Droplet generation device and droplet PCR system thereof

Abstract

The application discloses a kind of droplet generation device its droplet PCR system, droplet generation device includes droplet generation pipeline, inner phase input pipeline, inner phase pipeline fixing device, moving mechanism;Droplet generation pipeline is cuboid or trapezoidal body shape, opening is set on the top surface of droplet generation pipeline, opening is provided with rubber cover;The upper portion of droplet generation pipeline is provided with moving mechanism.The device can avoid cross contamination of different sample solutions to be measured by designing detachable inner phase input pipeline and sliding rail, and the detachable inner phase input pipeline can also realize the quick replacement of different samples, and the setting of sliding rail can quickly change the sample input port, to further improve the accuracy and efficiency of droplet PCR.The device of the application is simple in structure, easy to operate and can effectively improve the efficiency and stability of PCR reaction, and can greatly improve the work efficiency and reliability of detection results of droplet PCR multiple sample detection.

Description

Technical Field

[0001] This invention relates to the field of nucleotide PCR amplification device technology, specifically to a droplet generation device and its droplet PCR system. Background Technology

[0002] PCR (Polymerase Chain Reaction) is a rapid in vitro DNA amplification technique widely used in the early diagnosis of diseases. For example, it is used to detect pathogenic microorganisms and genetic diseases, and to track and analyze the expression of specific genes. The complete PCR process consists of three stages: In the high-temperature stage, the DNA in the sample solution is heated to around 95°C, causing hydrogen bonds to break and the template DNA double strand to open, becoming two single strands; next, in the low-temperature annealing stage, specific primers bind to the template according to the base complementarity pairing principle; finally, in the intermediate-temperature extension stage, through the catalytic action of DNA polymerase, starting with the primers and using four nucleotides as raw materials, a DNA strand complementary to the template is synthesized, thus completing one PCR cycle.

[0003] Currently, PCR technology has evolved to its third generation—Droplet Digital PCR (ddPCR). ddPCR is an absolute quantification technique for nucleic acid molecules based on a droplet platform. It combines PCR with droplet microfluidic technology, dropletizing the sample before PCR amplification. The sample is uniformly "distributed" into thousands of nanoliter-level droplets, each isolated from the others, forming an independent reaction system capable of performing PCR amplification independently. This allows for exponential amplification of DNA fragments in a short time. Each droplet is then analyzed individually, with droplets containing the target gene fragment labeled as 1 and those without as 0. Combined with Poisson distribution, the specific number or concentration of target gene molecules in the sample can be calculated. Therefore, ddPCR achieves highly sensitive, accurate, and specific absolute quantification of nucleic acid molecules.

[0004] Current PCR systems include static chamber systems and flow systems. Compared to static chamber systems, flow systems have higher thermal cycling efficiency and can effectively ensure the temperature homogeneity of sample solutions. Therefore, flow systems are widely used in current PCR amplification processes. Flow systems can be further divided into continuous and droplet systems, with droplet PCR systems having lower thermal inertia and allowing for precise absolute quantification and large-scale control compared to continuous systems. In droplet PCR, a nucleic acid suspension is typically mixed with PCR reaction reagents containing nucleic acid lysis reagents as the dispersed phase (i.e., the inner phase); bio-silicone oil serves as the continuous phase (i.e., the outer phase encapsulating the dispersed phase). Under the shearing action of the continuous phase, the PCR reaction mixture is broken into many tiny droplets, each containing a single DNA molecule, thus completing the nucleic acid encapsulation process. The droplets then enter a constant-temperature water bath at different temperature ranges for PCR amplification. After more than 40 cycles, the droplets will encapsulate millions of DNA molecules. In addition, after the cycle is completed, optical detection instruments are needed to record the amplification of DNA molecules, thereby detecting specific genes in the sample.

[0005] In the traditional nucleic acid encapsulation process, the mixed PCR reaction mixture is directly injected into the droplet-generating tube. However, for detecting different types of sample solutions, the tube usually needs to be cleaned before reuse, reducing the efficiency of the detection process. Secondly, incomplete tube cleaning can easily lead to cross-contamination. Therefore, designing a device that can effectively improve detection efficiency, avoid sample solution contamination, and reduce the influence of the external environment on the DNA amplification process is crucial for droplet PCR systems. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned problems by providing a droplet generation device and its droplet PCR system that are simple in structure, easy to operate, and avoid sample solution contamination.

[0007] To achieve its objective, the present invention employs the following technical solution:

[0008] The first aspect of the present invention provides a droplet generating device, including a droplet generating pipe, an inner phase input pipe, an inner phase pipe fixing device, and a moving mechanism; the droplet generating pipe is cuboid or trapezoidal in shape, and an opening is provided on the top surface of the droplet generating pipe, and a rubber cover is provided at the opening;

[0009] The moving mechanism is installed above the droplet generating pipe. The moving mechanism includes a moving plate, at least two limiting components, and at least two sets of parallel linear slide rails. The linear slide rails are installed on the top surface of the droplet generating pipe. Each set of linear slide rails includes a left slide rail and a right slide rail respectively located on the left and right sides of the rubber cover. A slider is slidably mounted on each linear slide rail. The moving plate has a mounting hole in the middle. The top of the slider is fixedly connected to the bottom surface of the moving plate. At least one set of limiting holes is provided on the moving plate on the left and right sides of the rubber cover. Each set of limiting holes includes multiple limiting holes arranged linearly. Each limiting component includes a limiting block and a limiting pin. The limiting block is fixedly installed on the top surface of the droplet generating pipe at a position corresponding to the limiting hole. The top of the limiting block has a limiting groove for the insertion of the limiting pin. The slider slides left and right in the linear slide rails, causing the moving plate to move left and right. When the moving plate moves to a set position, the limiting pin is inserted into the limiting hole and the limiting groove, thereby fixing the position of the moving plate.

[0010] The inner phase pipe fixing device is a cylindrical body with an internal cavity. It has a pipe insertion port at the top and a pipe extension port at the bottom. A pipe clamping device is located in the lower middle part to clamp the inner phase input pipe. The pipe clamping device includes two locking knobs and two clamping parts. The locking knobs are symmetrically arranged on the outside of the inner phase pipe fixing device, and the clamping parts are located inside the cavity of the inner phase pipe fixing device. The locking knobs and clamping parts are connected by bolts. The bottom of the inner phase pipe fixing device is inserted into the mounting hole of the moving plate to fix the position of the inner phase pipe fixing device.

[0011] The lower part of the inner phase input pipe is a puncture needle part that can pass through the rubber plug. The top of the inner phase input pipe is provided with an outwardly extending protrusion. The inner phase input pipe is inserted into the inner phase pipe fixing device. The protrusion rests on the edge of the pipe insertion port of the inner phase pipe fixing device to support the inner phase input pipe.

[0012] Preferably, the inner phase input pipe is funnel-shaped, including an upper liquid-holding section and a lower puncture needle section, and a pipe cap is provided at the top of the inner phase input pipe.

[0013] Preferably, the inner phase pipe fixing device is larger in the middle and smaller at the bottom, with the bottom used to insert into the mounting hole of the moving plate to fix the position of the inner phase pipe fixing device; the clamping part is made of rubber.

[0014] Preferably, the moving mechanism further includes a fixed plate, which is fixedly installed on the top surface of the droplet generating pipe, and the limiting block and the moving rail are fixedly installed on the fixed plate;

[0015] The left and right slide rails of each set of linear slide rails are on the same straight line.

[0016] Preferably, the droplet generation pipe is provided with an inlet and an outlet at both ends, which are respectively connected to an external phase injection pump and a PCR circulating water bath amplification device.

[0017] Preferably, the droplet generating device further includes a fixing frame for fixing the inner phase pipe fixing device. The fixing frame includes a base, a connecting rod, and a clamping rod respectively disposed on the left and right sides of the droplet generating device. The base is fixedly connected to the connecting rod, the connecting rod is fixedly connected to the clamping rod, and the clamping rods on the left and right sides clamp the inner phase pipe fixing device.

[0018] A second aspect of the present invention provides a droplet PCR system, comprising the droplet generation device described in any of the above claims, and further comprising a PCR circulating water bath amplification device and an optical detection device.

[0019] Preferably, the droplet PCR system further includes an inner phase injection pump and an outer phase injection pump. The inner phase injection pump is connected to the top of the inner phase input pipe via a pipe, and the outer phase injection pump is connected to the inlet end of the droplet generation pipe via a pipe.

[0020] Preferably, the PCR circulating water bath amplification device includes a constant temperature zone, a variable temperature circulation zone, and a reaction tube. The reaction tube is a continuous S-shaped tube that enters from the constant temperature zone, passes through the constant temperature zone and the variable temperature circulation zone in sequence, and then extends out to connect with the optical detection device.

[0021] Preferably, the optical detection device includes a laser emitter, a dichroic mirror, a focusing mirror, a detector, and a microdroplet channel. The microdroplet channel is connected to the reaction tube of a PCR circulating water bath amplification device. The focusing mirror, dichroic mirror, and detector are arranged sequentially below the microdroplet channel, and the laser emitter is located on one side of the dichroic mirror.

[0022] The beneficial effects of this invention are:

[0023] This device, with its detachable internal phase input pipe and sliding track, avoids cross-contamination between different sample solutions and reduces the impact of the external environment on the DNA amplification process. The detachable internal phase input pipe also allows for rapid replacement of different samples, and the sliding track enables quick changes to the sample input port, further improving the accuracy and efficiency of droplet PCR. This invention features a simple structure, is easy to operate, and effectively improves PCR reaction efficiency and stability, significantly enhancing the efficiency and reliability of multiple sample detection in droplet PCR. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the droplet generation device of the present invention;

[0025] Figure 2 yes Figure 1A schematic diagram showing the assembly and connection relationship between the inner phase input pipe and the inner phase pipe fixing device.

[0026] Figure 3 This is a schematic diagram of a PCR circulating water bath amplification device;

[0027] Figure 4 This is a schematic diagram of the optical detection device;

[0028] Figure 5 This is a schematic diagram of the droplet PCR system of the present invention;

[0029] The elements or structures indicated by the reference numerals in the attached drawings are:

[0030] Droplet generating device 1, droplet generating pipe 10, rubber cover 101, internal phase input pipe 11, puncture needle part 111, liquid holding part 113, protruding edge 112; internal phase pipe fixing device 12, pipe clamping device 121, locking knob 1211, clamping part 1212; moving mechanism 13, moving plate 131, left slide rail 132, right slide rail 133, slider 134, mounting hole 135, limiting hole 136, limiting block 137, limiting groove 137a, limiting pin 138;

[0031] PCR circulating water bath amplification device 2, constant temperature zone 21, variable temperature circulating zone 22, reaction tube 23;

[0032] Optical detection device 3, laser emitter 31, dichroic mirror 32, focusing mirror 33, detector 34, microdroplet channel 35;

[0033] Internal phase injection pump 4, external phase injection pump 5. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, but this does not limit the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0036] Example 1: Droplet generation device of the present invention

[0037] like Figure 1-2 The droplet generating device 1 of the present invention shown mainly consists of a droplet generating pipe 10, an inner phase input pipe 11, an inner phase pipe fixing device 12, and a moving mechanism 13. The droplet generating pipe 10 is rectangular or trapezoidal in shape, and an opening is provided on the top surface of the droplet generating pipe 10, with a rubber cover 101 provided at the opening.

[0038] A moving mechanism 13 is provided above the droplet generating pipe 10. The moving mechanism 13 includes a moving plate 131, at least two limiting components, and at least two sets of parallel linear slide rails. The linear slide rails are installed on the top surface of the droplet generating pipe 10. Each set of linear slide rails includes a left slide rail 132 and a right slide rail 133 respectively located on the left and right sides of the rubber cover 101 (i.e., each set of linear slide rails is interrupted at the rubber cover 101, crossing the rubber cover 101, and no slide rail is provided on the rubber cover 101). A slider 134 is slidably mounted on each linear slide rail. A mounting hole 135 is provided in the middle of the moving plate 131. The top end of the slider 134 is fixedly connected to the bottom surface of the moving plate 131. The slider 134 is mounted on the moving plate 131 on the rubber cover 10. At least one set of limiting holes is provided on each of the left and right sides of 1. Each set of limiting holes includes multiple limiting holes 136 arranged linearly. Each limiting component includes a limiting block 137 and a limiting pin 138. The limiting block 137 is fixedly installed on the top surface of the droplet generating pipe 10 at a position corresponding to the limiting hole 136 (the installation position of the limiting block 137 does not interfere with the slide rail). The top of the limiting block 137 is provided with a limiting groove 137a for the insertion of the limiting pin 138. The slider 134 slides left and right in the linear slide rail, driving the moving plate 131 to move left and right. When the moving plate 131 moves to the set position, the limiting pin 138 is inserted into the limiting hole 136 and the limiting groove 137a, thereby fixing the position of the moving plate 131.

[0039] The inner phase pipe fixing device 12 is a cylindrical body (it can be a cylindrical, square, or other cylindrical body, and the size of the upper, middle, and lower parts of the cylinder can be the same or different), and it has an internal cavity. The top has a pipe insertion port, and the bottom has a pipe extension port. The internal cavity extends through the top and bottom. The lower middle part of the inner phase pipe fixing device 12 is provided with a pipe clamping device 121 for clamping the inner phase input pipe 11. The pipe clamping device 121 includes two locking knobs 1211 and two clamping parts 1212. The locking knobs 1211 are symmetrically arranged on the outside of the inner phase pipe fixing device 12, and the clamping parts 1212 are arranged in the cavity of the inner phase pipe fixing device 12. The locking knobs 1211 and the clamping parts 1212 are connected by bolts. The bottom of the inner phase pipe fixing device 12 is inserted into the mounting hole 135 of the moving plate 131 to fix the position of the inner phase pipe fixing device 12.

[0040] The lower part of the inner phase input pipe 11 is a puncture needle part 111 that can pass through the rubber plug. The top of the inner phase input pipe 11 is provided with an outwardly extending protrusion 112. The inner phase input pipe 11 is inserted into the inner phase pipe fixing device 12. The protrusion 112 rests on the edge of the pipe insertion port of the inner phase pipe fixing device 12, thereby supporting the inner phase input pipe 11.

[0041] In some embodiments, the droplet generating pipe 10 is preferably trapezoidal in shape. The trapezoidal droplet generating pipe is placed horizontally, with the end with the larger diameter serving as the external phase inlet and the end with the smaller diameter serving as the droplet outlet. This facilitates the liquid flowing out of the outlet, causing the droplet velocity to gradually increase in the flow direction. There is a certain velocity difference between adjacent generated droplets, which can effectively prevent droplet co-aggregation at the outlet and avoid liquid stagnation and accumulation in the pipe, thus facilitating the rapid flow of liquid out of the outlet.

[0042] In some embodiments, the internal phase input pipe 11 is funnel-shaped, including an upper liquid-holding section 113 and a lower puncture needle section 111, and a pipe cap is provided at the top of the internal phase input pipe. The internal phase injection pump 4 pumps the internal phase liquid into the internal phase input pipe 11, where it is buffered and stored in the liquid-holding section 113, and then injected into the droplet generation pipe 10 through the puncture needle section 111.

[0043] In some embodiments, the inner phase pipe fixing device 12 is larger in the middle and smaller at the bottom, making it easier to insert into the mounting hole 135 of the moving plate 131 to fix the position of the inner phase pipe fixing device 12; the clamping part 1212 is made of rubber and is preferably U-shaped, which fits better with the pipe wall of the inner phase input pipe 11 and is less likely to damage the inner phase input pipe 11.

[0044] In some embodiments, the moving mechanism 13 further includes a fixing plate, which is fixedly installed on the top surface of the droplet generating pipe 10. The limiting block 137 and the moving rail are fixedly installed on the fixing plate. The limiting block 137 and the moving rail can be directly fixedly installed on the top surface of the droplet generating pipe 10, or they can be installed through the fixing plate, which makes it easier to install the entire device. When using the fixing plate, the fixing plate should not cover the rubber cover 101. Holes should be made at the corresponding positions of the rubber cover 101, or two fixing plates should be installed on both sides of the rubber cover 101 respectively. Preferably, the left slide rail 132 and the right slide rail 133 of each set of linear slide rails are on the same straight line, which is more conducive to the stability of the moving plate 131 and prevents tilting.

[0045] In some embodiments, the droplet generation pipe 10 is provided with an inlet and an outlet at both ends, which are respectively connected to the external phase injection pump 5 and the PCR circulating water bath amplification device 2.

[0046] In some embodiments, the droplet generating device 1 described above further includes a fixing frame for fixing the inner phase pipe fixing device 12. The fixing frame includes a base, a connecting rod, and a clamping rod respectively disposed on the left and right sides of the droplet generating device 1 (one set of base, connecting rod, and clamping rod is disposed on the left side, and one set of base, connecting rod, and clamping rod is disposed on the right side). The base is fixedly connected to the connecting rod, and the connecting rod is fixedly connected to the clamping rod. The clamping rods on the left and right sides clamp the inner phase pipe fixing device 12 to further ensure the stability of the system structure and prevent the inner phase pipe fixing device 12 from falling off.

[0047] Example 2: The droplet PCR system of the present invention

[0048] like Figure 1-5 The droplet PCR system of the present invention shown mainly consists of the droplet generation device 1, the PCR circulating water bath amplification device 2, and the optical detection device 3 as described in Example 1. The PCR circulating water bath amplification device 2 and the optical detection device 3 can adopt existing device structures.

[0049] In some embodiments, the droplet PCR system of the present invention further includes an inner phase injection pump 4 and an outer phase injection pump 5. The inner phase injection pump 4 is connected to the top of the inner phase input pipe 11 via a pipe, and the outer phase injection pump 5 is connected to the inlet end of the droplet generation pipe 10 via a pipe.

[0050] In some embodiments, the PCR circulating water bath amplification device 2 includes a constant temperature zone 21, a variable temperature circulation zone 22, and reaction tubes 23. The reaction tubes 23 are continuous S-shaped tubes that enter from the constant temperature zone 21, pass through the constant temperature zone 21 and the variable temperature circulation zone 22 sequentially, and then extend to connect to the optical detection device 3. The constant temperature zone 21 can be configured with a first constant temperature zone (e.g., a 55°C low-temperature zone) and a second constant temperature zone (e.g., a 95°C high-temperature zone) according to experimental needs. Experiments that do not require low-temperature pretreatment can use only one constant temperature zone. Both the constant temperature zone 21 and the variable temperature circulation zone 22 achieve their set temperatures via a water bath. The reaction solution flows through different temperature zones, and the length of the reaction tube in each temperature zone is set according to the reaction solution flow rate and the required processing time at that temperature, thereby achieving the required processing time at a specific temperature. Because the reaction tubes are continuous, after reaching the set time in one temperature zone, the process moves to the next set temperature zone. An exemplary PCR reaction is as follows: Droplets generated in the droplet generation channel first enter a low-temperature isothermal zone at 55°C, which is the pre-denaturation stage of the DNA molecules, lasting for 5 minutes. After this stage, they enter a high-temperature isothermal zone at 95°C, which is the hot-start stage, where hydrogen bonds in the DNA molecules break, lasting for 2.5 minutes. Finally, the droplets enter a temperature-variable cycling zone to begin their amplification process. In this stage, the cycling temperatures are 95°C and 65°C, with durations of 5 seconds and 30 seconds respectively, for a total of 45 cycles. After the reaction is complete, the reaction products are detected by an optical detection device.

[0051] In some embodiments, the optical detection device 3 includes a laser emitter 31, a dichroscope 32, a focusing lens 33, a detector 34, and a microdroplet channel 35. The microdroplet channel 35 is connected to the reaction tube 23 of the PCR circulating water bath amplification device 2. The focusing lens 33, dichroscope 32, and detector 34 are sequentially arranged below the microdroplet channel 35, and the laser emitter 31 is located on one side of the dichroscope 32. The excitation light generated by the laser emitter is reflected by the dichroscope and focused onto the microdroplet channel by the focusing lens. The microdroplets in the microdroplet channel are arranged sequentially in the channel and pass through the focusing position one by one. Each microdroplet is sequentially irradiated to generate excitation fluorescence. The fluorescence is collected by the detector arranged below and converted into an electrical signal that is easy to process and analyze by photoelectric devices, thereby detecting the number of positive and negative droplets and finally completing the analysis of the sample to be tested.

[0052] The working process of the droplet PCR system of the present invention is as follows:

[0053] Remove the limiting pin from the limiting hole, move the moving plate to the desired position, and then insert the limiting pin into the limiting hole and the limiting groove on the limiting block. The position of the moving plate is now fixed. Insert the inner phase input pipe into the inner phase pipe fixing device. The puncture needle of the inner phase input pipe passes through the rubber cap on the droplet generation pipe, and the needle tip is in the droplet generation pipe. Tighten the locking knob to clamp and fix the inner phase input pipe, preventing the puncture needle from being misaligned, which could contaminate the sample solution and affect droplet generation. Then adjust the clamping rod of the fixing frame to clamp the inner phase pipe fixing device, further stabilizing the structure and preventing the inner phase pipe fixing device from collapsing. Connect the pipes between the inner phase injection pump and the inner phase input pipe, and between the outer phase injection pump and the droplet generation pipe. Connect the droplet generation pipe outlet, the reaction tube of the PCR circulating water bath amplification device, and the microdroplet channel of the optical detection device through the pipes in sequence. Turn on the inner-phase injection pump and the outer-phase injection pump. The inner-phase injection pump pumps the sample solution (i.e., the inner phase) mixed with nucleic acid and PCR reaction reagents into the inner-phase input tube. The outer-phase injection pump pumps the bio-silicone oil (i.e., the outer phase) into the droplet generating device. The sample solution drips from the inner-phase input tube into the silicone oil in the droplet generating device. As the sample solution drips in and the continuous oil phase shears against it, the nucleic acid molecules are encapsulated inside the droplets and enter the reaction tube of the subsequent PCR circulating water bath amplification device for PCR amplification. After the PCR amplification is completed, the reaction products enter the optical detection device for detection, and the detection results are output.

[0054] Once the sample solution has completely drained, the old internal phase inlet tubing can be removed from the internal phase tubing fixing device. The limiting pin is removed, the moving plate is moved to another position, and the limiting pin is reinserted to fix the moving plate in place. A new internal phase inlet tubing is then installed, and another sample is pumped in, thus achieving rapid replacement. The internal phase inlet tubing slides to the new position via the moving plate, preventing contamination between different samples caused by puncturing the same rubber cap. This process effectively ensures that the internal environment of the device is not contaminated by external factors and avoids cross-contamination between different sample solutions.

Claims

1. A droplet generating device, characterized in that: It includes a droplet generating pipe, an inner phase input pipe, an inner phase pipe fixing device, and a moving mechanism; the droplet generating pipe is rectangular or trapezoidal in shape, and an opening is provided on the top surface of the droplet generating pipe, and a rubber cover is provided at the opening; The moving mechanism is installed above the droplet generating pipe. The moving mechanism includes a moving plate, at least two limiting components, and at least two sets of parallel linear slide rails. The linear slide rails are installed on the top surface of the droplet generating pipe. Each set of linear slide rails includes a left slide rail and a right slide rail respectively located on the left and right sides of the rubber cover. A slider is slidably mounted on each linear slide rail. The moving plate has a mounting hole in the middle. The top of the slider is fixedly connected to the bottom surface of the moving plate. At least one set of limiting holes is provided on the moving plate on the left and right sides of the rubber cover. Each set of limiting holes includes multiple limiting holes arranged linearly. Each limiting component includes a limiting block and a limiting pin. The limiting block is fixedly installed on the top surface of the droplet generating pipe at a position corresponding to the limiting hole. The top of the limiting block has a limiting groove for the insertion of the limiting pin. The slider slides left and right in the linear slide rails, causing the moving plate to move left and right. When the moving plate moves to a set position, the limiting pin is inserted into the limiting hole and the limiting groove, thereby fixing the position of the moving plate. The inner phase pipe fixing device is a cylindrical body with an internal cavity. It has a pipe insertion port at the top and a pipe extension port at the bottom. A pipe clamping device is located in the lower middle part to clamp the inner phase input pipe. The pipe clamping device includes two locking knobs and two clamping parts. The locking knobs are symmetrically arranged on the outside of the inner phase pipe fixing device, and the clamping parts are located inside the cavity of the inner phase pipe fixing device. The locking knobs and clamping parts are connected by bolts. The bottom of the inner phase pipe fixing device is inserted into the mounting hole of the moving plate to fix the position of the inner phase pipe fixing device. The lower part of the inner phase input pipe is a puncture needle part that can pass through the rubber plug. The top of the inner phase input pipe is provided with an outwardly extending protrusion. The inner phase input pipe is inserted into the inner phase pipe fixing device. The protrusion rests on the edge of the pipe insertion port of the inner phase pipe fixing device to support the inner phase input pipe.

2. The droplet generating device according to claim 1, characterized in that: The inner phase input pipe is funnel-shaped, including an upper liquid-holding section and a lower puncture needle section, and a pipe cap is provided at the top of the inner phase input pipe.

3. The droplet generating device according to claim 1, characterized in that: The inner phase pipe fixing device is larger in the middle and smaller at the bottom. The bottom is used to insert into the mounting hole of the moving plate to fix the position of the inner phase pipe fixing device; the clamping part is made of rubber.

4. The droplet generating device according to claim 1, characterized in that: The moving mechanism also includes a fixed plate, which is fixedly installed on the top surface of the droplet generating pipe, and the limiting block and the moving rail are fixedly installed on the fixed plate; The left and right slide rails of each set of linear slide rails are on the same straight line.

5. The droplet generating device according to claim 1, characterized in that: The droplet generation pipe is provided with an inlet and an outlet at both ends, which are connected to an external phase injection pump and a PCR circulating water bath amplification device through pipes respectively.

6. The droplet generating device according to claim 1, characterized in that: It also includes a fixing frame for fixing the inner phase pipe fixing device. The fixing frame includes a base, a connecting rod, and a clamping rod respectively disposed on the left and right sides of the droplet generating device. The base is fixedly connected to the connecting rod, and the connecting rod is fixedly connected to the clamping rod. The clamping rods on the left and right sides clamp the inner phase pipe fixing device.

7. A droplet PCR system, characterized in that: The device includes the droplet generation apparatus according to any one of claims 1 to 6, and further includes a PCR circulating water bath amplification apparatus and an optical detection apparatus.

8. The droplet PCR system according to claim 7, characterized in that: It also includes an internal phase injection pump and an external phase injection pump. The internal phase injection pump is connected to the top of the internal phase input pipe via a pipe, and the external phase injection pump is connected to the inlet end of the droplet generation pipe via a pipe.

9. The droplet PCR system according to claim 7, characterized in that: The PCR circulating water bath amplification device includes a constant temperature zone, a variable temperature circulating zone, and reaction tubes. The reaction tubes are continuous S-shaped tubes that enter from the constant temperature zone, pass through the constant temperature zone and the variable temperature circulating zone in sequence, and then extend to connect with the optical detection device.

10. The droplet PCR system according to claim 7, characterized in that: The optical detection device includes a laser emitter, a dichroic mirror, a focusing mirror, a detector, and a microdroplet channel. The microdroplet channel is connected to the reaction tube of a PCR circulating water bath amplification device. The focusing mirror, dichroic mirror, and detector are arranged sequentially below the microdroplet channel. The laser emitter is located on one side of the dichroic mirror.

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