Integrated digital PCR instrument and control method thereof

Abstract

The present invention provides an integrated digital PCR instrument and a control method thereof, wherein the integrated digital PCR instrument includes a chip loading module; a droplet generation module, which can transfer and store the sample in the sample loading chamber into a reaction chamber after generating microdroplets during the microdroplet generation process; a temperature cycle module, so that the microdroplets in the reaction chamber can be amplified; a fluorescence detection module, which can transfer the microdroplets in the reaction chamber to the fluorescence detection area and then to the sample loading chamber during the microdroplet detection process, and complete the microdroplet fluorescence detection in the process; a chip discarding module; a scheduling mechanism, which 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 discarding module under the control of the main control module, and can turn the integrated microdroplet chip in the temperature cycle module upside down by 180° and then transfer it to the fluorescence detection module. The present invention has a high degree of automation and integration, can improve the efficiency of detection and analysis operations, and reduce labor costs.

Description

Technical Field

[0001] The present invention belongs to the technical field of digital PCR analyzers, and in particular relates to an integrated digital PCR analyzer and a control method thereof. Background Art

[0002] Digital PCR is the latest quantitative technology. It is an absolute quantitative method based on the single-molecule PCR method for counting nucleic acid quantification. It mainly uses the microfluidics or droplet method in the current hot research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactor or droplet of the biochip. The number of nucleic acid templates in each reactor is less than or equal to 1. In this way, after the PCR cycle, the reactor with a nucleic acid molecule template will give a fluorescent signal, and the reactor without a template will not have a fluorescent signal. According to the relative proportion and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated. The traditional digital PCR analyzer has a single function and requires manual operation of multiple instruments to complete an amplification and detection work. With the development of society, the medical industry and scientific research fields have put forward higher requirements for high throughput, automation, high speed, and integration of 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 instrument and a control method thereof, so as to overcome the shortcomings of the digital PCR instrument in the prior art, which has a single function, requires manual operation of multiple instruments to cooperate to complete an amplification and detection work, and has low detection efficiency.

[0004] In order to solve the above problems, the present invention provides an integrated digital PCR instrument, including: a chip loading module, which is used to place an integrated micro-droplet chip after sample loading, wherein the integrated micro-droplet chip includes a chip body, and the chip body is configured with a sample loading chamber, a reaction chamber, and a fluorescence detection area; a droplet generation module, which is used to supply oil and gas to the integrated micro-droplet chip, and can generate micro-droplets from the sample in the sample loading chamber during the micro-droplet generation process and then transfer and store them in the reaction chamber; a temperature cycle module, which is used to form a heating and cooling cycle for the micro-droplets in the reaction chamber to amplify the micro-droplets in the reaction chamber; and a fluorescence detection module. The invention relates to a block, which can transfer the micro-droplets in the reaction chamber to the fluorescence detection area and then to the sample loading chamber during the micro-droplet detection process, and perform fluorescence detection on the micro-droplets in the fluorescence detection area; a chip discarding module, which is used to store the integrated micro-droplet chip after detection; a scheduling mechanism, which is used to transfer the integrated micro-droplet chip between the chip loading module, the droplet generation module, the temperature circulation module, the fluorescence detection module and the chip discarding module under the control of the main control module, and can flip the integrated micro-droplet chip in the temperature circulation module upside down by 180° before transferring it to the fluorescence detection module.

[0005] In some embodiments, the integrated micro-droplet chip includes a chip body, and a micro-droplet generation structure, an oil-liquid interface, and a gas-liquid interface are constructed in the chip body. The gas-liquid interface is connected to the reaction chamber, the sample loading chamber is connected to the micro-droplet generation structure, and the oil-liquid interface is connected to the micro-droplet generation structure; when the integrated micro-droplet chip is in the droplet generation module, the droplet generation module can form a first pressure difference between the sample loading chamber and the gas-liquid interface, and a second pressure difference between the oil-liquid interface and the gas-liquid interface, and the first pressure difference and the second pressure difference respectively drive the sample in the sample loading chamber to move forward and backward. The generated oil from the oil-liquid interface enters the micro-droplet generating structure, and the generated micro-droplets enter and are stored in the reaction chamber; when the integrated micro-droplet chip is in the fluorescence detection module, the fluorescence detection module can drive the detection pushing oil from the gas-liquid interface to enter the reaction chamber, so that the micro-droplets in the reaction chamber flow out of the reaction chamber to the micro-droplet generating structure, and the droplet generating module can also drive the detection separation oil from the oil-liquid interface to enter the micro-droplet generating structure, and the detection separation oil will flow out of the reaction chamber to the micro-droplet separation in the micro-droplet generating structure to form a queue and enter the fluorescence detection area.

[0006] In some embodiments, the micro-droplet generation structure includes an oil pipeline and a connecting pipeline, the oil pipeline and the connecting pipeline form a cross, the connecting pipeline includes a first pipeline located on the first side of the cross and connected to the reaction chamber and a second pipeline located on the second side of the cross and connected to the sample addition chamber, and the oil interface is connected to the oil pipeline.

[0007] In some embodiments, with the first side surface of the chip body being in a horizontal position as a reference, the reaction chamber and the sample loading chamber are located on the first side surface, and the connection interface between the reaction chamber and the first side surface of the chip body extends upward and forms a trumpet mouth that is smaller at the bottom and larger at the top.

[0008] In some embodiments, a gas-liquid pipeline extending from bottom to top is further constructed in the reaction chamber, the lower end of the gas-liquid pipeline is connected to the gas-liquid interface, and the upper end of the gas-liquid pipeline is higher than the upper end of the connection interface.

[0009] In some embodiments, a micro-droplet observation area is provided between the first pipe and the connection interface.

[0010] In some embodiments, the fluorescence detection zone is located on the second channel.

[0011] In some embodiments, the sample loading chamber includes an open chamber and a sealing cover sealed to an opening of the open chamber; and / or, the sample loading chamber is provided with a filter membrane or an exhaust hole.

[0012] In some embodiments, light oil is pre-placed in the reaction chamber before the micro-droplets enter the reaction chamber.

[0013] The present invention also provides a control method for an integrated digital PCR instrument, comprising the following steps:

[0014] A chip loading step, placing the integrated micro-droplet chip into a chip loading module;

[0015] A droplet generation step, transferring the integrated micro-droplet chip in the chip loading module to the droplet generation module, and controlling the sample in the sample loading chamber of the integrated micro-droplet chip to generate micro-droplets and store them in the reaction chamber;

[0016] a temperature cycle step, wherein after the integrated micro-droplet chip is turned upside down by 180°, a heating and cooling cycle is formed on the micro-droplets in the micro-droplets in the reaction chamber at the bottom of the integrated micro-droplet chip;

[0017] a fluorescence detection step, after the micro-droplets in the reaction chamber are amplified, controlling the micro-droplets in the reaction chamber to enter a fluorescence detection area and completing fluorescence detection;

[0018] In the chip disposal step, the integrated micro-droplet chip after the fluorescence detection is transferred to the chip disposal module.

[0019] The present invention provides an integrated digital PCR instrument and a control method thereof. Under the control of the main control module, the scheduling mechanism can coordinate the operation of the integrated micro-droplet chip among various functional modules, 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, which can improve the efficiency of detection and analysis operations and reduce labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the structure of an integrated digital PCR instrument according to an embodiment of the present invention;

[0021] Figure 2 for Figure 1 Schematic diagram of the three-dimensional structure of the integrated micro-droplet chip;

[0022] Figure 3 for Figure 2 Schematic diagram of the structure of the micro-droplet generation structure;

[0023] Figure 4 Schematic diagram of the microdroplet generation process;

[0024] Figure 5 Schematic diagram of microdroplets being stored in a reaction chamber after being generated;

[0025] Figure 6 for Figure 1 The integrated micro-droplet chip in the figure is turned upside down 180° and then inside the reaction chamber;

[0026] Figure 7 for Figure 6 Schematic diagram of the state after oil is introduced into the reaction chamber (the arrows in the figure show the direction of micro-droplets and oil flow);

[0027] Figure 8 The schematic diagram shows the state of the micro-droplets after being forced out of the reaction chamber;

[0028] Fig. 9 for Figure 1 Schematic diagram of the working optical path principle of the fluorescence detection module;

[0029] Fig.10 This is the detection result of the digital PCR instrument according to the embodiment of the present invention.

[0030] The reference numerals are as follows:

[0031] 1. Chip body; 11. Reaction chamber; 111. Connection interface; 112. Gas-liquid pipeline; 12. Sample adding chamber; 121. Opening chamber; 122. Sealing cover; 21. Oil-liquid pipeline; 22. First pipeline; 23. Second pipeline; 31. Oil-liquid interface; 32. Gas-liquid interface; 33. Fluorescence detection area; 34. Micro-droplet observation area; 4. Micro-droplets; 5. Detection of driving oil; 91. Excitation light source; 92. Excitation light; 93. Fluorescence; 94. Spectroscopic optical path; 95. Fluorescence detection unit; 100. Integrated micro-droplet chip; 200. Chip loading module; 300. Droplet generation module; 400. Temperature cycle module; 500. Fluorescence detection module; 600. Chip discarding module; 700. Scheduling mechanism; 800. Main control module. DETAILED DESCRIPTION

[0032] See also Figures 1 to 10As shown, according to an embodiment of the present invention, an integrated digital PCR instrument is provided, including: a chip loading module 200, for placing an integrated micro-droplet chip 100 after sample loading, wherein the integrated micro-droplet chip 100 includes a chip body 1, and the chip body 1 is configured with a sample loading chamber 12, a reaction chamber 11, and a fluorescence detection area 33; a droplet generation module 300, for supplying oil and gas (i.e., oil or pressurized gas) to the integrated micro-droplet chip 100, and can generate micro-droplets 4 from the sample in the sample loading chamber 12 during the micro-droplet generation process and then transfer and store them in the reaction chamber 11; a temperature cycle module 400, for forming a heating and cooling cycle for the micro-droplets 4 in the reaction chamber 11, so that the micro-droplets 4 in the reaction chamber 11 can be amplified; and a fluorescence detection module 500. 00, which can transfer the micro-droplets 4 in the reaction chamber 11 to the fluorescence detection area 33 and then to the sample addition chamber 12 during the micro-droplet detection process, and perform fluorescence detection on the micro-droplets 4 in the fluorescence detection area 33; a chip discarding module 600, which is used to store the integrated micro-droplet chip 100 after detection; a scheduling mechanism 700, which is used to transfer the integrated micro-droplet chip 100 between the chip loading module 200, the droplet generation module 300, the temperature circulation module 400, the fluorescence detection module 500, and the chip discarding module 600 under the control of the main control module 800, and can flip the integrated micro-droplet chip 100 in the temperature circulation module 400 upside down 180° and then transfer it 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 operation of the integrated micro-droplet chip 100 between various functional modules, realizing the integrated operation of chip loading, droplet generation, PCR amplification, fluorescence detection and analysis until chip disposal, with a high degree of automation and integration, which can improve the efficiency of detection and analysis operations and reduce labor costs. More importantly, the scheduling mechanism 700 in the present invention can be configured to flip the integrated micro-droplet chip 100 up and down 180°, so that the operation process of droplet generation, PCR amplification and fluorescence detection and analysis can be integrated on the same chip, that is, the integrated micro-droplet chip 100, and the degree of integration and automation can be further improved.

[0033] In some embodiments, the integrated micro-droplet chip 100 includes a chip body 1, in which a micro-droplet generation structure, an oil-liquid interface 31, and a gas-liquid interface 32 are constructed, the gas-liquid interface 32 is connected to the reaction chamber 11, the sample addition chamber 12 is connected to the micro-droplet generation structure, and the oil-liquid interface 31 is connected to the micro-droplet generation structure; when the integrated micro-droplet chip 100 is in the droplet generation module 300, the droplet generation module 300 can form a first pressure difference between the sample addition chamber 12 and the gas-liquid interface 32 (that is, the gas path of the droplet generation module 300 is connected to the sample addition chamber 12 and the gas-liquid interface 32), and form a second pressure difference between the oil-liquid interface 31 and the gas-liquid interface 32 (that is, the liquid path of the droplet generation module 300 is connected to the oil-liquid interface 31 and the gas-liquid interface 32). The first pressure difference and the second pressure difference respectively drive the sample in the sample addition chamber 12 and the generated oil of the oil-liquid interface 31 to enter the micro-droplet generation structure, and the generated micro-droplets 4 enter and are stored in the reaction chamber 11; when the integrated micro-droplet chip 100 is in the fluorescence detection module 500, the droplet generation module 300 can drive the detection pushing oil 5 from the gas-liquid interface 32 to enter the reaction chamber 11, so that the micro-droplets 4 in the reaction chamber 11 flow out of the reaction chamber 11 to the micro-droplet generation structure, and the droplet generation module 300 can also drive the detection separation oil from the oil-liquid interface 31 to enter the micro-droplet generation structure, and the detection separation oil will separate the micro-droplets 4 flowing out of the reaction chamber 11 to the micro-droplet generation structure into a queue and enter the fluorescence detection area 33. By time-division multiplexing of the micro-droplet generation structure (with the flipping of the integrated micro-droplet chip 100 as the time-division boundary), droplet generation, amplification and detection are integrated into one chip, realizing a fully integrated and fully closed digital PCR process. This not only inherits the advantages of uniform droplet size, unrestricted droplet number, and high fluorescence detection signal-to-noise ratio, but also overcomes the difficulties of the original chip structure being complex, generation and detection being completed in different chips, low integration, and difficulty in automation. This is an important technological breakthrough in the field of digital PCR.

[0034] As a specific implementation of the micro-droplet generation structure, the micro-droplet generation structure includes an oil pipeline 21 and a connecting pipeline, the oil pipeline 21 and the connecting pipeline form a cross, the connecting pipeline includes a first pipeline 22 located on the first side of the cross intersection and connected to the reaction chamber 11 and a second pipeline 23 located on the second side of the cross intersection and connected to the sample addition chamber 12, and the oil interface 31 is connected to the oil pipeline 21.

[0035] In some embodiments, see Figure 2As shown, with the first side of the chip body 1 in a horizontal position as a reference, the reaction chamber 11 and the sample loading chamber 12 are on the first side, and the connection interface 111 between the reaction chamber 11 and the first side of the chip body 1 extends upward, and forms a trumpet mouth with a small bottom and a large top. The connection interface 111 forming a trumpet mouth can facilitate the micro-droplets 4 to enter the reaction chamber 11 from the first pipe 22, and also facilitate the micro-droplets 4 to enter the first pipe 22 from the reaction chamber 11, thereby preventing the micro-droplets 4 from being detained. It should be noted that at this time, the reaction chamber 11 and the sample loading chamber 12 are both on the first side (specifically the top surface) of the chip body 1, and the micro-droplets 4 entering the reaction chamber 11 are all gathered at the connection interface 111. When performing PCR amplification, the chip body 1 needs to be inverted as a whole, that is, flipped 180°, so that the micro-droplets 4 can be in the reaction area of ​​the reaction chamber 11.

[0036] In some embodiments, a gas-liquid pipeline 112 extending from bottom to top is further constructed in the reaction chamber 11, and the lower end of the gas-liquid pipeline 112 is connected to the gas-liquid interface 32, and the upper end of the gas-liquid pipeline 112 is higher than the upper end of the connection interface 111. This can prevent the micro-droplets 4 generated by the micro-droplet generation structure from further flowing out of the gas-liquid pipeline 112 after entering the reaction chamber 11 when the reaction chamber 11 is under negative pressure.

[0037] In some embodiments, a micro-droplet observation area 34 is provided between the first pipe 22 and the connection interface 111, and the flow area of ​​the micro-droplet observation area 34 is much larger than the flow area of ​​the first pipe 22, that is, the micro-droplet observation area 34 is an area enlarged on the first pipe 22 (the width becomes larger), so that the flow rate of the micro-droplets 4 entering the area is reduced, which can facilitate external camera imaging, record the droplet morphology, and determine whether the state of the droplet generation process is normal.

[0038] In one embodiment, the fluorescence detection area 33 is located on the second pipe 23. On the second pipe 23, the micro-droplets 4 flowing out of the reaction chamber 11 can be separated into droplet queues with appropriate spacing under the action of the detection oil in the oil pipe 21 when passing through the cross point, thereby completing the fluorescence detection under the action of the external system.

[0039] As a specific implementation, the sample loading chamber 12 includes an open chamber 121 and a sealing cover 122 sealed to the opening of the open chamber 121, so that the operator can add the sample into the sample loading chamber 12. Furthermore, the sample loading chamber 12 is provided with a filter membrane or an exhaust hole, and when the sample loading chamber 12 becomes a waste liquid pool (that is, when the droplet chip is turned upside down), a certain amount of air is exhausted to prevent pressure accumulation in the sample loading chamber 12.

[0040] In some embodiments, before the micro-droplets 4 enter the reaction chamber 11, light oil (i.e., oil with a lower density) is pre-placed in the reaction chamber 11 to ensure that the light oil can always be on the top of the micro-droplets 4 in the reaction chamber 11, thereby solving the problem of micro-droplet evaporation during amplification and realizing heat-cover-free PCR.

[0041] According to an embodiment of the present invention, a control method of an integrated digital PCR instrument is also provided, comprising the following steps:

[0042] A chip loading step, placing the integrated micro-droplet chip 100 into the chip loading module 200;

[0043] Droplet generation step, transferring the integrated micro-droplet chip 100 in the chip loading module 200 to the droplet generation module 300, and controlling the sample in the sample loading chamber 12 of the integrated micro-droplet chip 100 to generate micro-droplets 4 and then store them in the reaction chamber 11;

[0044] A temperature cycle step, after the integrated micro-droplet chip 100 is turned upside down by 180°, a heating and cooling cycle is formed for the micro-droplets 4 in the micro-droplets in the reaction chamber 11 at the bottom of the integrated micro-droplet chip 100;

[0045] Fluorescence detection step, after the micro-droplets in the reaction chamber 11 are amplified, the micro-droplets in the reaction chamber 11 are controlled to enter the fluorescence detection area 33 and complete the fluorescence detection;

[0046] In the chip disposal step, the integrated micro-droplet chip after the fluorescence detection is transferred to the chip disposal module.

[0047] The working principle of the integrated digital PCR instrument of the present invention is further introduced below in conjunction with a specific embodiment.

[0048] In the first step, 30 microliters of the system (i.e., the sample) is added to the sample chamber 12. The 30 microliters of the PCR system includes 10 microliters of Bio-Rad's ddPCR Supermix for Probes, 5 microliters of GJB2 gene upstream and downstream primer reagents, and 5 microliters of a template containing 1 ng of genomic DNA. The integrated micro-droplet chip 100 is as follows: Figure 2 As shown, a chip contains 8 parallel independent droplet chip structures, each structure includes a sample loading chamber 12, an oil-liquid interface 31, a gas-liquid interface 32, a micro-droplet generation structure, and a reaction chamber 11. Then, the sealing cover 122 is tightly closed or glued to seal the sample loading chamber 12. It is best to have a filter membrane or a small-caliber exhaust hole in the sample loading chamber 12.

[0049] In the second step, the integrated micro-droplet chip with the sample added is manually placed in the chip loading module 200 in the integrated digital PCR instrument, and then the hatch is closed to start the instrument process.

[0050] 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 to the droplet generation module 300 .

[0051] In the fourth step, micro-droplets are prepared in the droplet generation module 300, such as Figure 3 and Figure 4 As shown. First, the generation oil Bio-Rad Generation Oil required for droplet generation is provided to the oil-liquid interface 31, and the generation oil contains a surfactant that can stabilize the droplets; a negative pressure of -200 mBar is provided to the gas-liquid interface 32, so that a pressure difference is formed between the sample addition chamber 12 and the oil-liquid interface 31. Among them, the sample addition chamber 12 is connected to the second pipe 23 in the micro-droplet generation structure, the oil-liquid interface 31 is connected to the oil-liquid pipe 21 in the micro-droplet generation structure, and the first pipe 22 and the gas-liquid interface 32 are both connected to the reaction chamber 11, wherein the first pipe 22 is connected to the connection interface 111, and the gas-liquid interface 32 is connected to the gas-liquid pipe 112. The various parts of the micro-droplet generation structure are shown in the figure. Figure 2 As shown, the oil pipeline 21 has two branches, which are respectively located on both sides of the second pipeline 23 and the first pipeline 22, and are both connected to the oil interface 31. Driven by the pressure difference, the reaction system enters the second pipeline 23, and the generated oil enters the oil pipeline 21 and intersects at the cross structure. Under the action of fluid shear force and surface tension, oil-in-water micro-droplets of uniform size are formed. The pipeline depth at the cross is about 70 microns, the width is 80 microns, and the droplet size is about 100 microns. The micro-droplets enter the first pipeline 22, and the flow rate is reduced after entering the micro-droplet observation area 34, forming a dense droplet community, which is convenient for camera imaging and recording. The schematic diagram of the droplet generation process is shown in the figure. Figure 4 The generated droplets flow through the first pipe 22 and reach the connection interface 111 of the reaction chamber 11. The bottom of the connection interface 111 has a slope structure (i.e., the aforementioned bell mouth), and the bottom of the slope is connected to the first pipe 22. When the droplet generation process is completed, the pressure difference applied to the chip interface is removed. At this time, the droplets should still be located below the gas-liquid pipe 112, as shown in FIG. Figure 5 shown.

[0052] In the fifth step, after the micro-droplet preparation is completed, the scheduling mechanism 700 takes the chip out of the droplet generation module 300 and turns the chip upside down to transfer the droplets from the connection interface 111 to the reaction area (ie, away from the connection interface 111). Figure 6 shown.

[0053] In the sixth step, the scheduling mechanism 700 places the flipped chip in the temperature cycle module 400 as described. The structure of the reaction zone should be designed with high heat transfer efficiency, such as a flat design with high depth and thin thickness. The temperature cycle module 400 heats and cools the reaction zone from the left and right sides, which not only ensures a short distance for temperature conduction, but also ensures a large contact area, thereby achieving efficient heat transfer. In this embodiment, the temperature cycle process is to first perform a pre-denaturation at 95°C for 10 minutes, followed by 40 temperature cycles, with 95°C for 5 seconds, 60°C for 15 seconds, and finally 4°C insulation in each cycle. In order to reduce evaporation, 30 microliters of low-density anti-volatile reagent (such as the light oil) can be placed in the reaction chamber 11 in advance.

[0054] Step 7: 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 for micro-droplet fluorescence detection. The fluorescence detection module 500 injects detection oil into the gas-liquid interface 32 to continuously fill the reaction chamber 11. In this process, the liquid level of the droplet rises continuously, and then is guided by the slope of the connection interface 111 and enters the first pipeline 22, as shown in FIG. Figure 7 As shown. When the droplets pass through the observation area in the first pipe 22, the camera can also be used to perform bright field imaging of the droplets, so as to evaluate the state of the droplets after the amplification reaction. At the same time, the fluorescence detection module 500 injects detection oil into the oil interface 31. The detection oil passes through the oil pipe 21 and merges with the droplet queue at the cross pipe, and separates the closely arranged droplets into droplet queues with appropriate spacing. The droplet queues pass through the fluorescence detection area 33 located in the second pipe 23 in turn, as shown. Figure 8 shown.

[0055] The position corresponding to the fluorescence detection area 33 is the fluorescence detection focus of the fluorescence detection module 500. The fluorescence detection process is as follows Fig. 9 As shown, the excitation light source 91 of the fluorescence detection module 500 focuses the excitation light 92, such as laser or LED narrow-band light with wavelengths of 488nm and 532nm, to the fluorescence detection area 33 through the split light path 94. When the droplets pass through the fluorescence detection area 33 in sequence, the fluorescence 93 excited in the droplets will also be received by the split light path 94 and distributed to the fluorescence detection unit 95 for collection, so as to obtain the fluorescence information of each droplet, such as Fig.10 As shown. The signal threshold is defined by using the fluorescence information of the droplets, the positive and negative properties of the droplets are distinguished, and the Poisson distribution model is used to calculate the number of copies of the target molecule in the sample. Finally, the droplets that have completed the fluorescence detection enter the sample loading chamber 12. Since the sample loading chamber 12 has been sealed by the sealing cover 122, it will not come into contact with the environment outside the chip, eliminating the possibility of aerosol contamination and realizing a fully closed digital PCR process.

[0056] In the eighth step, after completing the detection of all samples, the scheduling mechanism 700 takes out the chip from the fluorescence detection module 500 and transfers it to the chip discarding module 600, thus completing the entire digital PCR detection process.

[0057] It is easy for those skilled in the art to understand that the above-mentioned advantageous methods can be freely combined and superimposed without conflict.

[0058] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present invention, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. An integrated digital PCR instrument, characterized in that: include: A chip loading module (200) is used to place an integrated micro-droplet chip (100) on which sample loading has been completed, wherein the integrated micro-droplet chip (100) comprises a chip body (1), and the chip body (1) is provided with a sample loading chamber (12), a reaction chamber (11), and a fluorescence detection area (33); A droplet generation module (300) is used to supply oil and gas to the integrated micro-droplet chip (100), and is capable of generating micro-droplets (4) from the sample in the sample addition chamber (12) during the micro-droplet generation process and then transferring and storing the micro-droplets in the reaction chamber (11); A temperature cycle module (400) is used to form a heating and cooling cycle for the micro-droplets (4) in the reaction chamber (11), so as to achieve amplification of the micro-droplets (4) in the reaction chamber (11); A fluorescence detection module (500) capable of transferring the micro-droplets (4) in the reaction chamber (11) to the fluorescence detection area (33) and then to the sample addition chamber (12) during the micro-droplet detection process, and performing fluorescence detection on the micro-droplets (4) in the fluorescence detection area (33); A chip discarding module (600), used for storing the integrated micro-droplet chip (100) after detection; A scheduling mechanism (700) is used to transfer the integrated micro-droplet 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 discarding module (600) under the control of the main control module (800), and is capable of turning the integrated micro-droplet chip (100) in the temperature cycle module (400) upside down by 180° before transferring it to the fluorescence detection module (500); The integrated micro-droplet chip (100) comprises a chip body (1), wherein a micro-droplet generation structure, an oil-liquid interface (31), and a gas-liquid interface (32) are constructed in the chip body (1), wherein the gas-liquid interface (32) is communicated with the reaction chamber (11), the sample addition chamber (12) is communicated with the micro-droplet generation structure, and the oil-liquid interface (31) is communicated with the micro-droplet generation structure; When the integrated micro-droplet chip (100) is in the droplet generation module (300), the droplet generation module (300) is capable of forming a first pressure difference between the sample addition chamber (12) and the gas-liquid interface (32), and a second pressure difference between the oil-liquid interface (31) and the gas-liquid interface (32), the first pressure difference and the second pressure difference respectively driving the sample in the sample addition chamber (12) and the generated oil in the oil-liquid interface (31) to enter the micro-droplet generation structure, and the generated micro-droplets (4) enter and are stored in the reaction chamber (11); The micro-droplet generation structure comprises an oil pipeline (21) and a connecting pipeline, the oil pipeline (21) and the connecting pipeline intersect each other in a cross, the connecting pipeline comprises a first pipeline (22) located on a first side of the cross intersection and connected to the reaction chamber (11), and a second pipeline (23) located on a second side of the cross intersection and connected to the sample addition chamber (12), and the oil interface (31) is connected to the oil pipeline (21); The sample adding chamber (12) comprises an open chamber (121).

2. The integrated digital PCR instrument according to claim 1, characterized in that: When the integrated micro-droplet chip (100) is in the fluorescence detection module (500), the droplet generation module (300) is capable of driving the detection pushing oil (5) to enter the reaction chamber (11) from the gas-liquid interface (32), so that the micro-droplets (4) in the reaction chamber (11) flow out of the reaction chamber (11) to the micro-droplet generation structure. The droplet generation module (300) is also capable of driving the detection separation oil to enter the micro-droplet generation structure from the oil-liquid interface (31). The detection separation oil separates the micro-droplets (4) flowing out of the reaction chamber (11) to the micro-droplet generation structure into a queue and enters the fluorescence detection area (33).

3. The integrated digital PCR instrument according to claim 2, characterized in that: With the first side surface of the chip body (1) being in a horizontal position as a reference, the reaction chamber (11) and the sample addition chamber (12) are located on the first side surface, and a connection interface (111) between the reaction chamber (11) and the first side surface of the chip body (1) extends upwards and forms a trumpet mouth that is smaller at the bottom and larger at the top.

4. The integrated digital PCR instrument according to claim 3, characterized in that: A gas-liquid pipeline (112) extending from bottom to top is also constructed in the reaction chamber (11), the lower end of the gas-liquid pipeline (112) is in communication with the gas-liquid interface (32), and the upper end of the gas-liquid pipeline (112) is higher than the upper end of the connection interface (111).

5. The integrated digital PCR instrument according to claim 4, characterized in that: A micro-droplet observation area (34) is provided between the first pipeline (22) and the connection interface (111).

6. The integrated digital PCR instrument according to claim 1, characterized in that: The fluorescence detection area (33) is located on the second pipe (23).

7. The integrated digital PCR instrument according to claim 1, characterized in that: The sample adding chamber (12) comprises a sealing cover (122) which is sealed to the opening of the opening chamber (121); and / or the sample adding chamber (12) is provided with a filter membrane or an exhaust hole.

8. The integrated digital PCR instrument according to claim 1, characterized in that: Before the micro-droplets (4) enter the reaction chamber (11), light oil is pre-placed in the reaction chamber (11).

9. A control method for the integrated digital PCR instrument according to any one of claims 1 to 8, characterized in that: The steps include: A chip loading step, placing the integrated micro-droplet chip (100) into a chip loading module (200); A droplet generation step, transferring the integrated micro-droplet chip (100) in the chip loading module (200) to the droplet generation module (300), and controlling the sample in the sample loading chamber (12) of the integrated micro-droplet chip (100) to generate micro-droplets (4) which are then stored in the reaction chamber (11); a temperature cycle step, wherein after the integrated micro-droplet chip (100) is turned upside down by 180 degrees, a heating and cooling cycle is formed on the micro-droplets (4) in the micro-droplets in the reaction chamber (11) at the bottom of the integrated micro-droplet chip (100); A fluorescence detection step, after the micro-droplets in the reaction chamber (11) have been amplified, controlling the micro-droplets in the reaction chamber (11) to enter the fluorescence detection area (33) and complete the fluorescence detection; In the chip disposal step, the integrated micro-droplet chip after the fluorescence detection is transferred to the chip disposal module.

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