An integrated digital PCR system and methods of use thereof

Abstract

This invention discloses an integrated digital PCR system and its usage method. The system comprises a high-density microcavity array digital PCR chip with an integrated cavity, an optical detection module, a hot water circulation temperature control module, a fluid control module, and a central control module. When applied to nucleic acid sample detection, the digital PCR chip is first fixed on the stage of the optical detection module, and connecting tubes for the sample tubes and hot water tank are installed at each chip interface. Then, the central control module is activated, and relevant parameters of the control program are set. The control program then controls the fluid control module, hot water circulation temperature control module, and optical detection module to sequentially perform operations such as sample filling, sample discretization, hot water circulation amplification, chip scanning and recording, and data analysis. Based on the combination of the microcavity array and integrated cavity structure of the digital PCR chip, this system allows the entire digital PCR detection process to be completed on a single instrument, improving detection efficiency and result reliability.

Description

[0001] Divisional application This application is a divisional application of Chinese invention patent application No. 202010863328.1, filed on August 25, 2020, entitled "An integrated digital PCR system and its usage method". Technical Field

[0002] This invention belongs to the field of microfluidic chip analysis technology, specifically relating to an integrated digital PCR system and its usage method. Background Technology

[0003] Digital PCR (dPCR) is the third-generation quantitative nucleic acid detection technology following quantitative real-time PCR (qPCR). It is an absolute nucleic acid quantification technique with the following significant advantages compared to traditional PCR: it can achieve absolute nucleic acid quantification without relying on a standard curve; it has high tolerance to inhibitors; it can analyze complex mixtures; and it can detect trace amounts of nucleic acid samples, detect rare mutations in complex backgrounds, and identify subtle differences in expression levels. Its working principle involves breaking down the sample into thousands or even millions of droplets (each droplet acting as an independent PCR reaction unit) until each droplet contains one or zero target molecules. When all droplets undergo simultaneous PCR amplification, droplets containing target molecules will generate a positive signal due to target amplification, while droplets without target molecules will not show a signal. Finally, by statistically counting the positive signals and calculating using the Poisson distribution formula, the original concentration of the sample or the content of target molecules can be deduced. This technology is particularly suitable for applications such as copy number variation, mutation detection, and relative gene expression studies.

[0004] Due to its significant technological advantages, digital PCR technology has attracted widespread attention from research institutions and enterprises both domestically and internationally in recent years, leading to the development of numerous digital PCR instrument systems. Currently, typical commercially available digital PCR systems include Fluidigm's BioMark™ system, Life Technologies' QuantStudio™ system, Bio-Rad's QX100™ system, and RainDance Technologies' RainDrop™ system. These systems all require multiple instruments to complete the entire digital PCR detection process. For example, the core component of the BioMark™ system is a PDMS chip integrating a series of complex microchannels and microvalves. This system requires two instruments to complete the digital PCR detection process. First, an Integrated Fluid Pathway Controller (IFC Controller) is used in conjunction with the numerous microvalves integrated on the digital PCR chip to decompose the sample filled with the microchannel network into a large number of independent reaction units. Then, the digital PCR chip with the sample discretized is transferred to a Biomark Reader for thermal cycling amplification and PCR reaction result reading and analysis. The core component of the QuantStudio™ system is a silicon-based chip with numerous through-holes. This system requires three instruments to complete the digital PCR detection process. First, a DigitalPCR Chip Loader is used, which, based on the difference in wettability between hydrophilic through-holes and hydrophobic surfaces, uses surface tension to distribute the liquid sample into all through-holes. Then, the chip with the sample distributed is transferred to a ProFlex™ 2x Flat PCR system. The system performs thermal cycling amplification on a microfluidic chip, and finally transfers the amplified chip to a "chip analyzer" (QuantStudio™ 3D Digital PCR Instrument) for PCR reaction result reading and analysis. Unlike the BioMark™ and QuantStudio™ systems, the QX100™ and RainDrop™ systems utilize droplet-type microfluidic chips to break down samples into thousands of nano-scale droplets. After PCR amplification, each droplet is detected individually using a flow cytometer-like detector to obtain the results. The entire system consists of at least three instruments: a microdroplet generator, a thermal cycler, and a microdroplet analyzer. In recent years, many researchers have used these systems to conduct extensive research in areas such as personalized medicine for tumors and gene copy number variation. However, there are still some technical and cost limitations for the large-scale application of these systems.For example, all of the aforementioned systems require additional instruments for sample introduction or discretization. Furthermore, the discretized digital PCR chip requires manual transfer between instruments, leading to a relatively complex process, longer detection time, and increased risks of human error and sample contamination. Additionally, the digital PCR chips used in the BioMark™ and QuantStudio™ systems have low decomposition densities, resulting in relatively insufficient detection accuracy, limited measurable dynamic range, and high chip manufacturing costs. The QX100™ and RainDrop™ systems utilize droplet-type microfluidic chips for sample decomposition. To maintain the dispersion of the decomposition droplets, surfactants are required. These additives may affect or interfere with certain experimental systems, thus impacting the reliability of the results.

[0005] Therefore, there is an urgent need to develop new, integrated digital PCR systems to achieve fully automated, "one-stop" operation of the entire digital PCR process, including sample introduction, sample discretization, thermal cycling amplification, and result reading and analysis. This would simplify the digital PCR testing process, reduce operating costs, improve the reliability of test results, and further enhance the accuracy of testing, transforming digital PCR technology from a "high-end" and "professional" laboratory technique into a routine tool for ordinary laboratories and clinical testing. Summary of the Invention

[0006] In view of the above-mentioned shortcomings in the prior art, the present invention provides an integrated digital PCR system and its usage method, which solves the problems of complex usage, high operating cost, low detection efficiency and insufficient reliability of the existing digital PCR system.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows: an integrated digital PCR system, comprising a digital PCR chip, an optical detection module, a fluid control module, a hot water circulation temperature control module, and a central control module; The digital PCR chip is placed on a two-dimensional stepping scanning platform on the optical detection module and is connected to the hot water circulation temperature control module and the fluid control module. The fluid control module is also connected to the hot water circulation temperature control module and the central control module. The hot water circulation temperature control module and the optical detection module are both connected to the central control module.

[0008] Furthermore, the digital PCR chip is a high-density microcavity array structure with integrated cavities; The digital PCR chip includes a top glass cover, a thin film layer, a cavity layer, and a bottom glass substrate; The top glass cover is provided with a sample inlet through hole, a sample outlet through hole, a first water inlet through hole and a first water outlet through hole; The thin film layer is provided with a polydimethylsiloxane microcavity array structure, a sample inlet, a waste liquid outlet, a second inlet through-hole, and a second outlet through-hole. The polydimethylsiloxane microcavity array structure is formed by several parallel microchannels connected in series with several microcavities. The two ends of the microchannels are respectively connected to the sample inlet and the waste liquid outlet. The second inlet through-hole and the second outlet through-hole are respectively located on both sides of the polydimethylsiloxane microcavity array structure. The side of the thin film layer where the polydimethylsiloxane microcavity array structure is provided is a structured surface, and the other side is a non-structured surface. The cavity layer is provided with a third inlet, a third outlet and a cavity region, and the projected area of ​​the cavity region on the thin film layer covers the polydimethylsiloxane microcavity array structure region thereon. The first water inlet through hole, the second water inlet through hole and the third water inlet are one-to-one correspondences and interconnected with each other; the first water outlet through hole, the second water outlet through hole and the third water outlet are one-to-one correspondences and interconnected with each other; the sample inlet through hole corresponds to the sample inlet and is interconnected with each other; the sample outlet through hole corresponds to the waste liquid outlet and is interconnected with each other. The top glass cover is bonded to the structural surface of the thin film layer to form a closed microchannel system, and the non-structural surface and cavity layer of the thin film layer are bonded to the bottom glass substrate to form a closed cavity. The sample inlet port of the digital PCR chip is connected to the outlet of the sample tube via a tube, and a first valve is provided on the tube.

[0009] Furthermore, the polydimethylsiloxane microcavity array structure includes at least 1000 microcavities, and the geometry and size of each microcavity are identical; The polydimethylsiloxane microcavity array structure is a non-permeable structure, and the polydimethylsiloxane film at the bottom of each microcavity constitutes an isolation membrane between the microcavity and the cavity layer, and the thickness of the isolation membrane is 20~200 micrometers.

[0010] Furthermore, the cavity layer has a transparent structure and is made by etching polydimethylsiloxane, double-sided tape or film, and the thickness of the cavity layer is 30~1000 micrometers.

[0011] Furthermore, the optical detection module includes a two-dimensional stepping scanning platform, a bright-field optical component, and a fluorescence microscopy optical component; The bright-field optical component is positioned above the two-dimensional step-scan platform, and the fluorescence microscopy optical component is positioned below the two-dimensional step-scan platform. The two-dimensional step-scan platform is connected to the central control module. The two-dimensional stepping scanning platform is used to place the digital PCR chip; The bright-field optical element is used to observe the integrity of sample introduction into the digital PCR chip; The fluorescence microscopy optical component is used for PCR result detection.

[0012] Furthermore, the fluid control module includes a fluid control circuit, a vacuum pump, a second valve, and a waste liquid bottle; The fluid control circuit is connected to the control terminal of the first valve and the central control module, respectively. The fluid control circuit is also connected to the vacuum pump, the second valve, and the three-way valve in the hot water circulation temperature control module. The air inlet of the vacuum pump is connected to the air outlet of the waste liquid bottle through a pipe. The output port of the second valve is connected to the feed inlet of the waste liquid bottle through a pipe. The two input ports of the second valve are connected to the sample outlet through hole and the first water outlet of the digital PCR chip, respectively.

[0013] Furthermore, the hot water circulation temperature control module includes a first constant temperature water tank, a second constant temperature water tank, a third constant temperature water tank, a three-way valve, and a temperature control circuit; The input terminal of the temperature control circuit is connected to the central control module, and the output terminal of the temperature control circuit is connected to the temperature control terminals of the first constant temperature water tank, the second constant temperature water tank, and the third constant temperature water tank, respectively. The three input terminals of the three-way valve are connected to the first constant temperature water tank, the second constant temperature water tank, and the third constant temperature water tank, respectively, through a connecting pipe. The output terminal of the three-way valve is connected to the water inlet on the digital PCR chip through a connecting pipe. The control terminal of the three-way valve is connected to the fluid control circuit.

[0014] A method for using an integrated digital PCR system includes the following steps: S1. Preparation: Fix the digital PCR chip on the two-dimensional stepping scanning platform, and assemble and connect each tube according to its interface requirements. At the same time, start the central control module, set the parameters for regulating fluid control and temperature control, and start the temperature control circuit. According to the digital PCR detection requirements, control the temperature of the constant temperature tank in the hot water circulation temperature control module. S2, Sample filling: Adjust the valve opening status based on the digital PCR detection requirements, and use a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. S3. Sample Discretization: Based on the requirements of digital PCR detection, adjust the valve opening status, use a vacuum pump to introduce the oil phase in the sample tube into the digital PCR chip, remove the sample liquid in each microchannel, and isolate the oil phase from the sample liquid in each microcavity. S4. Thermal cycling amplification: Based on the digital PCR detection requirements, the valve opening status is adjusted, and water in the constant temperature water tank at the preset temperature is continuously driven by the vacuum pump through the integrated cavity of the digital PCR chip, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. S5. Image Processing and Data Analysis: Controls the closure of all valves and vacuum pumps, and activates the optical detection module. The optical detection module scans and acquires microscopic images of the digital PCR chip, and the central control module stitches the microscopic images together to obtain a fluorescence image of the entire microcavity reaction area of ​​the digital PCR chip. After image processing and data analysis, the final detection result is obtained.

[0015] Furthermore, the PCR detection requirements in step S1 include conventional digital PCR detection and isothermal digital PCR detection; When the testing requirement is routine digital PCR testing, the three constant temperature water tanks in the hot water circulation temperature control module are heated to the preset temperature and maintained stably. When the testing requirement is isothermal digital PCR testing, the two constant temperature water tanks in the hot water circulation temperature control module are controlled to the preset temperature and kept stable, while the other constant temperature water tank is left empty; among the two temperature-controlled constant temperature water tanks, one is a constant temperature hot water tank and the other is a constant temperature cold water tank.

[0016] Furthermore, in step S2: When the testing requirement is routine digital PCR testing, the sample filling process is as follows: The system controls the opening of the first valve and the opening of the second valve towards the first outlet, and uses a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. When the testing requirement is isothermal digital PCR testing, the sample filling process is as follows: The second valve is controlled to open towards the first outlet, and the air in each microchannel and microcavity of the digital PCR chip is extracted using a vacuum pump. Then, the first valve is controlled to open, the second valve is controlled to open towards the first outlet, and the three-way valve is controlled to open towards the constant temperature cold water tank, so that the sample liquid in the sample tube is drawn into the digital PCR chip until the sample liquid fills all the microchannels and microcavities of the digital PCR chip. In step S3: When the testing requirement is routine digital PCR testing, the valve opening status is adjusted by controlling the first valve to open and the second valve to open towards the sample outlet. When the testing requirement is isothermal digital PCR testing, the valve opening status is adjusted as follows: the first valve is opened, the second valve is opened simultaneously in both inlet directions, and the three-way valve is opened towards the constant temperature cold water tank. In step S4: When the testing requirement is conventional digital PCR, the thermal cycling amplification method is as follows: The three-way valve is controlled to open towards the three constant temperature water tanks and the second valve is opened towards the first outlet. The vacuum pump drives the three hot waters of different temperatures in the three constant temperature water tanks to circulate through the integrated cavity of the digital PCR chip in a set order and cycle, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. When the detection requirement is isothermal digital PCR detection, the thermal cycling amplification method is as follows: The second valve is controlled to open towards the first outlet and the three-way valve is opened towards the constant temperature hot water tank. The constant temperature water in the constant temperature hot water tank is continuously passed through the cavity area by the vacuum pump, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction.

[0017] The beneficial effects of this invention are as follows: The integrated digital PCR system and its usage method provided by this invention have the advantages of simple operation, low cost, high detection efficiency, and good result reliability, specifically reflected in the following aspects: (1) The integrated digital PCR system can realize the fully automated and “foolproof” operation of the entire digital PCR detection process, including sample injection, sample discretization, thermal cycling amplification and result reading and analysis, on a single instrument. This greatly reduces the complexity of digital PCR detection operation, reduces the probability of human error and sample contamination, improves the reliability of detection results, and avoids the dependence of digital PCR detection operation on professional technicians. (2) Based on the gas permeability of polydimethylsiloxane (PDMS) film, the negative pressure injection method can first fill the microchannel network and microcavity array of the digital PCR chip, and then remove the excess sample in the filling microchannel. Only the sample in the filling microchannel is wasted. Compared with the existing digital PCR system, the sample utilization rate is greatly improved. (3) The thermal cycling amplification method based on integrated microcavity hot water circulation, compared with the existing thermal cycling amplification method based on hot plate heating and cooling, does not require complex and precise rapid heating and cooling temperature control design, avoids time-consuming heating and cooling processes, and the integrated structure also avoids the problem of uneven temperature caused by incomplete contact and bonding between the chip and the hot plate, which can greatly reduce the complexity of the temperature control module, shorten the thermal cycling amplification time, and improve the uniformity of the chip thermal cycling reaction. (4) The integrated cavity structure of the chip can also utilize the water molecule permeability of the PDMS film to play a water compensation role during the thermal cycling amplification process, avoiding the water loss problem caused by the high temperature evaporation of the reaction liquid in the microcavity due to thermal cycling, and further ensuring the reliability of the PCR reaction. Attached Figure Description

[0018] Figure 1 The integrated digital PCR system provided by this invention is shown in the structural diagram.

[0019] Figure 2 An exploded view of the digital PCR chip structure provided by this invention.

[0020] Figure 3 This is a schematic diagram of the digital PCR chip assembly provided by the present invention.

[0021] Figure 4 This is a top view schematic diagram of the digital PCR chip structure provided by the present invention.

[0022] Figure 5 This is a flowchart illustrating a method of using the integrated digital PCR system provided by the present invention.

[0023] Figure 6 This is a schematic diagram illustrating the application process of the integrated digital PCR system provided by the present invention in conventional digital PCR.

[0024] Figure 7 The image shows the detection results obtained by applying the integrated digital PCR system provided by this invention to conventional digital PCR.

[0025] Figure 8 This is a schematic diagram illustrating the application process of the integrated digital PCR system provided by the present invention in isothermal digital PCR.

[0026] The components include: 1. Digital PCR chip; 2. Optical detection module; 3. Fluid control module; 4. Hot water circulation temperature control module; 5. Central control module; 6. Sample tube; 7. First valve; 1-1. Top glass cover; 1-2. Thin film layer; 1-3. Cavity layer; 1-4. Bottom glass substrate; 1-11. Sample inlet through-hole; 1-12. Sample outlet through-hole; 1-13. First water inlet through-hole; 1-14. First water outlet through-hole; 1-21. Polydimethylsiloxane microcavity array structure; 1-211. Microchannel; 1-212. Microcavity; 1-22. Sample inlet. ; 1-23, Waste liquid outlet; 1-24, Second inlet through hole; 1-25, Second outlet through hole; 1-31, Third inlet; 1-32, Third outlet; 1-33, Cavity area; 2-1, Light source; 2-2, First convex lens; 2-3, First reflecting mirror; 2-4, Second convex lens; 2-5, First electronic shutter; 2-6, Two-dimensional stepping scanning platform; 2-7, Microscope objective; 2-8, Dichroic mirror; 2-9, Third convex lens; 2-10, Second electronic shutter; 2-11, Mercury lamp; 2-12, Second reflecting mirror; 2-13, Image sensor; 2-14 Excitation filter; 2-15 Emission filter; 3-1 Fluid control circuit; 3-2 Vacuum pump; 3-3 Second valve; 3-4 Waste liquid bottle; 4-1 First constant temperature water tank; 4-2 Second constant temperature water tank; 4-3 Third constant temperature water tank; 4-4 Three-way valve; 4-5 Temperature control circuit. Detailed Implementation

[0027] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0028] Example 1: like Figure 1 As shown, an integrated digital PCR system includes a digital PCR chip 1, an optical detection module 2, a fluid control module 3, a hot water circulation temperature control module 4, and a central control module 5. The digital PCR chip 1 is placed on the two-dimensional stepping scanning platform 2-6 on the optical detection module 2, and is connected to the hot water circulation temperature control module 4 and the fluid control module 3. The fluid control module 3 is also connected to the hot water circulation temperature control module 4 and the central control module 5 respectively. The hot water circulation temperature control module 4 and the optical detection module 2 are both connected to the central control module 5.

[0029] The digital PCR chip 1 is used to automatically decompose and quantitatively distribute the input sample without the need for complex macro-micro interfaces, thus reducing the complexity and operating cost of the digital PCR system. The optical detection module 2 can programmatically switch between bright-field and fluorescence microscopy observation modes and can programmatically autofocus to accurately acquire reaction images in the digital PCR chip 1. The hot water circulation temperature control module 4 contains three constant-temperature water tanks, which are programmed to achieve heating, cooling, or constant-temperature control through a temperature control circuit 4-5 to provide suitable temperature conditions for the sample amplification reaction in the digital PCR chip 1. The fluid control module 3 is used to fill and discretize the sample in the digital PCR chip 1 and control the order and time of liquid flow from different constant-temperature water tanks through the digital PCR chip 1, providing environmental support for the sample reaction in the digital PCR chip 1.

[0030] like Figure 2-4 As shown, the digital PCR chip 1 in this embodiment is a high-density microcavity array structure with integrated cavities; Digital PCR chip 1 includes a top glass cover 1-1, a thin film layer 1-2, a cavity layer 1-3, and a bottom glass substrate 1-4; The top glass cover 1-1 is provided with a sample inlet through hole 1-11, a sample outlet through hole 1-12, a first water inlet through hole 1-13 and a first water outlet through hole 1-14; The thin film layer 1-2 is provided with a polydimethylsiloxane microcavity array structure 1-21, a sample inlet 1-22, a waste liquid outlet 1-23, a second water inlet through hole 1-24, and a second water outlet through hole 1-25. The polydimethylsiloxane microcavity array structure 2-1 is formed by several parallel microchannels 2-211 connected in series with several microcavities 2-212. The two ends of the microchannels are connected to the sample inlet 1-22 and the waste liquid outlet 1-23, respectively. The second water inlet through hole 1-24 and the second water outlet through hole 1-25 are respectively located on both sides of the polydimethylsiloxane microcavity array structure 1-21. One side of the thin film layer 1-2 where the polydimethylsiloxane microcavity array structure 1-21 is provided is a structured surface, and the other side is a non-structured surface. The cavity layer 1-3 is provided with a third inlet 1-31, a third outlet 1-32 and a cavity region 1-33. The projected area of ​​the cavity region 1-33 on the thin film layer 1-2 covers the polydimethylsiloxane microcavity array structure 1-21 region thereon, and the projected area is slightly larger than the polydimethylsiloxane microcavity array structure 1-21 region. The first inlet through hole 1-13, the second inlet through hole 1-24, and the third inlet 1-31 correspond one-to-one and are interconnected; the first outlet through hole 1-14, the second outlet through hole 1-25, and the third outlet 1-32 correspond one-to-one and are interconnected; the sample inlet through hole 1-11 corresponds to the sample inlet 1-22 and is interconnected; and the sample outlet through hole 1-12 corresponds to the waste liquid outlet 1-23 and is interconnected. The top glass cover 1-1 and the structural surfaces of the thin film layer 1-2 are bonded to form a closed microchannel system. The non-structural surfaces of the thin film layer 1-2, the cavity layer 1-3, and the bottom glass substrate 1-4 are bonded to form a closed cavity. Figure 3 In the assembly of the digital PCR chip 1, the first inlet through hole 1-13, the second inlet through hole 1-24 and the third inlet 1-31 together form the inlet, the first outlet through hole 1-14, the second outlet through hole 1-25 and the third outlet 1-32 together form the outlet, the sample inlet through hole 1-11 and the sample inlet 1-22 together form the sample inlet, and the sample outlet through hole 1-12 and the waste liquid outlet 1-23 together form the sample outlet; In addition, the sample inlet port 1-11 of the digital PCR chip 1 is connected to the outlet of the sample tube 6 through a tube, and a first valve 7 is provided on the tube.

[0031] Specifically, the polydimethylsiloxane microcavity array structure 1-21 includes at least 1000 microcavities 2-212, and the geometry and size of each microcavity 2-212 are consistent. This arrangement is to ensure that the digital PCR chip 1 can realize basic digital PCR analysis. The polydimethylsiloxane microcavity array structure 1-21 is a non-permeable structure, and the polydimethylsiloxane film at the bottom of each microcavity 2-212 constitutes the isolation membrane between the microcavity 2-212 and the cavity layer 1-3. The thickness of the isolation membrane is 20~200 micrometers. When setting the thickness of the isolation membrane, if the isolation membrane is too thin, the deformation of the membrane will be too large when a vacuum negative pressure is applied to the cavity layer 1-3, which will affect the sample filling effect. If it is too thick, the air permeation rate will be slow, which will result in a slow negative pressure sample injection rate.

[0032] The aforementioned cavity layers 1-3 are fabricated using polydimethylsiloxane, double-sided tape, or film. The thickness of the cavity layers 1-3 is 30-1000 micrometers. The functions of the cavity layers 1-3 include connecting to a vacuum source to achieve bubble-free sample introduction through negative pressure; serving as a hot water circulation pool to realize thermal cycling amplification reaction; and serving as a water replenishment pool to prevent moisture evaporation from the microcavity array reaction system during thermal cycling amplification.

[0033] like Figure 1As shown, the optical detection module 2 in this embodiment includes a two-dimensional stepping scanning platform 2-6, a bright-field optical component, and a fluorescence microscopy optical component; the bright-field optical component is disposed above the two-dimensional stepping scanning platform 2-6, the fluorescence microscopy optical component is disposed below the two-dimensional stepping scanning platform 2-6, and the two-dimensional stepping scanning platform 2-6 is connected to the central control module 5. Among them, the two-dimensional stepping scanning platform 2-6 is used to place the digital PCR chip 1; the bright field optical element is used to observe the integrity of the sample introduction of the digital PCR chip 1; and the fluorescence microscopy optical component is used for PCR result detection.

[0034] Specifically, the bright-field optical assembly includes a light source 2-1, a first convex lens 2-2, a first reflector 2-3, a second convex lens 2-4, and a first electronic shutter 2-5; wherein, the light source 2-1, the first convex lens 2-2, the first reflector 2-3, the second convex lens 2-4, and the first electronic shutter 2-5 are all disposed above the two-dimensional stepping scanning platform 2-6, and the light emitted by the light source 2-1 passes through the first convex lens 2-2, the first reflector 2-3, the second convex lens 2-4, and the first electronic shutter 2-5 in sequence to illuminate the digital PCR chip 1; The fluorescence microscopy optical components include a microscope objective 2-7, a dichroic mirror 2-8, an excitation filter 2-14, an emission filter 2-15, a third convex lens 2-9, a second electronic shutter 2-10, a mercury lamp 2-11, a second reflector 2-12, and an image sensor 2-13. The light emitted from the mercury lamp 2-11 is sequentially transmitted through the second electronic shutter 2-10, the third convex lens 2-9, the excitation filter 2-14, and the dichroic mirror 2-8 to illuminate the digital PCR chip 1. The imaging results of the digital PCR chip 1 by the microscope objective 2-7 are sequentially transmitted through the dichroic mirror 2-8, the emission filter 2-15, the second reflector 2-12, and the image sensor 2-13 to the central control module 5. The first electronic shutter 2-5 and the second electronic shutter 2-10 are both connected to the central control module 5. The image sensor 2-13 is a CCD element or a CMOS element.

[0035] It should be noted that the above-described optical detection module structure is only one possible structure to achieve the above functions, and any bright-field optical components and fluorescence microscopy optical components that can achieve the above functions are within the scope of protection claimed in this application.

[0036] like Figure 1 As shown, the fluid control module 3 in this embodiment includes a fluid control circuit 3-1, a vacuum pump 3-2, a second valve 3-3, and a waste liquid bottle 3-4; The fluid control circuit 3-1 is connected to the control terminal of the first valve 7 and the central control module 5, respectively. The fluid control circuit 3-1 is also connected to the vacuum pump 3-2, the second valve 3-3, and the three-way valve 4-4 in the hot water circulation temperature control module 4. The air inlet of the vacuum pump 3-2 is connected to the outlet of the waste liquid bottle 3-4 through a connecting pipe. The output port of the second valve 3-3 is connected to the inlet of the waste liquid bottle 3-4 through a connecting pipe. The two input ports of the second valve 3-3 are connected to the sample outlet through hole 1-12 and the first water outlet of the PCR chip, respectively.

[0037] The vacuum pump is used to extract air and liquid that needs to be removed from the digital PCR chip. The waste liquid bottle is used to hold the waste liquid discharged from the digital PCR chip and the wastewater solution during the thermal cycle. The fluid control circuit is used to control the operation of the vacuum pump, the second valve, and the three-way valve in the hot water circulation temperature control module. The second valve, in addition to... Figure 1 The dual-branch valve shown, with one branch connected to the sample outlet of the digital PCR chip and the other branch connected to the water outlet of the digital PCR chip, can also be composed of two independent valves, as long as independent control of the two fluid paths can be achieved. Similarly, the three-way valve can be replaced by three independent valves connected to three constant temperature water tanks respectively, as long as independent control of the three fluid paths can be guaranteed. It should be noted that this embodiment only uses the term "fluid control circuit" to limit the functions it needs to achieve. In the prior art, any circuit that can achieve the above functions is within the scope of protection claimed in this application. Therefore, there is no need to describe its specific circuit structure in this embodiment.

[0038] like Figure 1 As shown, the hot water circulation temperature control module 4 in this embodiment includes a first constant temperature water tank 4-1, a second constant temperature water tank 4-2, a third constant temperature water tank 4-3, a three-way valve 4-4, and a temperature control circuit 4-5. The input terminal of the temperature control circuit 4-5 is connected to the central control module 5. The output terminal of the temperature control circuit 4-5 is connected to the temperature control terminals of the first constant temperature water tank 4-1, the second constant temperature water tank 4-2, and the third constant temperature water tank 4-3, respectively. The three input terminals of the three-way valve 4-4 are connected to the first constant temperature water tank 4-1, the second constant temperature water tank 4-2, and the third constant temperature water tank 4-3, respectively, through a connecting pipe. The output terminal of the three-way valve 4-4 is connected to the water inlet on the digital PCR chip 1 through a connecting pipe. The control terminal of the three-way valve 4-4 is connected to the fluid control circuit 3-1.

[0039] The three-way valve is used to control the conduction of the three constant temperature water tanks under the control of the fluid control circuit, while the temperature control circuit heats the three constant temperature water tanks according to the control of the central control module. Similarly, the temperature control circuit in this embodiment is set based on its function. In the prior art, any circuit that can achieve the above function is within the scope of protection claimed in this application. There is no need to describe its specific circuit structure in this embodiment.

[0040] Example 2: like Figure 5 As shown, this embodiment provides a method for using the digital PCR system in Embodiment 1 above, including the following steps: S1. Preparation: Fix the digital PCR chip on the two-dimensional stepping scanning platform, and assemble and connect each tube according to its interface requirements. At the same time, start the central control module, set the parameters for regulating fluid control and temperature control, and start the temperature control circuit. According to the digital PCR detection requirements, control the temperature of the constant temperature tank in the hot water circulation temperature control module. S2, Sample filling: Adjust the valve opening status based on the digital PCR detection requirements, and use a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. After the vacuum pump is started, the air permeability of the PDMS film is used to extract air from each microtube and microcavity in the digital PCR chip, creating a negative pressure, which in turn draws the sample liquid in the sample tube into the digital PCR chip. S3. Sample Discretization: Based on the requirements of digital PCR detection, adjust the valve opening status, use a vacuum pump to introduce the oil phase in the sample tube into the digital PCR chip, remove the sample liquid in each microchannel, and isolate the oil phase from the sample liquid in each microcavity. S4. Thermal cycling amplification: Based on the digital PCR detection requirements, the valve opening status is adjusted, and water in the constant temperature water tank at the preset temperature is continuously driven by the vacuum pump through the integrated cavity of the digital PCR chip, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. The integrated cavity in the digital PCR chip is the cavity region in the cavity layer. Because the cavity region is integrated into the digital PCR chip and is a component of the digital PCR chip, it is also called the integrated cavity of the digital PCR chip. S5. Image Processing and Data Analysis: Controls the closure of all valves and vacuum pumps, and activates the optical detection module. The optical detection module scans and acquires microscopic images of the digital PCR chip, and the central control module stitches the microscopic images together to obtain a fluorescence image of the entire microcavity reaction area of ​​the digital PCR chip. After image processing and data analysis, the final detection result is obtained.

[0041] Specifically, the PCR testing requirements for step S1 above include conventional digital PCR testing and isothermal digital PCR testing; When the testing requirement is routine digital PCR testing, the three constant temperature water tanks in the hot water circulation temperature control module are heated to the preset temperature and maintained stably. When the testing requirement is isothermal digital PCR testing, the two constant temperature water tanks in the hot water circulation temperature control module are controlled to the preset temperature and kept stable, while the other constant temperature water tank is left empty; among the two temperature-controlled constant temperature water tanks, one is a constant temperature hot water tank and the other is a constant temperature cold water tank.

[0042] In step S2 above: When the testing requirement is routine digital PCR testing, the sample filling process is as follows: The system controls the opening of the first valve and the opening of the second valve towards the first outlet, and uses a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. When the testing requirement is isothermal digital PCR testing, the sample filling process is as follows: The second valve is controlled to open towards the first outlet, and the air in each microchannel and microcavity of the digital PCR chip is extracted using a vacuum pump. Then, the first valve is controlled to open, the second valve is controlled to open towards the first outlet, and the three-way valve is controlled to open towards the constant temperature cold water tank, so that the sample liquid in the sample tube is drawn into the digital PCR chip until the sample liquid fills all the microchannels and microcavities of the digital PCR chip. In step S3 above: When the testing requirement is routine digital PCR testing, the valve opening status is adjusted by controlling the first valve to open and the second valve to open towards the sample outlet. When the testing requirement is isothermal digital PCR testing, the valve opening status is adjusted as follows: the first valve is opened, the second valve is opened simultaneously in both inlet directions, and the three-way valve is opened towards the constant temperature cold water tank. In step S4 above: When the testing requirement is conventional digital PCR, the thermal cycling amplification method is as follows: The three-way valve is controlled to open to the three constant temperature water tanks and the second valve is opened to the first outlet. The vacuum pump drives the three hot waters of different temperatures in the three constant temperature water tanks to circulate through the integrated cavity of the digital PCR chip in a set order and cycle, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. When the detection requirement is isothermal digital PCR detection, the thermal cycling amplification method is as follows: The second valve is controlled to open towards the first outlet and the three-way valve is opened towards the constant temperature hot water tank. The constant temperature water in the constant temperature hot water tank is continuously passed through the cavity area by the vacuum pump, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction.

[0043] It should be noted that in the above process, when each step mentions the control of valves and vacuum pumps, except for the valves or vacuum pumps that are controlled to be opened, the other valves or vacuum pumps are in the closed state. For example, in step S2, when the first valve and the second valve are controlled to open towards the first outlet and the vacuum pump is started, the three-way valve and the second valve are in the closed state towards the sample outlet.

[0044] Example 3: like Figure 6 As shown, the specific steps for performing routine digital PCR detection using the system of this invention, taking the detection of EGFR L858R mutation in the peripheral blood of a lung cancer patient as an example, are as follows: (1) Preparation of chips, reagents and samples An integrated cavity digital PCR chip containing 20,000 microcavities was fabricated using photolithography molding combined with PDMS casting and oxygen plasma surface treatment bonding. A pair of specific primers targeting the EGFR L858R mutation were synthesized and prepared. Circulating DNA was extracted from peripheral blood serum of lung cancer patients, and polymerase, primers, probes, buffer, etc. were added to a sample tube storing the circulating DNA to prepare a sample solution. Paraffin oil was added to the sample solution.

[0045] (2) Chip assembly The digital PCR chip prepared in step (1) is placed and fixed on a two-dimensional stepping scanning platform. The chip inlet is connected to the sample tube prepared in step (1) through a microtube and a valve. The chip water inlet is connected to three constant temperature water tanks through a microtube and a three-branch switching valve. The chip outlet and water outlet are connected to a vacuum pump through a microtube and a two-branch switching valve. (3) System control parameter settings Start the central control module and use the software interface to set the temperatures of the three constant temperature water tanks to T1=95°C, T2=60°C, and T3=50°C, respectively. Set the relevant parameters for the fluid control process as follows: 1) Valve 1 and Valve 2-II open, Valve 2-I and Valve 3 closed, duration 3 min; 2) Valve 1 and Valve 2-I open, Valve 2-II and Valve 3 closed, duration 5 min; 3) Valve 3-III and Valve 2-II open, Valve 1, Valve 2-I, Valve 3-I and Valve 3-II closed, duration 10 min; 4) Valve 3-I and Valve 2-II open, Valve 1, Valve 2-I, Valve 3-II and Valve 3-III closed, duration 10 min; 5) 45 cycles: Valve 3-I and Valve 2-II open, Valve 1, Valve 2-I, Valve 3-II and Valve 3-III closed, duration 40 s; Valve 3-II and Valve 2-II open, Valve 1, Valve 2-I, Valve 3-I and Valve 3-III closed, duration 30 s. (Wherein, valve 1 corresponds to the first valve; valve 2 corresponds to the second valve, and valves 2-I and 2-II correspond to the sample outlet and the first water outlet directions of the second valve connected to the digital PCR chip, respectively; valve 3 corresponds to the three-way valve, and valves 3-I, 3-II, and 3-III correspond to the directions of the three-way valve connected to the three constant temperature water tanks, respectively.) (4) Chip filling and sample discretization The temperature control circuit is activated until each constant temperature water tank is heated to the preset constant temperature. After each constant temperature water tank is heated to the preset temperature, the fluid control circuit is activated. The microvalve connecting the sample tube to the digital PCR chip inlet and the microvalve connecting the digital PCR chip outlet to the waste bottle are opened using the preset program of the control software. Other microvalve is kept closed. The vacuum pump is activated to draw the sample liquid from the sample tube into the digital PCR chip. Within 3 minutes, the sample liquid fills the entire microchannel network and microcavity array of the digital PCR chip. After the filling is completed, the microvalve connecting the sample tube to the digital PCR chip inlet and the microvalve connecting the digital PCR chip outlet to the waste bottle are opened using the preset program of the control software. Other microvalve is kept closed. The vacuum pump is used to introduce the upper oil phase from the sample tube into the digital PCR chip to remove excess sample liquid from the filling channels. The oil phase introduced within 5 minutes will completely replace the sample liquid in the filling channels, thereby isolating the sample liquid in each microcavity and realizing the discretization of the sample liquid.

[0046] (5) Thermal cycling amplification reaction After the sample filling and sample liquid discretization are completed, the three different temperatures of hot water are circulated through the chip integrated microcavity by the fluid control circuit and the control software preset program, so that the sample liquid in the microcavity array above the integrated microcavity can carry out thermal cycling amplification reaction. The specific fluid control is as follows: step (3) fluid control process 3)-5), that is, the thermal cycling curve of the PCR reaction is: 50°C for 10 minutes, 95°C for 10 minutes, and then 45 cycles of 95°C for 45 seconds and 60°C for 40 seconds.

[0047] (6) Signal reading and analysis After the thermal cycling amplification reaction is completed, the fluorescence path of the optical module is activated and automatically focused using the central control module software interface. The scanning start point and range are set, and the two-dimensional scanning moving platform of the optical module is controlled by the control software to scan and record fluorescence images of the microcavity array region of the digital PCR chip. The software automatically stitches the obtained scanned fluorescence images to obtain an overall fluorescence image of the microcavity array region of the digital PCR chip. Finally, the positive signal of the fluorescence image is extracted by the software to analyze the EGFR L858R mutation status of lung cancer patients.

[0048] like Figure 7 The figure shows the detection results obtained using the above method. It can be seen from the figure that this system can achieve high sensitivity and accurate absolute quantification of rare mutations in cancer patients, and the chip design based on 20,000 microcavities can achieve a high dynamic measurement range (10-104 copies / μL).

[0049] Example 4: like Figure 8 As shown, the specific steps for isothermal digital PCR detection using the system of this invention, taking Listeria detection as an example, are as follows: (1) Preparation of chips, reagents and samples An integrated cavity digital PCR chip containing 20,000 microcavities was fabricated using a combination of photolithography molding, PDMS casting, and oxygen plasma surface treatment bonding. A pair of specific primers were synthesized for Listeria monocytogenes. The sample tube containing the sample to be tested was placed in an ice water box, and then primers, magnesium acetate, TwistDx exo reagent, buffer, etc. were added to the sample tube to prepare a sample solution. Paraffin oil was added to the sample solution.

[0050] (2) Chip assembly The digital PCR chip prepared in step (1) is placed and fixed on a two-dimensional stepping scanning platform. The chip inlet is connected to the sample tube prepared in step (1) through a microtube and a single-way valve. The chip water inlet is connected to three constant temperature water tanks through a microtube and a three-branch switching valve. The chip outlet and water outlet are connected to a vacuum pump through a microtube and a two-branch switching valve. (3) System control parameter settings Start the central control module and use the software interface to set the temperatures of the three constant temperature water tanks as T1=0°C, T2=39°C, and T3=room temperature (empty, without water). Set the relevant parameters of the fluid control process as follows: 1) Valve 2-II open, valves 1, 2-I, and 3 closed, duration 5 minutes; 2) Valve 1, 2-II, and 3-I open, valves 2-I, 3-II, and 3-III closed, duration 3 minutes; 3) Valve 1, 2-I, 2-II, and 3-I open, valves 3-II and 3-III closed. 4) Close valves 2-II and 3-II, close valves 1, 2-I, 3-I, and 3-III, for 25 minutes; (wherein, valve 1 corresponds to the first valve; valve 2 corresponds to the second valve, and valves 2-I and 2-II correspond to the sample outlet and the first water outlet through-hole direction of the second valve connected to the digital PCR chip, respectively; valve 3 corresponds to the three-way valve, and valves 3-I, 3-II, and 3-III correspond to the constant temperature cold water tank, constant temperature hot water tank, and empty constant temperature water tank connected to the three-way valve, respectively). (4) Chip filling and sample discretization The temperature control circuit is activated until each constant-temperature water tank heats or cools to the preset constant temperature. Once the water temperature in each tank reaches the preset temperature, the fluid control circuit is activated. Using the preset program in the control software, the microvalve connecting the digital PCR chip outlet to the waste liquid bottle is opened, while other microvalves remain closed. The vacuum pump is then activated, utilizing the permeability of the PDMS membrane to establish negative pressure within the microchannel network and microcavity array of the digital PCR chip. After 5 minutes, the microvalve connecting the digital PCR chip inlet to the sample tube is opened, drawing the sample solution from the sample tube into the digital PCR chip through the established negative pressure. Simultaneously, the microvalve connecting the digital PCR chip inlet to the 0°C... The micro-valve of the water tank fills the chip integrated cavity with cold water, temporarily inhibiting the PCR amplification reaction of the sample liquid filled in the chip. After the filling is completed, the micro-valve connecting the digital PCR chip outlet and the waste bottle is opened by the preset program of the control software. The upper oil phase in the sample tube is introduced into the digital PCR chip by the vacuum pump to remove the excess sample liquid in the filling channel. The oil phase filled in within 5 minutes will completely replace the sample liquid in the filling channel, thereby isolating the sample liquid in each microcavity and realizing the discretization of the sample liquid.

[0051] (5) Thermal cycling amplification reaction After the sample filling and sample liquid discretization are completed, the micro-valve connecting the digital PCR chip inlet to the 39°C water tank and the micro-valve connecting the digital PCR chip outlet to the waste liquid bottle are opened by the fluid control circuit and the control software preset program, while keeping other micro-valve closed, so that 39°C hot water can continuously flow through the chip integrated cavity, so that the sample liquid in the micro-cavity array located on the integrated micro-cavity can undergo isothermal PCR reaction.

[0052] (6) Signal reading and analysis After 25 minutes, the PCR reaction was completed. The optical module fluorescence path was activated and automatically focused using the central control module software interface. The scanning start point and range were set, and the optical module's two-dimensional scanning moving platform was controlled by the control software to scan and record fluorescence images of the microcavity array region of the digital PCR chip. The software automatically stitched the obtained scanned fluorescence images to obtain an overall fluorescence image of the microcavity array region of the digital PCR chip. Finally, the positive signal of the fluorescence image was extracted by the software to analyze the concentration of Listeria in the sample, providing assistance for food safety and environmental monitoring.

[0053] In the description of this invention, it should be understood that the terms "center," "thickness," "upper," "lower," "horizontal," "top," "bottom," "inner," "outer," and "radial," etc., indicating orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying the relative importance or the number of technical features implicitly specified. Therefore, a feature defined by "first," "second," and "third" may explicitly or implicitly include one or more of that feature.

Claims

1. An integrated digital PCR system, characterized in that, It includes a digital PCR chip (1), an optical detection module (2), a fluid control module (3), a hot water circulation temperature control module (4), and a central control module (5). The digital PCR chip (1) is placed on the two-dimensional stepping scanning platform (2-6) on the optical detection module (2) and connected to the hot water circulation temperature control module (4) and the fluid control module (3). The fluid control module (3) is also connected to the hot water circulation temperature control module (4) and the central control module (5) respectively. The hot water circulation temperature control module (4) and the optical detection module (2) are both connected to the central control module (5). The digital PCR chip (1) includes a top glass cover (1-1), a thin film layer (1-2), a cavity layer (1-3), and a bottom glass substrate (1-4). The top glass cover (1-1) is provided with an inlet through hole (1-11), an outlet through hole (1-12), a first water inlet through hole (1-13), and a first water outlet through hole (1-14). The thin film layer (1-2) is provided with a polydimethylsiloxane microcavity array structure (1-21), a sample inlet (1-22), a waste liquid outlet (1-23), a second water inlet through hole (1-24), and a second water outlet through hole (1-25). The polydimethylsiloxane microcavity array structure (1-21) is formed by several parallel microchannels (1-211) connected in series with several microcavities (1-212). The two ends of the microchannels (1-211) are respectively connected to the sample inlet (1-22) and the waste liquid outlet (1-23). ​​The second water inlet through hole (1-24) and the second water outlet through hole (1-25) are respectively located on both sides of the polydimethylsiloxane microcavity array structure (1-21). One side of the thin film layer (1-2) where the polydimethylsiloxane microcavity array structure (1-21) is provided is a structured surface, and the other side is a non-structured surface. The cavity layer (1-3) is provided with a third inlet (1-31), a third outlet (1-32) and a cavity region (1-33), and the projected area of ​​the cavity region (1-33) on the thin film layer (1-2) covers the polydimethylsiloxane microcavity array structure (1-21) region thereon. The first inlet through hole (1-13), the second inlet through hole (1-24), and the third inlet (1-31) correspond one-to-one and are interconnected. The first outlet through hole (1-14), the second outlet through hole (1-25), and the third outlet (1-32) correspond one-to-one and are interconnected. The sample inlet through hole (1-11) corresponds to the sample inlet (1-22) and is interconnected. The sample outlet through hole (1-12) corresponds to the waste liquid outlet (1-23) and is interconnected. The top glass cover (1-1) is bonded to the structural surface of the thin film layer (1-2) to form a closed microchannel system, and the non-structural surface of the thin film layer (1-2), the cavity layer (1-3), and the bottom glass substrate (1-4) are bonded to form a closed cavity; The sample inlet port (1-11) of the digital PCR chip (1) is connected to the outlet of the sample tube (6) through a tube, and a first valve (7) is provided on the tube. The fluid control module (3) includes a fluid control circuit (3-1), a vacuum pump (3-2), a second valve (3-3), and a waste liquid bottle (3-4). The fluid control circuit (3-1) is connected to the control terminal of the first valve (7) and the central control module (5) respectively; The fluid control circuit (3-1) is also connected to the vacuum pump (3-2), the second valve (3-3), and the three-way valve (4-4) in the hot water circulation temperature control module (4). The air inlet of the vacuum pump (3-2) is connected to the air outlet of the waste liquid bottle (3-4) through a pipe. The output port of the second valve (3-3) is connected to the feed inlet of the waste liquid bottle (3-4) through a pipe. The two input ports of the second valve (3-3) are connected to the sample outlet through hole (1-12) and the first water outlet of the digital PCR chip (1) respectively. After the vacuum pump is started, the air permeability of the thin film layer is used to extract air from the digital PCR chip, creating a negative pressure. The vacuum pump is used to extract air and liquid that needs to be removed from the digital PCR chip. The cavity layer (1-3) serves several purposes, including connecting to a vacuum source to achieve bubble-free sample injection through negative pressure, acting as a hot water circulation pool to realize thermal cycling amplification reaction, and acting as a water replenishment pool to prevent moisture evaporation from the microcavity array reaction system during thermal cycling amplification.

2. The integrated digital PCR system according to claim 1, characterized in that, The polydimethylsiloxane microcavity array structure (1-21) includes at least 1000 microcavities, and each microcavity has the same geometry and size; The polydimethylsiloxane microcavity array structure (1-21) is a non-permeable structure, and the polydimethylsiloxane film at the bottom of each microcavity (1-212) constitutes an isolation membrane between the microcavity (1-212) and the cavity layer (1-3), and the thickness of the isolation membrane is 20~200 micrometers.

3. The integrated digital PCR system according to claim 1, characterized in that, The cavity layer (1-3) has a transparent structure and is made by etching polydimethylsiloxane, double-sided tape or film. The thickness of the cavity layer (1-3) is 30~1000 micrometers.

4. The integrated digital PCR system according to claim 1, characterized in that, The optical detection module (2) includes a two-dimensional step-scanning platform (2-6), a bright-field optical component, and a fluorescence microscopy optical component; The bright-field optical component is positioned above the two-dimensional step-scan platform (2-6), the fluorescence microscopy optical component is positioned below the two-dimensional step-scan platform (2-6), and the two-dimensional step-scan platform (2-6) is connected to the central control module (5). The two-dimensional stepping scanning platform (2-6) is used to place the digital PCR chip (1); The bright-field optical components are used to observe the integrity of the sample introduced into the digital PCR chip (1); The fluorescence microscopy optical component is used for PCR result detection.

5. The integrated digital PCR system according to claim 1, characterized in that, The digital PCR chip (1) is a high-density microcavity array structure with integrated cavities.

6. The integrated digital PCR system according to claim 1, characterized in that, The hot water circulation temperature control module (4) includes a first constant temperature water tank (4-1), a second constant temperature water tank (4-2), a third constant temperature water tank (4-3), a three-way valve (4-4), and a temperature control circuit (4-5). The input terminal of the temperature control circuit (4-5) is connected to the central control module (5), and the output terminal of the temperature control circuit (4-5) is connected to the temperature control terminals of the first constant temperature water tank (4-1), the second constant temperature water tank (4-2), and the third constant temperature water tank (4-3), respectively. The three input terminals of the three-way valve (4-4) are connected to the first constant temperature water tank (4-1), the second constant temperature water tank (4-2), and the third constant temperature water tank (4-3) through a connecting pipe, respectively. The output terminal of the three-way valve (4-4) is connected to the water inlet on the digital PCR chip (1) through a connecting pipe, and the control terminal of the three-way valve (4-4) is connected to the fluid control circuit (3-1).

7. A method of using an integrated digital PCR system, characterized in that, Includes the following steps: S1. Preparation: Fix the digital PCR chip onto the two-dimensional stepping scanning platform, and assemble and connect each tube according to its interface requirements. At the same time, start the central control module, set the parameters for regulating fluid control and temperature control, and start the temperature control circuit. According to the digital PCR detection requirements, control the temperature of the constant temperature water tank in the hot water circulation temperature control module. The digital PCR chip includes a top glass cover, a thin film layer, a cavity layer, and a bottom glass substrate. S2, Sample filling: Adjust the valve opening status based on the digital PCR detection requirements, and use a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. S3. Sample Discretization: Based on the requirements of digital PCR detection, adjust the valve opening status, use a vacuum pump to introduce the oil phase in the sample tube into the digital PCR chip, remove the sample liquid in each microchannel, and isolate the oil phase from the sample liquid in each microcavity. S4. Thermal cycling amplification: Based on the digital PCR detection requirements, the valve opening status is adjusted, and water in the constant temperature water tank at the preset temperature is continuously driven by the vacuum pump through the integrated cavity of the digital PCR chip, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. S5. Image processing and data analysis: Control all valves and vacuum pumps to close, and start the optical detection module. The optical detection module scans and acquires microscopic images of the digital PCR chip. The central control module stitches the microscopic images together to obtain a fluorescence image of the entire microcavity reaction area of ​​the digital PCR chip. After image processing and data analysis, the final detection result is obtained. After the vacuum pump is started, the air permeability of the thin film layer is used to extract air from the digital PCR chip, creating a negative pressure. The vacuum pump is used to extract air and liquid that needs to be removed from the digital PCR chip. The cavity layer serves several purposes, including connecting to a vacuum source to achieve bubble-free sample injection through negative pressure, acting as a hot water circulation pool to realize thermal cycling amplification reaction, and acting as a water replenishment pool to prevent moisture evaporation from the microcavity array reaction system during thermal cycling amplification. In step S2: When the testing requirement is routine digital PCR testing, the sample filling process is as follows: The system controls the opening of the first valve and the opening of the second valve towards the first outlet, and uses a vacuum pump to extract air from each microchannel and microcavity in the digital PCR chip, thereby drawing the sample liquid in the sample tube into the digital PCR chip until the sample liquid fills all the microchannels and microcavities in the digital PCR chip. When the testing requirement is isothermal digital PCR testing, the sample filling process is as follows: The second valve is controlled to open towards the first outlet, and the air in each microchannel and microcavity of the digital PCR chip is extracted using a vacuum pump. Then, the first valve is opened, the second valve is opened towards the first outlet, and the three-way valve is opened towards the constant temperature cold water tank, so that the sample liquid in the sample tube is drawn into the digital PCR chip until the sample liquid fills all the microchannels and microcavities of the digital PCR chip.

8. The method according to claim 7, characterized in that, The PCR detection requirements in step S1 include conventional digital PCR detection and isothermal digital PCR detection; When the testing requirement is routine digital PCR testing, the three constant temperature water tanks in the hot water circulation temperature control module are heated to the preset temperature and maintained stably. When the testing requirement is isothermal digital PCR testing, the two constant temperature water tanks in the hot water circulation temperature control module are controlled to the preset temperature and kept stable, while the other constant temperature water tank is left empty; among the two temperature-controlled constant temperature water tanks, one is a constant temperature hot water tank and the other is a constant temperature cold water tank.

9. The method according to claim 7, characterized in that, In step S3: When the testing requirement is routine digital PCR testing, the valve opening status is adjusted by controlling the first valve to open and the second valve to open towards the sample outlet. When the testing requirement is isothermal digital PCR testing, the valve opening status is adjusted as follows: the first valve is opened, the second valve is opened simultaneously in both inlet directions, and the three-way valve is opened towards the constant temperature cold water tank.

10. The method according to claim 7, characterized in that, In step S4: When the detection requirement is conventional digital PCR, the thermal cycling amplification method is as follows: The three-way valve is controlled to open towards the three constant temperature water tanks and the second valve is opened towards the first outlet. The vacuum pump drives the three hot waters of different temperatures in the three constant temperature water tanks to circulate through the integrated cavity of the digital PCR chip in a set order and cycle, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction. When the detection requirement is isothermal digital PCR detection, the thermal cycling amplification method is as follows: The second valve is controlled to open towards the first outlet and the three-way valve is opened towards the constant temperature hot water tank. The constant temperature water in the constant temperature hot water tank is continuously passed through the cavity area by the vacuum pump, so that the sample liquid in each microcavity of the digital PCR chip can achieve thermal cycling nucleic acid amplification reaction.

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