Digital PCR method for flow multi-index detection

Abstract

The present invention provides a digital PCR method for flow multi-index detection, comprising: placing an integrated microdroplet chip containing a sample to be tested in a digital PCR instrument, and making the reaction chamber located in a temperature control cavity; controlling the operation of a droplet generation module to generate microdroplets, and storing the generated microdroplets in the reaction chamber; controlling the operation of a PCR amplification module to make the temperature of the microdroplets in the reaction chamber reach a preset target temperature; controlling a fluorescence detection module to perform fluorescence detection on the microdroplets flowing out of the reaction chamber to obtain the fluorescence information of the flowing microdroplets, and simultaneously recording the preset target temperature corresponding to the obtained fluorescence information, and ensuring that during the fluorescence detection process, the reaction chamber is always located in the temperature control cavity, and the preset target temperature is set according to the detection rule in a gradient manner. The present invention can ensure that the microdroplets in the reaction chamber are constantly adjusted to be at the preset target temperature in real time according to the detection rule in a gradient manner, greatly improving the detection throughput and enhancing the detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of digital PCR instrument device design, and particularly relates to a digital PCR method for flow multi-index detection. Background Art

[0002] Digital PCR is the latest quantitative technology, which is nucleic acid quantification based on single-molecule PCR method for counting, and is an absolute quantification method. It mainly adopts the microfluidic or droplet-based method in the current popular research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactors or droplets of a biochip. The number of nucleic acid templates in each reactor is less than or equal to 1. After PCR cycling, the reactor with one nucleic acid molecule template will give a fluorescence signal, and the reactor without a template will have no fluorescence signal. According to the relative proportion and the volume of the reactor, the copy number of templates in the sample can be accurately calculated.

[0003] Digital PCR technology has the advantages of high sensitivity and accurate quantification, and has great application prospects in the fields of life science and molecular diagnosis. However, digital PCR technology is limited by the spectral distribution of fluorescent dyes. Only several fluorescent probes can be placed in a single PCR reaction system at the same time, which only corresponds to the detection of several nucleic acid sequences, and the multi-index detection ability is insufficient, making it difficult to meet the requirement of simultaneously distinguishing dozens of target sequences.

[0004] To improve the multi-index detection ability of digital PCR, one approach is to combine the melting curve technique, that is, to control the temperature change of the droplets to be detected during the detection, and cooperate with the sequence design of the fluorescent probe, so that single or multiple fluorescent probes show different fluorescence intensities at different temperatures, thus realizing the detection of more indexes under the same type of fluorescent dye. However, since the temperature of the droplets needs to be changed in real time during the detection process, the existing technical routes are all based on the platform of imaging digital PCR, that is, after the droplets are generated, they are spread out in the chip cavity, and then amplification and fluorescence imaging detection are completed in the cavity. This method has certain limitations. First of all, the imaging of different fluorescences by the imaging method is not completed synchronously. Therefore, the temperature points cannot change continuously, and every time a temperature point is reached, it is necessary to wait for the temperature to balance before imaging different fluorescence channels in turn. Limited by the temperature control speed and the fluorescence imaging speed, usually a single sample needs to be detected for more than 10 minutes, the detection speed is slow, and the throughput is low. Secondly, limited by the area of the chip cavity, the sample loading volume of a single sample is small and the total number of droplets is small, resulting in limited detection sensitivity and a small nucleic acid quantification range. These all limit the application of this technology in life science research and clinical diagnosis. That is to say, there are the following deficiencies in imaging digital PCR: the imaging of different fluorescences by the imaging method is not completed synchronously, so the temperature points cannot change continuously, and every time a temperature point is reached, it is necessary to wait for the temperature to balance before imaging different fluorescence channels in turn. Limited by the temperature control speed and the fluorescence imaging speed, usually a single sample needs to be detected for more than 10 minutes, the detection speed is slow, and the throughput is low; limited by the area of the chip cavity, the sample loading volume of a single sample is small and the total number of droplets is small, resulting in limited detection sensitivity and a small nucleic acid quantification range.

[0005] Flow-through fluorescence detection can detect all fluorescence channels of the droplets at the same time, and is not restricted by the spreading of the imaging method. The detection speed is fast, and the sample loading volume and the number of droplets are not restricted, which can overcome the problems of slow detection speed, limited sensitivity and small quantification range in the existing multi-index detection technology of digital PCR combined with temperature.

[0006] The applicant's previously filed invention patent application No. 202111381197.4 discloses an integrated digital PCR instrument and its control method, which has the technical advantages of high automation and integration levels, can improve the efficiency of detection and analysis operations, and reduce labor costs. In this technical solution, through the scheduling of a manipulator (i.e., a scheduling mechanism), the chip is sequentially scheduled between a droplet generation module, a flipping module, a PCR amplification module, a detection module, and a waste bin. Multiple chips work simultaneously to form an assembly line working mode. The chip sequentially completes the four digital PCR steps of droplet generation, chip flipping, droplet amplification, and droplet fluorescence detection. When using the flow cytometry method to detect sample droplets in this technical solution, since the temperature cycling module (i.e., the amplification module) and the fluorescence detection module are separately provided independently, the scheduling mechanism needs to transfer the microfluidic chip to the fluorescence detection module for detection after the temperature is constant, which makes it impossible to achieve continuous gradient constant temperature control of the sample droplets. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a digital PCR method for flow cytometry multi-index detection, which effectively overcomes the deficiency that the amplification module in the digital PCR instrument in the related art of the prior art cannot achieve continuous gradient constant temperature control of sample droplets.

[0008] To solve the above problems, the present invention provides a digital PCR method for flow cytometry multi-index detection, which is carried out using a digital PCR instrument. The digital PCR instrument includes a droplet generation module, a PCR amplification module, and a fluorescence detection module. Among them, the droplet generation module is used to generate micro-droplets in an integrated micro-droplet chip and store them in the reaction chamber it has, and the reaction chamber is located in the temperature control cavity of the PCR amplification module. The digital PCR method includes the following steps:

[0009] Place the integrated micro-droplet chip containing the sample to be tested in the digital PCR instrument, and make the reaction chamber located in the temperature control cavity.

[0010] Control the droplet generation module to operate to generate micro-droplets, and the generated micro-droplets are stored in the reaction chamber.

[0011] Control the PCR amplification module to operate so that the temperature of the micro-droplets in the reaction chamber is at a preset target temperature.

[0012] Control the fluorescence detection module to perform fluorescence detection on the micro-droplets flowing out of the reaction chamber to obtain the fluorescence information of the flowing micro-droplets, and synchronously record the preset target temperature corresponding to the obtained fluorescence information, and ensure that during the fluorescence detection process, the reaction chamber is always located in the temperature control cavity, and the preset target temperature is adjusted and set according to the detection rules in a gradient manner.

[0013] In some embodiments, the digital PCR instrument further includes an integration module and a rotation mechanism. The droplet generation module and the PCR amplification module are both located within the integration module. The rotation mechanism is used to drive the integration module to turn upside down by 180°; the digital PCR method further includes the following steps:

[0014] When the integrated microdroplet chip is placed in the digital PCR instrument, the bottom wall of the reaction chamber faces upward and remains in this state for the generation of microdroplets.

[0015] After the microdroplets are generated, control the rotation mechanism to operate and drive the integration module to turn upside down by 180° so that the bottom wall of the reaction chamber faces downward.

[0016] In some embodiments, the integration module includes a first working platform and a second working platform. The first working platform has a plurality of chip placement grooves for positioning and placing the integrated microdroplet chip. The second working platform is arranged parallel to the first working platform and has a working state close to the first working platform and a chip picking and placing state away from the first working platform. The droplet generation module and the PCR amplification module are located on the second working platform. When the second working platform is in the working state, the droplet generation module is respectively pressed and docked with the chip oil hole and the chip gas-liquid hole of the integrated microdroplet chip so as to be able to form microdroplets within the integrated microdroplet chip, and the reaction chamber of the integrated microdroplet chip is located within the temperature control cavity of the PCR amplification module. The rotation mechanism is used to drive the first working platform and the second working platform to turn upside down by 180° synchronously when the second working platform is in the working state.

[0017] In some embodiments, guiding slide rods are vertically arranged at the four corner regions corresponding to each other between the first working platform and the second working platform. A first return spring is sleeved on the outer periphery of the guiding slide rod. The first return spring is clamped between the first working platform and the second working platform. It further includes a pressing structure. The pressing structure can overcome the elastic force of the first return spring under the action of a downward pressure so that the second working platform is switched from the chip picking and placing state to the working state.

[0018] In some embodiments, the droplet generation module includes a generation pressing block. The length extension direction of the generation pressing block is parallel to the width direction of the second working platform. The two ends of the length of the generation pressing block are connected to the second working platform via connecting lugs. A first generation hole and a second generation hole are formed on the generation pressing block. The droplet generation module further includes a generation cover plate. The generation cover plate has a closing position for sealing the top surface of the chip oil hole and the chip gas-liquid hole when generating micro-droplets and realizing the press connection and communication between the gas path on the generation cover plate and the chip gas-liquid hole.

[0019] In some embodiments, the PCR amplification module includes a semiconductor heating element. A heat conducting block is connected to the first end of the semiconductor heating element. The temperature control cavity is formed on the end surface of the heat conducting block facing away from the semiconductor heating element. A heat dissipation structure is connected to the second end of the semiconductor heating element.

[0020] In some embodiments, the heat dissipation structure includes a first heat dissipation block in contact connection with the second end and a second heat dissipation block in contact connection with the first heat dissipation block. The heat dissipation area of the second heat dissipation block is larger than that of the first heat dissipation block.

[0021] In some embodiments, the heat dissipation structure further includes a third heat dissipation block and a heat conducting pipe. The first end of the heat conducting pipe is between the first heat dissipation block and the second heat dissipation block. The second end of the heat conducting pipe is in heat exchange connection with the third heat dissipation block.

[0022] In some embodiments, a heat insulation plate is annularly arranged at the connection position between the first heat dissipation block and the semiconductor heating element; and / or, a temperature measuring probe and a temperature fuse are arranged in the heat conducting block.

[0023] In some embodiments, the chip placement grooves are sequentially arranged at intervals along the length direction of the first working platform. Each of the droplet generation modules and the PCR amplification modules is arranged in one-to-one correspondence with each of the chip placement grooves.

[0024] A digital PCR method for flow multi-index detection provided by the present invention is based on the technical route of flow fluorescence detection. In the detection process, when droplets pass through the fluorescence detection point, different fluorescences are simultaneously excited and detected, without the need to switch filter plates for sequential detection as in the imaging method. Therefore, it can be detected under continuously changing temperatures, avoiding the waiting time required for temperature equilibrium and the time for switching between different fluorescence channels. The detection time is compressed from more than 10 minutes in the existing method to 1 minute, and the speed is increased by 10 times, significantly improving the detection throughput. At the same time, the generated droplets are stored in the reaction chamber without the need to form a single-layer flat layout, so it is not limited by the chip area. A 50-μL system can be loaded, generating more than 100,000 droplets. Compared with the sample loading volume of less than 25 μL and the total number of droplets of less than 30,000 in the traditional imaging method, the sensitivity and quantitative range are significantly improved. More importantly, the PCR amplification module in this application is always tightly wrapped around the reaction chamber during the outflow of micro-droplets, which can ensure that the micro-droplets in the reaction chamber are constantly adjusted to the preset target temperature in real time according to the detection rules, greatly improving the detection throughput and detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic structural diagram of the compact digital PCR instrument according to the embodiment of the present invention (the overall structure omits some structures such as the outer shell);

[0026] Figure 2 is Figure 1 a schematic three-dimensional structure diagram of the integrated micro-droplet chip in

[0027] Figure 3 is Figure 1 a schematic three-dimensional structure diagram of the integrated module in

[0028] Figure 4 is Figure 3 a partial enlarged view of part A in

[0029] Figure 5 is Figure 1 a schematic three-dimensional structure diagram of the droplet generation module in

[0030] Figure 6 is a schematic three-dimensional structure diagram of the PCR amplification module;

[0031] Figure 7 is a schematic three-dimensional structure diagram after the flipping mechanism is assembled;

[0032] Figure 8 is a schematic three-dimensional structure diagram of the fluorescence detection module and the detection crimping module assembled in the instrument;

[0033] Figure 9 is Figure 8Schematic three-dimensional structure diagram of the detection and crimping module therein;

[0034] Figure 10 is Figure 8 Schematic three-dimensional structure diagram of the fluorescence detection module therein;

[0035] Figure 11 Schematic three-dimensional structure diagram of the instrument in the state before micro-droplet generation;

[0036] Figure 12 Schematic three-dimensional structure diagram of the instrument in the state of micro-droplet generation;

[0037] Figure 13 Schematic three-dimensional structure diagram of the instrument in the state of 180°-flipped PCR amplification;

[0038] Figure 14 Schematic partial three-dimensional structure diagram in the state of fluorescence detection;

[0039] Figure 15 Schematic flow chart of the digital PCR method for multi-index detection by flow cytometry according to the embodiment of the present invention;

[0040] Figure 16 Schematic diagram of state switching at different preset target temperatures for the digital PCR method for multi-index detection by flow cytometry according to the embodiment of the present invention;

[0041] Figure 17 Micro-droplet detection results of multi-index detection at different temperature gradients;

[0042] Figure 18 Theoretical fluorescence change curve of multi-index detection at different temperature gradients.

[0043] The reference numerals are shown as:

[0044] 1. First working platform; 11. Vertical plate; 12. Connecting plate; 121. Guide slide bar; 122. First return spring; 123. Pressing rod; 124. Roller; 125. Towing rope; 126. Linear bearing; 13. Chip placement groove; 131. Guide spring; 2. Second working platform; 3. Droplet generation module; 31. Generate first hole; 32. Generate second hole; 33. Generate pressing block; 34. Generate cover plate; 35. Electromagnet; 36. Rotating shaft; 37. Second return spring; 38. Guide rod; 39. Third return spring; 310. Cover plate gasket; 4. PCR amplification module; 41. Semiconductor heating element; 42. Heat insulation plate; 43. Heat conducting block; 44. First heat sink; 47. Third heat sink; 48. Second heat sink; 49. Heat conducting pipe; 5. Rotating mechanism; 51. Rotary motor; 52. Transmission component; 6. Fluorescence detection module; 61. Objective lens; 62. Optical path component; 63. Lifting motor; 64. Translational motor; 7. Detection and crimping module; 70. Detection oil circuit; 71. Detection and crimping block; 72. Moving block; 73. First crimping motor; 74. Second crimping motor; 75. First gasket; 76. Air cavity; 77. Light source; 100. Integrated micro-droplet chip; 101. Chip oil and liquid hole; 102. Chip gas and liquid hole; 103. Reaction chamber; 104. Fluorescence detection area; 105. Sampling cavity; 201. Device bottom plate; 2021. Longitudinal movement guide rail; 2022. Longitudinal movement motor. Detailed implementation manner

[0045] Refer to in combination Figures 1 to 18As shown, according to an embodiment of the present invention, a structurally compact digital PCR instrument is provided, which includes an integration module (not labeled in the figure) and a rotation mechanism 5 (not labeled in the figure). The integration module includes a first working platform 1 and a second working platform 2. The first working platform 1 has a plurality of chip placement grooves 13 for positioning and placing an integrated microdroplet chip 100. It can be understood that each chip placement groove 13 positions and places an integrated microdroplet chip 100. The second working platform 2 is arranged parallel to the first working platform 1 and has a working state close to the first working platform 1 and a chip picking and placing state away from the first working platform 1. The second working platform 2 has a droplet generation module 3 and a PCR amplification module 4. When the second working platform 2 is in the working state, the droplet generation module 3 is respectively pressed and docked with the chip oil hole 101 and the chip gas-liquid hole 102 of the integrated microdroplet chip 100 to form microdroplets in the integrated microdroplet chip 100. The formed microdroplets will be stored in the reaction chamber 103 of the integrated microdroplet chip 100, and the reaction chamber 103 of the integrated microdroplet chip 100 is located in the temperature control cavity (not shown and not labeled) of the PCR amplification module 4. The rotation mechanism 5 is used to drive the first working platform 1 and the second working platform 2 to synchronously flip 180° when the second working platform 2 is in the working state, so that the reaction chamber 103 is upside down. At this time, it is ensured that the microdroplets stored in the reaction chamber 103 can be at the bottom of the reaction chamber 103 under the action of their own weight, thereby ensuring that the temperature control cavity of the PCR amplification module 4 can more efficiently control the temperature of the microdroplets (i.e., the reaction solution).

[0046] In this technical solution, the droplet generation module 3 and the PCR amplification module 4 are integrated on the second working platform 2. At the same time, the second working platform 2 has a working state and a chip picking and placing state and can switch between the two states. The synchronous flipping of the first working platform 1 and the second working platform 2 is realized by the rotation drive of the rotation mechanism 5, without using the scheduling mechanism in the prior art to switch the integrated microdroplet chip 100 between different working modules or to flip it, making the structure of the digital PCR instrument more compact and the instrument volume smaller, thus having stronger portability, and at the same time, the manufacturing cost is relatively reduced.

[0047] Specifically refer to Figure 7 As shown, the shape of the aforementioned chip placement groove 13 matches the bottom surface shape of the integrated microdroplet chip 100. As a preferred embodiment, a corresponding guiding spring 131 is arranged on one side of the aforementioned chip placement groove 13 away from the operation side of the operator, so that the position of the chip can be more reliable when placing the chip, and when taking out the chip, it can apply force to the end of the chip to facilitate the chip to pop out of the chip placement groove 13 and thus facilitate the removal of the chip.

[0048] In some embodiments, the integrated module further includes vertical plates 11 which are arranged at intervals left and right. There are two vertical plates 11 which are placed vertically at intervals. The two ends of the length of the first working platform 1 are pivotally mounted (for example, rotationally connected through a rolling rotating shaft) at the top positions of the two vertical plates 11. The bottom positions of the two vertical plates 11 are connected into one body through a connecting plate 12 to improve the structural reliability between the two vertical plates 11. The rotating mechanism 5 includes a rotary motor 51 and a transmission component 52. The rotary motor 51 is fixedly connected to one of the vertical plates 11. The rotary motor 51 is drivingly connected to one end of the first working platform 1 through the transmission component 52 to drive the first working platform 1 to rotate by a preset angle. In a specific embodiment, the aforementioned rotary motor 51 and transmission component 52 can be realized by using a commercially available rotary drive component.

[0049] In this technical solution, the rotating mechanism 5 can drive the first working platform 1 to turn over 180° up and down, so that the top surface of the integrated micro-droplet chip 100 faces upward to generate micro-droplets. After the micro-droplets are generated, it rotates so that the first working platform 1 and the second working platform 2 turn over up and down synchronously, and then the top surface of the integrated micro-droplet chip 100 faces downward, so that the micro-droplets in the reaction chamber 103 can be at the bottom of the reaction chamber 103 under the action of their own weight, thereby ensuring the reliable temperature control of the micro-droplets in the reaction chamber 103 by the PCR amplification module 4. At the same time, the rotating mechanism 5 can also drive the first working platform 1 to swing back and forth, that is, rotate the aforementioned preset angle (generally ±10° is sufficient) during the amplification process, so as to accelerate the uniform process of the temperature field through fluid convection, improve the heat transfer efficiency and temperature uniformity in the micro-droplet sample, and also reduce the temperature uniformity requirement of the temperature control module itself, thereby improving the efficiency of the amplification link.

[0050] In some embodiments, specifically refer to Figure 8 As shown, the structure-compact digital PCR instrument further includes a device bottom plate 201 and a longitudinal movement component (not labeled in the figure). The longitudinal movement component includes a longitudinal movement guide rail 2021 and a longitudinal movement motor 2022. The bottom end of the integrated module is slidably connected between the longitudinal movement guide rail 2021 and the device bottom plate 201, and the longitudinal movement motor 2022 can drive the entire integrated module to reciprocally move linearly along the guiding direction of the longitudinal movement guide rail 2021. The guiding direction of the longitudinal movement guide rail 2021 is a horizontal direction perpendicular to the length direction of the first working platform 1.

[0051] In this technical solution, the longitudinal movement component can drive the integrated module to adjust its position in the horizontal longitudinal direction of the instrument, so that the integrated module can adjust its corresponding position with the corresponding processing modules (such as the subsequent detection and crimping module 7 and the fluorescence detection module 6) in different process flows, simplifying the structural design of the processing modules. For example, there is no need for the processing modules to control the displacement in the longitudinal direction, that is, the control is simplified.

[0052] Further referring to Figure 8 As shown, in some embodiments, the compact digital PCR instrument further includes a fluorescence detection module 6 on the first side of the integrated module and a detection and crimping module 7 on the second side of the integrated module. The first side and the second side are respectively one of the top side and the bottom side of the first operation platform 1. In a specific embodiment, with Figure 8 the shown orientation as a reference, the aforementioned first side is also the upper side of the integrated module, and the second side is also the lower side of the integrated module. The fluorescence detection module 6 can be controlled to move up and down to be away from or close to the first operation platform 1 to achieve position correspondence with the fluorescence detection area 104 of the integrated micro-droplet chip 100 and perform fluorescence detection. The detection and crimping module 7 can be controlled to move up and down to be close to or away from the second operation platform 2 to achieve tight docking with the generation first hole 31 and the generation second hole 32 in the droplet generation module 3. Furthermore, the detection oil in the detection oil circuit 70 (specifically, it can be understood as a detection oil bottle) can enter the reaction chamber 103 to float the amplified micro-droplets to the fluorescence detection area 104 and enter the sample addition chamber 105 of the integrated micro-droplet chip 100. The fluorescence detection module 6 and the detection and crimping module 7 can be controlled to move to be position-corresponding to each integrated micro-droplet chip 100.

[0053] In this technical solution, by respectively arranging the fluorescence detection module 6 and the detection and crimping module 7 on the upper and lower sides of the integrated module, the fluorescence detection function of the micro-droplets of the PCR instrument of the present invention can be integrated, realizing the function compounding of the instrument.

[0054] Specifically referring to Figure 9 As shown, in some embodiments, the detection and crimping module 7 includes an air chamber 76 and a crimping component (not labeled in the figure) connected to the air chamber 76. The air chamber 76 is used to provide negative pressure for the droplet generation module 3 and positive pressure for the detection and crimping module 7. Among them, during the operation of the droplet generation module 3, negative pressure is provided for the micro-droplet generation process, and during the operation of the fluorescence detection module 6, positive pressure is provided for the supply of detection oil.

[0055] In this technical solution, the air chamber 76 can serve as the pressure vessel of the instrument and at the same time can also serve as the assembly carrier of the crimping component, which can further simplify the structural design of the instrument and improve the structural compactness of the instrument.

[0056] Continue to refer to Figure 9 As shown, in some embodiments, the crimping assembly includes a detection crimping block 71 and a moving block 72. The detection crimping block 71 is slidably connected to the moving block 72 and can be driven to lift and lower by a first crimping motor 73. The moving block 72 is slidably connected to the air chamber 76 and can be driven by a second crimping motor 74 to traverse along the length direction of the first working platform 1, so as to realize the sequential docking of the crimping assembly to each of the plurality of integrated micro-droplet chips 100. It can be understood that corresponding guide rails are also provided in the crimping assembly to guide the smooth lifting and traversing of the detection crimping block 71; in a preferred embodiment, a light source 77 is further provided on the moving block 72 to provide a light field for the camera of the fluorescence detection module 6. Setting the light source 77 on the moving block 72 makes the relative position of the light source 77 and the detection crimping block 71 stable, and the two move synchronously, simplifying the control.

[0057] In some embodiments, the detection crimping block 71 is formed with a first detection through hole and a second detection through hole corresponding to the chip oil hole 101 and the chip gas-liquid hole 102 respectively. The detection oil enters the reaction chamber 103 through the first detection through hole and the second detection through hole. The detection crimping block 71 is provided with a first gasket 75 to form a seal at the crimping position when the detection crimping block 71 is crimped with the droplet generation module 3.

[0058] Combined with reference to Figure 3 and Figure 4 As shown, in some embodiments, guide slide bars 121 are vertically provided at the four corner regions corresponding to each other between the first working platform 1 and the second working platform 2. In a specific embodiment, the guide slide bars 121 are slidably connected to the first working platform 1 and / or the second working platform 2 through linear bearings 126. A first return spring 122 is sleeved on the outer periphery of the guide slide bars 121. The first return spring 122 is clamped between the first working platform 1 and the second working platform 2 to be able to apply a return elastic force for separating the first working platform 1 and the second working platform 2 from each other. It further includes a pressing-down structure (not labeled in the figure). The pressing-down structure can overcome the elastic force of the first return spring 122 under the action of a downward pressure, so that the second working platform 2 is switched from the chip picking and placing state to the working state. The aforementioned downward pressure can be specifically realized by an operator pressing down manually.

[0059] In this technical solution, by applying a force to the downward pressing structure to overcome the elastic force of the first return spring 122, the first working platform 1 and the second working platform 2 are brought closer, and thus the droplet generation module 3 and the integrated micro-droplet chip 100 are pressed together. When the downward pressing structure is reversely forced, that is, an upward pulling force is applied, the first return spring 122 can assist the second working platform 2 to switch from the working state to the chip picking and placing state, and prevent the downward pressing structure from falling under its own weight, facilitating the picking and placing of the chip.

[0060] Specifically, refer to Figure 4 As shown, in some embodiments, the downward pressing structure includes a pressing rod 123 hinged to the two ends of the length of the second working platform 2 and a roller 124. A traction rope 125 (such as a steel wire) is connected between the pressing rod 123 and the first working platform 1. The traction rope 125 forms a wrap angle on the roller 124 (that is, the traction rope 125 partially wraps the outer circumferential surface of the roller 124). When the pressing rod 123 is pressed downward toward the first working platform 1, the traction rope 125 can pull the first working platform 1 and the second working platform 2 to approach each other and be in the working state. Preferably, when the pressing rod 123 is pressed downward to the lowest position toward the first working platform 1, the connection point of the traction rope 125 and the second working platform 2 is on the side of the center point of the roller 124 close to the first working platform 1.

[0061] In this technical solution, through the direction-changing guiding action of the roller 124 and the pulling connection action of the rope, the state of the second working platform 2 can be driven to switch when the position of the pressing rod 123 changes. The structure is simple. Especially after the pressing rod 123 is pressed down, since the connection point of the traction rope 125 and the pressing rod 123 is below the center point of the roller 124, the structure is self-locked, and there is no need to separately set a corresponding locking device, further simplifying the structural design.

[0062] Specifically, refer to Figure 5 As shown, the droplet generation module 3 includes a generation pressing block 33. The length extension direction of the generation pressing block 33 is parallel to the width direction of the second working platform 2, and the two ends of the length of the generation pressing block 33 are connected to the second working platform 2 via connecting lugs (not labeled in the figure). The first generation hole 31 and the second generation hole 32 are both formed on the generation pressing block 33. The droplet generation module 3 further includes a generation cover plate 34. The generation cover plate 34 has a covering position where the top surface of the chip oil hole 101 and the chip gas-liquid hole 102 are sealed by a cover plate gasket 310 when generating micro-droplets, and the gas path (not labeled in the figure) on the generation cover plate 34 is press-connected and communicated with the chip gas-liquid hole 102.

[0063] In this technical solution, both ends of the forming block 33 are connected to the second working platform 2 via connecting lugs, with a reliable and simple structure.

[0064] In some embodiments, the forming cover plate 34 is hinged to the forming block 33 via a rotating shaft 36. An electromagnet 35 is provided on the forming block 33. When the electromagnet 35 is powered on, the forming cover plate 34 is attracted and rotates around the rotating shaft 36 to be in the covering position. In this technical solution, the attraction of the forming cover plate 34 is achieved through the electromagnet 35, and both the structure and control are relatively simple.

[0065] In some embodiments, there are two electromagnets 35, and the two electromagnets 35 are respectively located at both ends of the length of the forming block 33. Suction blocks corresponding to the positions of the respective electromagnets 35 are respectively provided at both ends of the length of the forming cover plate 34. The two electromagnets 35 being respectively located at both ends of the length of the forming block 33 can ensure the smooth closing of the forming cover plate 34 and can ensure the reliable sealing of the forming cover plate 34.

[0066] In some embodiments, a second return spring 37 is sleeved on the rotating shaft 36 for switching the forming cover plate 34 from the covering position to the open position when the electromagnet 35 is powered off. In this technical solution, by providing the second return spring 37, the forming cover plate 34 can be opened by its own elastic force when the electromagnet 35 is powered off. The aforementioned second return spring 37 can specifically be a torsion spring.

[0067] In some embodiments, a hand-held protrusion (not shown in the figure) is provided on one side edge of the forming cover plate 34 away from the rotating shaft 36, facilitating an operator to apply force to the hand-held protrusion to assist in opening or closing the forming cover plate 34. A cover plate sealing gasket 310 is provided on one side end surface of the forming cover plate 34 facing the forming block 33 and is arranged around each of the forming first holes 31 and forming second holes 32.

[0068] In some embodiments, both ends of the length of the forming block 33 are connected to the second working platform 2 via the connecting lugs in a vertically slidable manner. A guide rod 38 is provided between the top surface of the forming block 33 and the bottom surface of the second working platform 2. A third return spring 39 is sleeved outside the guide rod 38, and the third return spring 39 is clamped between the bottom surface of the second working platform 2 and the top surface of the forming block 33. In this technical solution, while both ends of the forming block 33 are connected to the second working platform 2 via the connecting lugs in a vertically slidable manner, the third return spring 39 clamped between the second working platform 2 and the forming block 33 can buffer and adjust the horizontal state of the forming block 33, thereby ensuring the crimping sealing performance of the forming block 33 on the integrated micro-droplet chip 100.

[0069] For specific referenceFigure 6 As shown, in some embodiments, the PCR amplification module 4 includes a semiconductor heating element 41. A heat conduction block 43 is connected to the first end of the semiconductor heating element 41. The temperature control cavity is formed on the end face of the heat conduction block 43 facing away from the semiconductor heating element 41. A heat dissipation structure (not labeled in the figure) is connected to the second end of the semiconductor heating element 41. The aforementioned heat conduction block 43 can evenly transfer the heat of the semiconductor heating element 41 to the temperature control cavity, so as to evenly control the temperature of the micro-droplets in the reaction chamber 103. The aforementioned semiconductor heating element 41 can specifically adopt a Peltier element. According to actual needs, multiple Peltier elements can be used.

[0070] In some embodiments, the heat dissipation structure includes a first heat dissipation block 44 in contact connection with the second end and a second heat dissipation block 48 in contact connection with the first heat dissipation block 44. The heat dissipation area of the second heat dissipation block 48 is larger than that of the first heat dissipation block 44, so as to quickly dissipate the heat at the first end of the semiconductor heating element 41.

[0071] In some embodiments, the heat dissipation structure further includes a third heat dissipation block 47 and a heat conduction tube 49. The aforementioned heat conduction tube 49 can specifically adopt a heat pipe. There is a heat conduction medium in the heat pipe, which has a high heat transfer efficiency. The first end of the heat conduction tube 49 is between the first heat dissipation block 44 and the second heat dissipation block 48, and the second end of the heat conduction tube 49 is in heat exchange connection with the third heat dissipation block 47. In this technical solution, the heat conduction tube 49 can quickly transfer and conduct the heat at the first heat dissipation block 44 and the second heat dissipation block 48 to the third heat dissipation block 47 for dissipation. The position of the third heat dissipation block 47 can be flexibly selected by adjusting the length of the heat conduction tube 49, thereby greatly increasing the heat dissipation area and achieving the purpose of quickly dissipating heat.

[0072] In some embodiments, a heat insulation plate 42 is annularly arranged at the connection position between the first heat dissipation block 44 and the semiconductor heating element 41 to prevent cold and heat from short-circuiting and mixing, reducing energy consumption; and / or, a temperature measurement probe (not shown in the figure) and a temperature fuse (not shown in the figure) are arranged in the heat conduction block 43. The temperature probe can detect the real-time temperature at the heat conduction block 43, and the temperature fuse can cut off the power supply of the semiconductor heating element 41 when the real-time temperature of the heat conduction block 43 exceeds a preset value, thereby achieving the purpose of constant temperature control.

[0073] In some embodiments, the chip placement grooves 13 are sequentially arranged at intervals along the length direction of the first working platform 1. Each of the droplet generation modules 3 and the PCR amplification modules 4 is arranged in one-to-one correspondence with each of the chip placement grooves 13, so as to improve the processing throughput of the instrument.

[0074] According to an embodiment of the present invention, there is also provided a control method for the above-mentioned compact digital PCR instrument, including the following steps:

[0075] Place the integrated microdroplet chip 100 carrying the sample solution into the chip placement groove 13;

[0076] Control the second working platform 2 to switch from the chip picking and placing state to the working state;

[0077] Control the droplet generation module 3 to operate to generate microdroplets in the integrated microdroplet chip 100, and store the microdroplets in the reaction chamber 103 of the integrated microdroplet chip 100;

[0078] After the microdroplets are generated, control the first working platform 1 and the second working platform 2 to flip 180° synchronously, and then control the PCR amplification module 4 to operate to amplify the microdroplets in the reaction chamber 103;

[0079] After the amplification is completed, control the detection and crimping module 7 and the fluorescence detection module 6 to move towards the integrated microdroplet chip 100. After the generation first hole 31 and the generation second hole 32 of the detection and crimping module 7 are docked with the droplet generation module 3, control the detection and crimping module 7 to operate to drive the amplified microdroplets from the reaction chamber 103 through the fluorescence detection area 104 of the integrated microdroplet chip 100 into the sample addition cavity 105 of the integrated microdroplet chip 100, and control the fluorescence detection module 6 to perform fluorescence detection in the fluorescence detection area 104.

[0080] In this technical solution, the droplet generation module 3 and the PCR amplification module 4 are integrated on the second working platform 2. At the same time, the second working platform 2 has a working state and a chip picking and placing state and can switch between the two states, and the synchronous flipping of the first working platform 1 and the second working platform 2 is realized by the rotation drive of the rotating mechanism 5. There is no need to use the scheduling mechanism in the prior art to switch the integrated microdroplet chip 100 between different working modules or to flip it, making the structure of the digital PCR instrument more compact, the instrument volume smaller, thus having stronger portability, and at the same time the manufacturing cost is relatively reduced.

[0081] In some embodiments, the control method further includes: during the operation of the PCR amplification module 4, controlling the first working platform 1 and the second working platform 2 to swing reciprocally synchronously, which can accelerate the uniform process of the temperature field through fluid convection, improve the heat transfer efficiency and temperature uniformity in the micro-droplet sample, and also reduce the requirement for the temperature uniformity of the temperature control module itself, thereby improving the efficiency of the amplification step; and / or, after the fluorescence detection is completed, controlling the first working platform 1 and the second working platform 2 to rotate reversely and synchronously by 180°, and then controlling to switch the second working platform 2 from the working state to the chip picking and placing state.

[0082] Specifically refer to Figure 15 and Figure 16 As shown, according to an embodiment of the present invention, there is also provided a digital PCR method for flow-through multi-index detection, which is performed using the above digital PCR instrument; the digital PCR method includes the following steps:

[0083] Place the integrated micro-droplet chip 100 containing the sample to be tested in the digital PCR instrument, and make the reaction chamber 103 located in the temperature control cavity.

[0084] Control the droplet generation module 3 to operate to generate micro-droplets, and the generated micro-droplets are stored in the reaction chamber 103.

[0085] Control the PCR amplification module 4 to operate so that the temperature of the micro-droplets in the reaction chamber 103 is at a preset target temperature.

[0086] Control the fluorescence detection module 6 to perform fluorescence detection on the micro-droplets flowing out of the reaction chamber 103 to obtain the fluorescence information of the flowing micro-droplets, and synchronously record the preset target temperature corresponding to the obtained fluorescence information, and ensure that during the fluorescence detection process, the reaction chamber 103 is always located in the temperature control cavity, and the preset target temperature is adjusted and set according to the detection rules in a gradient manner.

[0087] In this technical solution, based on the technical route of flow cytometry fluorescence detection, during the detection process, when the droplets pass through the fluorescence detection point, different fluorescences are simultaneously excited and detected, without the need to switch filter plates for sequential detection as in the imaging method. Therefore, it can be detected under continuously changing temperatures, avoiding the waiting time required for temperature equilibrium and the time for switching between different fluorescence channels. The detection time is compressed from over 10 minutes in the existing method to 1 minute, with the speed increased by 10 times, significantly improving the detection throughput. At the same time, the generated droplets are stored in the reaction chamber 103 and do not need to form a single-layer flat layout. Therefore, it is not limited by the chip area and can sample a 50-μL system to generate more than 100,000 droplets, significantly improving the sensitivity and quantitative range compared with the sample loading volume of less than 25 μL and the total number of droplets of less than 30,000 in the traditional imaging method. More importantly, the PCR amplification module in this application always tightly surrounds and wraps the reaction chamber 103 during the outflow of the microdroplets, ensuring that the microdroplets in the reaction chamber 103 can be adjusted in real time according to the detection rules and gradients to be constantly maintained at the preset target temperature, greatly improving the detection throughput and enhancing the detection efficiency.

[0088] When the integrated microdroplet chip 100 can be driven to flip 180° up and down, the digital PCR method further includes the following steps:

[0089] When the integrated microdroplet chip 100 is placed in the digital PCR instrument, the bottom wall of the reaction chamber 103 is oriented upward and maintained in this state for the generation of microdroplets.

[0090] After the microdroplets are generated, control the rotation mechanism 5 to operate and drive the integrated module to flip 180° up and down so that the bottom wall of the reaction chamber 103 faces downward, which can ensure sufficient heat transfer and temperature adjustment of the microdroplets in the reaction chamber 103 by the PCR amplification module.

[0091] The following further elaborates on the digital PCR method for flow cytometry multi-index detection of the present invention in conjunction with a specific embodiment:

[0092] Put the microdroplet chip containing the sample to be tested (i.e., the aforementioned integrated microdroplet chip 100, the same below) into the flipping module (i.e., the overall component composed of the aforementioned integrated module and the rotation mechanism 5, the same below), as Figure 11 , when the flipping module is in the open state, the chip can be placed in or taken out of the flipping module.

[0093] Change the flipping module to the compressed state, as Figure 12, in the pressed state, the droplet generation module is hermetically connected to the micro-droplet chip. The droplet generation module can provide driving air pressure and micro-droplet generation oil to achieve droplet generation. In the pressed state, the amplification module is in close contact with the reaction tube of the micro-droplet chip (i.e., the reaction chamber 103, the same below), and can be used as a temperature control unit in the nucleic acid amplification process to continuously control the temperature of the micro-droplets in the reaction tube to achieve nucleic acid amplification.

[0094] When the instrument is started, droplet generation begins, and all automated processes are controlled by the control module for all other modules. The specific pressure that the droplet generation module can provide, the micro-droplet generation oil and the sample in the micro-droplet chip enter the chip pipeline respectively under the action of air pressure, interact with each other in the pipeline to generate micro-droplets with uniform size, and are collected in the reaction tube of the micro-droplet chip to complete droplet generation.

[0095] The flipping module flips the chip by 180°, as Figure 3 , and through the action of gravity, the generated micro-droplets are collected in the heating area of the reaction tube.

[0096] The amplification module is started to control the temperature of the micro-droplets in the reaction tube. In this embodiment, the temperature cycling process is to first perform a pre-denaturation at 95°C for 10 minutes, and then 40 temperature cycles, with 95°C for 10 seconds and 60°C for 25 seconds in each cycle (i.e., the aforementioned detection rule gradient).

[0097] After amplification is completed, droplet fluorescence detection begins. The droplet generation pressure module provides specific air pressure and micro-droplet detection oil for the fluorescence detection module. Under the action of air pressure, the micro-droplet detection oil enters the micro-droplet chip and drives the droplets to be detected in the reaction tube into the chip pipeline to form a droplet queue. The fluorescence detection module aligns the fluorescence detection point with the chip pipeline through which the droplet queue passes. When the droplet queue passes through the fluorescence detection point, the fluorescence excitation and detection of each droplet are completed. At the same time, the amplification module controls the temperature of the droplets to be detected in real time, which can be continuously variable temperature or stepped variable temperature. The detected droplet fluorescence signal is converted into a value and recorded by the control module, and the droplet temperature at the time of detection is also recorded synchronously. The detected droplet fluorescence signal and temperature information are plotted into a one-dimensional fluorescence scatter plot, as Figure 17 , and quantitative information of different templates can be obtained for different temperature changes. Its theoretical fluorescence change curve is as Figure 18 . By analyzing the data of different fluorescence channels at different temperatures, the copy number content of each target template corresponding to each temperature can be calculated, so as to calculate the copy number of all target templates in the original sample and achieve multi-index detection of digital PCR.

[0098] It is easy for those skilled in the art to understand that, on the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed.

[0099] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. The above is only the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can still be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.

Claims

1. A digital PCR method for flow multi-index detection, characterized in that: A digital PCR instrument is used for the PCR, the digital PCR instrument comprising a droplet generation module (3), a PCR amplification module (4) and a fluorescence detection module (6), wherein the droplet generation module (3) is used to generate microdroplets in an integrated microdroplet chip (100) and store the microdroplets in a reaction chamber (103) thereof, and the reaction chamber (103) is located in a temperature-controlled chamber of the PCR amplification module (4); the digital PCR method comprises the following steps: Placing the integrated micro-droplet chip (100) containing the sample to be tested in the digital PCR instrument, and placing the reaction chamber (103) in the temperature-controlled chamber; Controlling the droplet generation module (3) to operate to generate micro-droplets, wherein the generated micro-droplets are stored in the reaction chamber (103); Controlling the PCR amplification module (4) to operate so that the temperature of the micro-droplets in the reaction chamber (103) is at a preset target temperature; Controlling the fluorescence detection module (6) to perform fluorescence detection on the micro-droplets flowing out of the reaction chamber (103) to obtain fluorescence information of the flowing micro-droplets, and synchronously recording a preset target temperature corresponding to the obtained fluorescence information, and ensuring that during the fluorescence detection process, the reaction chamber (103) is in the temperature control chamber, and the preset target temperature is adjusted and set according to the detection rule gradient; The digital PCR instrument further comprises an integrated module and a rotating mechanism (5), wherein the droplet generation module (3) and the PCR amplification module (4) are both located in the integrated module, and the rotating mechanism (5) is used to drive the integrated module to flip up and down by 180°; the digital PCR method further comprises the following steps: When the integrated micro-droplet chip (100) is placed in the digital PCR instrument, the bottom wall of the reaction chamber (103) is directed upwards and maintained in this state to generate micro-droplets; After the micro-droplets are generated, the rotating mechanism (5) is controlled to drive the integrated module to flip up and down 180 degrees so that the bottom wall of the reaction chamber (103) faces downward.

2. The digital PCR method according to claim 1, characterized in that: The integrated module comprises a first operating platform (1) and a second operating platform (2); the first operating platform (1) has a plurality of chip placement grooves (13) for positioning and placing the integrated micro-droplet chip (100); the second operating platform (2) is arranged in parallel with the first operating platform (1) and has a working state close to the first operating platform (1) and a chip placement state far from the first operating platform (1); the droplet generation module (3) and the PCR amplification module (4) are located on the second operating platform (2); when the second operating platform (2) is in the In the working state, the droplet generation module (3) is pressed and docked with the chip oil-liquid hole (101) and the chip gas-liquid hole (102) of the integrated micro-droplet chip (100) so as to form micro-droplets in the integrated micro-droplet chip (100), and the reaction chamber (103) of the integrated micro-droplet chip (100) is located in the temperature control chamber of the PCR amplification module (4). The rotating mechanism (5) is used to drive the first working platform (1) and the second working platform (2) to synchronously flip 180 degrees when the second working platform (2) is in the working state.

3. The digital PCR method according to claim 2, characterized in that: A guide slide bar (121) is vertically arranged at each of the four corresponding corner areas of the first working platform (1) and the second working platform (2); a first return spring (122) is sleeved on the outer periphery of the guide slide bar (121); the first return spring (122) is clamped between the first working platform (1) and the second working platform (2); and further comprises a downward pressing structure; the downward pressing structure can overcome the elastic force of the first return spring (122) under the action of downward pressure so that the second working platform (2) switches from the chip placement state to the working state.

4. The digital PCR method according to claim 3, characterized in that: The droplet generation module (3) comprises a generation block (33), the length extension direction of the generation block (33) is parallel to the width direction of the second working platform (2), and the two ends of the length of the generation block (33) are connected to the second working platform (2) via connecting ears, and the generation block (33) is formed with a first generation hole (31) and a second generation hole (32). The droplet generation module (3) also comprises a generation cover plate (34), and the generation cover plate (34) has a covering position for sealing the top surface of the chip oil-liquid hole (101) and the chip gas-liquid hole (102) when generating micro-droplets and realizing the pressure connection between the gas path on the generation cover plate (34) and the chip gas-liquid hole (102).

5. The digital PCR method according to claim 2, characterized in that: The PCR amplification module (4) comprises a semiconductor heating element (41), a first end of the semiconductor heating element (41) being connected to a heat conducting block (43), the temperature control cavity being formed on an end surface of the heat conducting block (43) facing away from the semiconductor heating element (41), and a second end of the semiconductor heating element (41) being connected to a heat dissipation structure.

6. The digital PCR method according to claim 5, characterized in that: The heat dissipation structure comprises a first heat dissipation block (44) contacting and connected to the second end, and a second heat dissipation block (48) contacting and connected to the first heat dissipation block (44), wherein the heat dissipation area of ​​the second heat dissipation block (48) is greater than the heat dissipation area of ​​the first heat dissipation block (44).

7. The digital PCR method according to claim 6, characterized in that: The heat dissipation structure further comprises a third heat dissipation block (47) and a heat conduction pipe (49), wherein a first end of the heat conduction pipe (49) is located between the first heat dissipation block (44) and the second heat dissipation block (48), and a second end of the heat conduction pipe (49) is connected to the third heat dissipation block (47) for heat exchange.

8. The digital PCR method according to claim 7, characterized in that: A heat insulation plate (42) is arranged in a circle at the connection position between the first heat dissipation block (44) and the semiconductor heating element (41); and / or a temperature measuring probe and a temperature fuse are arranged in the heat conduction block (43).

9. According to the digital PCR method of claim 2, each of the chip placement grooves (13) is arranged in sequence along the length direction of the first working platform (1), and each of the droplet generation modules (3) and the PCR amplification modules (4) are arranged in a one-to-one correspondence with each of the chip placement grooves (13).

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