup in the sample addition chamber 12.

[0024] In some embodiments, before the microdroplets 4 enter the reaction chamber 11, a light oil (i.e., an oil liquid with a relatively low density) is pre-placed in the reaction chamber 11 to ensure that the light oil is always located at the top of the microdroplets 4 in the reaction chamber 11, thereby solving the problem of evaporation of the microdroplets during amplification and realizing heat-free lid PCR.

[0025] According to an embodiment of the present invention, there is further provided a method for controlling an integrated digital PCR device, the method including a chip loading step, a droplet generation step, a temperature cycling step, a fluorescence detection step, and a chip discarding step. In the chip loading step, the integrated microdroplet 100 is placed in a chip loading module 200 . In the droplet generation step, the integrated micro-droplet chip 100 in the chip loading module 200 is transferred into the droplet generation module 300, and the sample in the sample addition chamber 12 of the integrated micro-droplet chip 100 is controlled to generate micro-droplets 4, which are then stored in the reaction chamber 11. In the temperature cycle step, the integrated micro-droplet chip 100 is turned upside down by 180°, and then a heating and cooling cycle is formed on the micro-droplet 4 in the micro-droplet in the reaction chamber 11 located at the bottom of the integrated micro-droplet chip 100. In the fluorescence detection step, after the amplification of the microdroplets in the reaction chamber 11 is completed, the microdroplets in the reaction chamber 11 are controlled to enter the fluorescence detection region 33, thereby completing the fluorescence detection. In the tip disposal step, the integrated micro-droplet chip after the completion of the fluorescence detection is transferred into a tip disposal module.

[0026] The working principle of the integrated digital PCR device of the present invention will be further explained below with reference to specific examples.

[0027] In the first step, 30 microliters of system (i.e., sample) is added to the sample addition chamber 12. The 30 microliter PCR system contains 10 microliters of Bio-Rad's ddPCR Supermix for Probes, 5 microliters of GJB2 gene upstream and downstream primer reagents, and 5 microliters of template containing 1 ng of genomic DNA. The entire integrated droplet chip 100 is shown in Figure 2. One chip contains eight parallel and independent droplet chip structures, each of which includes a sample addition chamber 12, an oil / liquid port 31, an air / liquid port 32, a droplet generation structure, and a reaction chamber 11. Next, the sealing cover 122 is sealed or glued to seal the sample addition chamber 12. The sample addition chamber 12 preferably has a filtration membrane or a small-diameter exhaust hole.

[0028] In the second step, the integrated microdroplet chip with the added sample is manually placed into the chip loading module 200 of the integrated digital PCR device, after which the door is closed and the device process is started.

[0029] In the third step, after the scheduling mechanism 700 scans and confirms the status of the integrated micro-droplet chip 100, the scheduling mechanism 700 grabs the integrated micro-droplet chip 100 from the chip loading module 200 and transfers it into the droplet generation module 300.

[0030] In the fourth step, as shown in Figures 3 and 4, microdroplets are prepared in the droplet generation module 300. First, Bio-Rad Generation Oil, the generation oil required for droplet generation, is supplied to the oil-liquid port 31. The generation oil contains a surfactant that can stabilize the droplets. Negative pressure of -200 mBar is applied to the gas-liquid port 32. As a result, a pressure difference is created between the gas-liquid port 32 and the sample addition chamber 12 and the oil-liquid port 31. The sample addition chamber 12 is connected to the second conduit 23 in the microdroplet generation structure, the oil-liquid port 31 is connected to the oil-liquid conduit 21 in the microdroplet generation structure, and the first conduit 22 and the gas-liquid port 32 are both connected to the reaction chamber 11. Here, the first conduit 22 is connected to the connection port 111, and the gas-liquid port 32 is connected to the gas-liquid conduit 112. The components of the microdroplet generation structure are shown in Figure 2. The oil-liquid conduit 21 has two branches. The two branches are located on either side of the second conduit 23 and the first conduit 22, respectively, and both are connected to the oil-liquid port 31. Driven by a pressure difference, the reaction system enters the second conduit 23, and the resulting oil enters the oil-liquid conduit 21. The two conduits merge in a cross-shaped configuration, where, due to the effects of fluid shear and surface tension, uniformly sized water-in-oil microdroplets are formed. The cross-shaped conduit is approximately 70 micrometers deep and 80 micrometers wide, with droplets of approximately 100 micrometers in size. The droplets enter the first conduit 22 and then enter the microdroplet observation area 34, where their flow rate slows, forming a dense droplet community that can be easily imaged and recorded by a camera. A diagram of the droplet generation process is shown in Figure 4. The generated droplets flow through the first conduit 22 and reach the connection port 111 of the reaction chamber 11. The bottom of the connection port 111 has a slope structure (i.e., the aforementioned trumpet mouth), and the bottom of the slope communicates with the first conduit 22. When the droplet generation process is completed, the pressure difference applied to the chip port is removed. At this time, the droplet should still be located below the gas-liquid conduit 112, as shown in FIG. 5.

[0031] In the fifth step, after the preparation of the microdroplets is completed, the scheduling mechanism 700 removes the chip from the droplet generation module 300, turns the chip upside down as shown in FIG. 6, and transfers the droplets from the connection port 111 to the reaction area (i.e., away from the connection port 111).

[0032] In step 6, the scheduling mechanism 700 places the inverted chip into the temperature cycling module 400. The structure of the reaction area should be designed for high heat transfer efficiency, such as a flat design with a deep depth and a thin thickness. The temperature cycling module 400 heats and cools the reaction area from both the left and right sides, ensuring not only a short heat transfer distance but also a large contact area, achieving efficient heat transfer. In this example, the temperature cycling process involves a 10-minute pre-denaturation at 95°C, followed by 40 temperature cycles, each cycle consisting of 5 seconds at 95°C, 15 seconds at 60°C, and a final 4°C hold. To reduce evaporation, 30 microliters of a low-density volatile inhibitor (e.g., the aforementioned diesel) can be pre-placed in the reaction chamber 11.

[0033] In the seventh step, after the amplification reaction is completed, the scheduling mechanism 700 transfers the chip from the temperature cycle module 400 to the fluorescence detection module 500 to perform microdroplet fluorescence detection. The fluorescence detection module 500 injects detection oil into the gas-liquid port 32 to continuously fill the reaction chamber 11. As shown in FIG. 7, during this process, the droplet level continues to rise and, guided by the slope of the connection port 111, enters the first conduit 22. As the droplets pass through the observation area in the first conduit 22, a camera can be used to perform bright-field imaging of the droplets to evaluate their status after the amplification reaction. Simultaneously, the fluorescence detection module 500 injects detection oil into the oil-liquid port 31. The detection oil passes through the oil-liquid conduit 21 and merges with the droplet train at the cross conduit, separating the closely spaced droplets into droplet trains with appropriate spacing. The droplet train passes through the fluorescence detection region 33 located in the second pipeline 23 in order, as shown in FIG.

[0034] The position corresponding to the fluorescence detection region 33 is the fluorescence detection focus of the fluorescence detection module 500. The fluorescence detection process is shown in FIG. 9. The excitation light source 91 of the fluorescence detection module 500 focuses excitation light 92, such as a laser or LED narrow-band light with wavelengths of 488 nm and 532 nm, onto the fluorescence detection region 33 via a spectral light path 94. As the droplets pass through the fluorescence detection region 33, the fluorescence 93 excited within the droplets is also received by the spectral light path 94 and distributed to and collected by the fluorescence detection unit 95, thereby obtaining fluorescence information for each droplet, as shown in FIG. 10. The fluorescence information of the droplets is used to determine a signal threshold to distinguish between negative and positive droplets, and a Poisson distribution model is used to calculate the copy number of the target molecule in the sample. Finally, the droplets that have undergone fluorescence detection enter the sample addition chamber 12. The sample addition chamber 12 is sealed by the sealing cover 122, so that it does not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a completely sealed digital PCR process.

[0035] In the eighth step, after the detection of all samples is completed, the scheduling mechanism 700 removes the chip from the fluorescence detection module 500 and transfers it into the chip disposal module 600 to complete the entire digital PCR detection process.

[0036] Those skilled in the art will readily understand that the above advantageous methods can be freely combined or superimposed as long as no contradiction occurs.

[0037] The above is only a preferred embodiment of the present invention, and the present invention is not limited thereto. As long as it falls within the spirit and principle of the present invention, all modifications, equivalent replacements, improvements, etc., made should be included within the protection scope of the present invention. It is obvious to those skilled in the art that the above is only a preferred embodiment of the present invention, and some improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications are also included within the protection scope of the invention. [Explanation of symbols]

[0038] 1, chip body; 11, reaction chamber; 111, connection port; 112, gas-liquid channel; 12, sample addition chamber; 121, opening chamber; 122, sealing cover; 21, oil-liquid channel; 22, first channel; 23, second channel; 31, oil-liquid port; 32, gas-liquid port; 33, fluorescence detection area; 34, microdroplet observation area; 4, microdroplet; 5, detection oil; 91, excitation light source; 92, excitation light; 93, fluorescence; 94, spectroscopic light path; 95, fluorescence detection unit; 100, integrated microdroplet chip; 200, chip loading module; 300, droplet generation module; 400, temperature cycle module; 500, fluorescence detection module; 600, chip disposal module; 700, scheduling mechanism; 800, main control module.

Claims

1. An integrated digital PCR device comprising: a chip loading module (200), a droplet generation module (300), a temperature cycle module (400), a fluorescence detection module (500), a chip disposal module (600), and a scheduling mechanism (700); The chip loading module (200) is used to place an integrated micro-droplet chip (100) to which a sample has been added, and the integrated micro-droplet chip (100) includes a chip body (1), and the chip body (1) is configured with a sample addition chamber (12), a reaction chamber (11), and a fluorescence detection region (33); The droplet generation module (300) is used to supply oil, liquid, or pressurized gas to the integrated micro-droplet chip (100), and during the micro-droplet generation process, micro-droplets (4) are generated from the sample in the sample addition chamber (12), and then transferred to and stored in the reaction chamber (11); the temperature cycle module (400) is used to generate a heating and cooling cycle for the microdroplets (4) in the reaction chamber (11) in order to amplify nucleic acids in the microdroplets (4) in the reaction chamber (11); The fluorescence detection module (500) can transfer the microdroplets (4) in the reaction chamber (11) to the fluorescence detection region (33) and then to the sample addition chamber (12) during the microdroplet detection process, and perform fluorescence detection on the microdroplets (4) located in the fluorescence detection region (33); The chip disposal module (600) is used to store the integrated micro-droplet chip (100) after detection; The scheduling mechanism (700) is used to transfer the integrated micro-droplet chip (100) between the chip loading module (200), droplet generation module (300), temperature cycle module (400), fluorescence detection module (500), and chip disposal module (600) under the control of a main control module (800), and the integrated micro-droplet chip (100) in the temperature cycle module (400) can be flipped upside down by 180° before being transferred into the fluorescence detection module (500).

2. The integrated micro-droplet chip (100) includes a chip body (1), in which a micro-droplet generating structure, an oil-liquid port (31), and a gas-liquid port (32) are configured, the gas-liquid port (32) is in communication with the reaction chamber (11), the sample addition chamber (12) is in communication with the micro-droplet generating structure, and the oil-liquid port (31) is in communication with the micro-droplet generating structure; When the integrated micro-droplet chip (100) is located in the droplet generating module (300), the droplet generating module (300) can form a first pressure difference between the sample addition chamber (12) and the gas-liquid port (32), and a second pressure difference between the oil-liquid port (31) and the gas-liquid port (32), and the first pressure difference and the second pressure difference drive the sample in the sample addition chamber (12) and the generated oil in the oil-liquid port (31), respectively, to enter the micro-droplet generating structure, and the generated micro-droplets (4) enter the reaction chamber (11) and are stored; 2. The integrated digital PCR device of claim 1, wherein when the integrated micro-droplet chip (100) is located within the fluorescence detection module (500), the droplet generating module (300) drives the detection pushing oil (5) to enter the reaction chamber (11) through the gas-liquid port (32) to cause the micro-droplets (4) in the reaction chamber (11) to flow out from the reaction chamber (11) to the micro-droplet generating structure; the droplet generating module (300) can also drive the detection separating oil to enter the micro-droplet generating structure through the oil-liquid port (31), and the detection separating oil separates the micro-droplets (4) that flow out from the reaction chamber (11) into the micro-droplet generating structure to form a line, and causes them to enter the fluorescence detection region (33).

3. The integrated digital PCR device of claim 2, characterized in that the microdroplet generation structure includes an oil-liquid conduit (21) and a communicating conduit, the oil-liquid conduit (21) and the communicating conduit intersect in a cross shape, the communicating conduit includes a first conduit (22) located on a first side of the cross intersection and communicating with the reaction chamber (11), and a second conduit (23) located on a second side of the cross intersection and communicating with the sample addition chamber (12), and the oil-liquid port (31) communicates with the oil-liquid conduit (21).

4. The integrated digital PCR device of claim 3, characterized in that the first side of the chip body (1) is located in a horizontal orientation, the reaction chamber (11) and the sample addition chamber (12) are located on the first side, and the connection port (111) between the reaction chamber (11) and the first side of the chip body (1) extends upward and forms a trumpet-shaped mouth that is small at the bottom and large at the top.

5. The integrated digital PCR device of claim 4, characterized in that a gas-liquid conduit (112) extending from bottom to top is configured within the reaction chamber (11), the lower opening of the gas-liquid conduit (112) communicates with the gas-liquid port (32), and the upper opening of the gas-liquid conduit (112) is higher than the upper opening of the connection port (111).

6. The integrated digital PCR device according to claim 5, characterized in that a microdroplet observation region (34) is located between the first conduit (22) and the connection port (111).

7. The integrated digital PCR device according to claim 3, wherein the fluorescence detection region (33) is located in the second channel (23).

8. The integrated digital PCR device of claim 1, characterized in that the sample addition chamber (12) includes an open chamber (121) and a sealed cover (122) sealed to the opening of the open chamber (121), and / or the sample addition chamber (12) is provided with a filtration membrane or an exhaust hole.

9. 2. The integrated digital PCR device of claim 1, wherein light oil is pre-placed in the reaction chamber (11) before the microdroplets (4) enter the reaction chamber (11).

10. A method for controlling an integrated digital PCR device, comprising: a chip loading step; a droplet generating step; a temperature cycling step; a fluorescence detecting step; and a chip discarding step; In the chip loading step, the integrated microdroplet chip (100) is placed in a chip loading module (200); In the droplet generation step, the integrated micro-droplet chip (100) in the chip loading module (200) is transferred into the droplet generation module (300), and the sample in the sample addition chamber (12) of the integrated micro-droplet chip (100) is controlled to generate micro-droplets (4), which are then stored in the reaction chamber (11); In the temperature cycle step, the integrated micro-droplet chip (100) is turned upside down by 180°, and then a heating and cooling cycle is formed on the micro-droplet (4) in the micro-droplet in the reaction chamber (11) located at the bottom of the integrated micro-droplet chip (100); In the fluorescence detection step, after the amplification of the nucleic acid in the microdroplets in the reaction chamber (11) is completed, the microdroplets in the reaction chamber (11) are controlled to enter a fluorescence detection region (33) to complete the fluorescence detection; The method for controlling an integrated digital PCR device is characterized in that in the chip disposal step, the integrated micro-droplet chip after the completion of the fluorescence detection is transferred into a chip disposal module.

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