Digital PCR (polymerase chain reaction) method for flow type multi-index detection

Abstract

The invention provides a digital PCR (Polymerase Chain Reaction) method for flow type multi-index detection, which comprises the following steps: placing an integrated micro-droplet chip containing a sample to be detected in a digital PCR instrument, and positioning a reaction chamber in a temperature control cavity; the droplet generation module is controlled to operate to generate micro-droplets, and the generated micro-droplets are stored in the reaction chamber; controlling a PCR amplification module to operate so as to enable the temperature of the micro-droplets in the reaction chamber to be at a preset target temperature; controlling the fluorescence detection module to perform fluorescence detection on the micro-droplets flowing out of the reaction chamber to obtain fluorescence information of the flowing micro-droplets, synchronously recording a preset target temperature corresponding to the obtained fluorescence information, and ensuring that the reaction chamber is located in the temperature control cavity in the fluorescence detection process; the preset target temperature is adjusted and set according to the detection rule gradient. According to the invention, the micro-droplets in the reaction chamber can be controlled and adjusted in real time according to the detection rule gradient to be constantly at the preset target temperature, the detection flux is greatly improved, and the detection efficiency is improved.

Description

Technical Field

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

[0002] Digital PCR is the latest quantitative technology. It is based on the single-molecule PCR method to count nucleic acid quantification. It is an absolute quantitative method. It mainly adopts the microfluidics or droplet method in the current hot research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactor or droplet of the biochip. The number of nucleic acid templates in each reactor is less than or equal to 1. In this way, after the PCR cycle, the reactor with a nucleic acid molecule template will give a fluorescent signal, and the reactor without a template will not have a fluorescent signal. According to the relative proportion and the volume of the reactor, the number of template copies in the sample can be accurately calculated.

[0003] Digital PCR technology has the advantages of high sensitivity and precise quantification, and has great application prospects in the fields of life sciences and molecular diagnosis. However, digital PCR technology is limited by the spectral distribution of fluorescent dyes. A single PCR reaction system can only place several fluorescent probes at the same time, which only corresponds to the detection of several nucleic acid sequences. The multi-index detection capability is insufficient, and it is difficult to meet the requirements of distinguishing dozens of target sequences at the same time.

[0004] In order to improve the multi-index detection capability of digital PCR, one route is to combine the melting curve technology, that is, to control the temperature change of the droplet to be tested during the detection, and cooperate with the sequence design of the fluorescent probe, so that a single or multiple fluorescent probes show different fluorescence intensities at different temperatures, thereby realizing the detection of more indicators under the same type of fluorescent dye. However, since the temperature of the droplet needs to be changed in real time during the detection process, the existing technical routes are all based on the platform of digital PCR using imaging method, that is, the droplet is flattened in the chip cavity after generation, and then the amplification and fluorescence imaging detection are completed in the cavity. This method has certain limitations. First, the imaging method is not completed synchronously for different fluorescences, so the temperature point cannot change continuously and each time a temperature point is reached, it is necessary to wait for the temperature to balance before imaging the different fluorescence channels in turn. Limited by the temperature control speed and the fluorescence imaging speed, a single sample often needs to be detected for more than 10 minutes, the detection speed is slow, and the throughput is low. Secondly, due to the area of ​​the chip cavity, the loading volume of a single sample is small and the total number of droplets is small, resulting in limited detection sensitivity and a small range of nucleic acid quantification. This limits the application of this technology in life science research and clinical diagnosis. That is, imaging-based digital PCR has the following shortcomings: the imaging of different fluorescences is not completed synchronously, so the temperature point cannot change continuously and each time a temperature point is reached, it is necessary to wait for the temperature to be balanced before imaging different fluorescence channels in turn. Limited by the temperature control speed and the fluorescence imaging speed, a single sample often requires more than 10 minutes to be tested, with a slow detection speed and low throughput; limited by the area of ​​the chip cavity, the 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 fluorescence detection can detect all fluorescence channels of droplets simultaneously and is not limited by the tiling of imaging methods. It has a fast detection speed and is not restricted by the sample volume and the number of droplets. It can overcome the problems of slow detection speed, limited sensitivity and small quantitative range in the existing multi-index detection technology of digital PCR combined with temperature.

[0006] The invention patent with patent application number 202111381197.4 previously filed by the applicant discloses an integrated digital PCR instrument and its control method, which has a high degree of automation and integration, can improve the efficiency of detection and analysis operations, and reduce labor costs. The technical advantages, in this technical scheme, are dispatched by a manipulator (that is, a scheduling mechanism) to dispatch the chip in turn between the droplet generation module, the flipping module, the PCR amplification module, the detection module and the waste box. Multiple chips work at the same time to form an assembly line working mode. The chip completes the four digital PCR links of droplet generation, chip flipping, droplet amplification, and droplet fluorescence detection in turn. When the flow method is used to detect the sample droplets in this technical scheme, since the temperature cycle module (that is, the amplification module) and the fluorescence detection module are independently set, the scheduling mechanism is required 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 multi-index detection, which effectively overcomes the deficiency that the amplification module in the digital PCR instrument in the related technology in the prior art cannot achieve continuous gradient constant temperature control of the sample droplets.

[0008] In order to solve the above problems, the present invention provides a digital PCR method for flow multi-index detection, which is performed using a digital PCR instrument, wherein the digital PCR instrument includes a droplet generation module, a PCR amplification module, and a fluorescence detection module, wherein the droplet generation module is used to generate microdroplets in an integrated microdroplet chip and store them in a reaction chamber thereof, and the reaction chamber is in a temperature control chamber of the PCR amplification module; the digital PCR method includes the following steps: Placing the integrated micro-droplet chip containing the sample to be tested in the digital PCR instrument, and placing the reaction chamber in the temperature-controlled chamber; Controlling the droplet generation module to operate to generate micro-droplets, and the generated micro-droplets are stored in the reaction chamber; Controlling the PCR amplification module to operate so that the temperature of the micro-droplets in the reaction chamber is at a preset target temperature; The fluorescence detection module is controlled to perform fluorescence detection on the micro-droplets flowing out of the reaction chamber to obtain fluorescence information of the flowing micro-droplets, and a preset target temperature corresponding to the obtained fluorescence information is synchronously recorded, and it is ensured that during the fluorescence detection process, the reaction chamber is in the temperature control cavity, and the preset target temperature is set according to the gradient adjustment of the detection rule.

[0009] In some embodiments, the digital PCR instrument further includes an integrated module and a rotating mechanism, the droplet generation module and the PCR amplification module are both located in the integrated module, and the rotating mechanism is used to drive the integrated module to flip 180° up and down; the digital PCR method further includes the following steps: When the integrated micro-droplet chip is placed in the digital PCR instrument, the bottom wall of the reaction chamber is directed upward and maintained in this state to generate micro-droplets; After the micro-droplets are generated, the rotating mechanism is controlled to drive the integrated module to flip up and down 180 degrees so that the bottom wall of the reaction chamber faces downward.

[0010] In some embodiments, the integrated module includes a first working platform and a second working platform, the first working platform having a plurality of chip placement grooves for positioning and placing the integrated micro-droplet chip, the second working platform being arranged in parallel with the first working platform and having 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 on the second working platform, and when the second working platform is in the working state, the droplet generation module is respectively pressed and docked with the chip oil-liquid holes and the chip gas-liquid holes of the integrated micro-droplet chip so as to form micro-droplets in the integrated micro-droplet chip, and the reaction chamber of the integrated micro-droplet chip is in the temperature control cavity of the PCR amplification module, and the rotating mechanism is used to drive the first working platform and the second working platform to synchronously flip 180° when the second working platform is in the working state.

[0011] In some embodiments, guide slide bars are vertically arranged at the four corresponding corner areas of the first working platform and the second working platform, and a first return spring is sleeved on the outer periphery of the guide slide bar. The first return spring is clamped between the first working platform and the second working platform, and also includes a downward pressure structure. The downward pressure structure can overcome the elastic force of the first return spring under the action of downward pressure so that the second working platform switches from the chip placement state to the working state.

[0012] In some embodiments, the droplet generation module includes a generation block, the length extension direction of the generation block is parallel to the width direction of the second working platform, and the two ends of the length of the generation block are connected to the second working platform via connecting ears, and a first generation hole and a second generation hole are formed on the generation block. The droplet generation module also includes a generation cover plate, and the generation cover plate has a covering position for sealing the top surface of the chip oil-liquid hole and the chip gas-liquid hole when generating micro-droplets and realizing the pressure-connected connection between the air path on the generation cover plate and the chip gas-liquid hole.

[0013] In some embodiments, the PCR amplification module includes a semiconductor heater, a first end of the semiconductor heater is connected to a heat conductive block, the temperature control cavity is formed on the end surface of the heat conductive block facing away from the semiconductor heater, and a second end of the semiconductor heater is connected to a heat dissipation structure.

[0014] In some embodiments, the heat dissipation structure includes a first heat dissipation block contacting and connected to the second end and a second heat dissipation block contacting and connected to the first heat dissipation block, and a heat dissipation area of ​​the second heat dissipation block is greater than a heat dissipation area of ​​the first heat dissipation block.

[0015] In some embodiments, the heat dissipation structure further includes a third heat dissipation block and a heat pipe, wherein a first end of the heat pipe is located between the first heat dissipation block and the second heat dissipation block, and a second end of the heat pipe is connected to the third heat dissipation block for heat exchange.

[0016] In some embodiments, a heat insulation plate is provided in a circle at the connection position between the first heat sink block and the semiconductor heating element; and / or a temperature measuring probe and a temperature fuse are provided in the heat conducting block.

[0017] In some embodiments, the chip placement grooves are arranged in sequence and at intervals along the length direction of the first working platform, and the droplet generation modules and PCR amplification modules are arranged in one-to-one correspondence with the chip placement grooves.

[0018] The present invention provides a digital PCR method for flow multi-index detection, which is based on the technical route of flow fluorescence detection. In the detection link, when the droplets pass through the fluorescence detection point, different fluorescences are synchronously excited and detected, and there is no need to switch filters for sequential detection like the imaging method. Therefore, detection can be performed under the condition of continuous temperature change, avoiding the waiting time required for temperature equilibrium and the time for switching different fluorescence channels, and compressing the detection time from more than 10 minutes in the existing method to 1 minute, increasing the speed by 10 times, and significantly improving the detection throughput; at the same time, the generated droplets are stored in the reaction chamber, and there is no need to form a single layer of tiling, so it is not limited by the chip area, and a 50 microliter system can be loaded to generate more than 100,000 droplets, which is significantly improved compared with the loading volume of less than 25 microliters and the total number of droplets of less than 30,000 in the traditional imaging method. The sensitivity and quantitative range are significantly improved; and more importantly, the PCR amplification module in the present application is always tightly wrapped with the reaction chamber during the outflow of the microdroplets, which can ensure that the microdroplets in the reaction chamber are constantly controlled and adjusted at the preset target temperature according to the detection rule gradient in real time, greatly improving the detection flux and improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a schematic diagram of the structure of a compact digital PCR instrument according to an embodiment of the present invention (the overall structure omits the outer shell and other parts); Figure 2 for Figure 1 Schematic diagram of the three-dimensional structure of the integrated micro-droplet chip; Figure 3 for Figure 1 Schematic diagram of the three-dimensional structure of the integrated module (including the rotating mechanism); Figure 4 yes Figure 3 A partial enlarged view of the middle A; Figure 5 for Figure 1 Schematic diagram of the three-dimensional structure of the droplet generation module; Figure 6 It is a schematic diagram of the three-dimensional structure of the PCR amplification module; Figure 7 It is a schematic diagram of the three-dimensional structure after the flip mechanism is assembled; Figure 8 It is a schematic diagram of the three-dimensional structure of the fluorescence detection module and the detection crimping module assembled in the instrument; Fig. 9 for Figure 8 A schematic diagram of the three-dimensional structure of the detection crimping module; Fig.10 for Figure 8 Schematic diagram of the three-dimensional structure of the fluorescence detection module; Fig.11 It is a schematic diagram of the three-dimensional structure of the instrument before the micro-droplet is generated; Fig.12 It is a schematic diagram of the three-dimensional structure of the instrument in the state of micro-droplet generation; Fig.13 This is a schematic diagram of the three-dimensional structure of the instrument in the PCR amplification state when flipped 180°; Fig.14 It is a schematic diagram of the local three-dimensional structure under the state of fluorescence detection; Fig.15 A schematic diagram of the flow chart of the digital PCR method for flow-based multi-index detection according to an embodiment of the present invention; Fig.16 A schematic diagram of state switching at different preset target temperatures using a digital PCR method for flow multi-index detection according to an embodiment of the present invention; Fig.17 The micro-droplet detection results of multi-index detection under different temperature gradients; Fig.18 This is the theoretical fluorescence change curve of multi-index detection under different temperature gradients.

[0020] The reference numerals are: 1. First working platform; 11. Vertical plate; 12. Connecting plate; 121. Guide slide bar; 122. First reset spring; 123. Pressure bar; 124. Roller; 125. Traction 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 pressure block; 34. Generate cover plate; 35. Electromagnet; 36. Rotating shaft; 37. Second reset spring; 38. Guide bar; 39. Third reset spring; 310. Cover plate sealing pad; 4. PCR amplification module; 41. Semiconductor heating element; 42. Heat insulation board; 43. Heat conduction block; 44. First heat dissipation block; 47. The third heat sink; 48. The second heat sink; 49. The heat pipe; 5. The rotating mechanism; 51. The rotary motor; 52. The transmission assembly; 6. The fluorescence detection module; 61. The objective lens; 62. The optical path assembly; 63. The lifting motor; 64. The translation motor; 7. The detection crimping module; 70. The detection oil circuit; 71. The detection crimping block; 72. The moving block; 73. The first crimping motor; 74. The second crimping motor; 75. The first sealing gasket; 76. The air cavity; 77. The light source; 100. The integrated micro-droplet chip; 101. The chip oil-liquid hole; 102. The chip air-liquid hole; 103. The reaction chamber; 104. The fluorescence detection area; 105. The sample loading chamber; 201. The device bottom plate; 2021. The longitudinal guide rail; 2022. The longitudinal motor. DETAILED DESCRIPTION

[0021] See also Figures 1 to 18As shown, according to an embodiment of the present invention, a compact digital PCR instrument is provided, including an integrated module (not labeled in the figure) and a rotating mechanism 5 (not labeled in the figure), the integrated 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 micro-droplet chip 100, it can be understood that each chip placement groove 13 is used to position and place an integrated micro-droplet chip 100, the second working platform 2 is arranged in parallel with the first working platform 1 and has a working state close to the first working platform 1 and a chip placement 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 and the integrated micro-droplet chip 100 are placed. The chip oil-liquid hole 101 and the chip gas-liquid hole 102 of the chip 100 are respectively pressed and docked to form micro-droplets in the integrated micro-droplet chip 100, and the formed micro-droplets will be stored in the reaction chamber 103 of the integrated micro-droplet chip 100, and the reaction chamber 103 of the integrated micro-droplet chip 100 is in the temperature control cavity (not shown and not labeled) 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° when the second working platform 2 is in the working state, so that the reaction chamber 103 is turned upside down, at this time, it is ensured that the micro-droplets 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 micro-droplets (that is, the reaction liquid).

[0022] 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 placement state and can switch between the two states. The rotation drive of the rotating mechanism 5 realizes the synchronous flipping of the first working platform 1 and the second working platform 2. There is no need to use the scheduling mechanism in the prior art to switch or flip the integrated micro-droplet chip 100 between different working modules, so that the structure of the digital PCR instrument is more compact and the instrument size is smaller, so it has stronger portability, and the manufacturing cost is relatively reduced.

[0023] See Figure 7 As shown, the shape of the aforementioned chip placement groove 13 matches the bottom surface shape of the integrated micro-droplet chip 100. As a preferred embodiment, a corresponding guide spring 131 is provided on the side of the aforementioned chip placement groove 13 away from the operating side of the operator, so that the position of the chip can be more reliable when the chip is placed, and when the chip is taken out, force can be applied to the end of the chip to facilitate the chip to be ejected from the chip placement groove 13 and thus facilitate the removal of the chip.

[0024] In some embodiments, the integrated module also includes a vertical plate 11 spaced apart on the left and right, and the vertical plates 11 have two and are placed vertically spaced apart. The first working platform 1 is pivoted at both ends of its length (for example, connected by a rolling shaft) and mounted on the top positions of the two vertical plates 11. The bottom ends of the two vertical plates 11 are connected as a whole 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 assembly 52. ​​The rotary motor 51 is fixedly connected to one of the vertical plates 11. The rotary motor 51 is driven and connected to one end of the first working platform 1 via the transmission assembly 52 to drive the first working platform 1 to rotate by a preset angle. In a specific embodiment, the rotary motor 51 and the transmission assembly 52 can be implemented using a rotary drive assembly available on the market.

[0025] In this technical solution, the rotating mechanism 5 can drive the first working platform 1 to flip up and down by 180°, so that the top surface of the integrated micro-droplet chip 100 faces upward to generate micro-droplets, and after the micro-droplets are generated, the first working platform 1 and the second working platform 2 are rotated to flip up and down synchronously, so that the top surface of the integrated micro-droplet chip 100 faces downward to achieve that the micro-droplets in its reaction chamber 103 can be at the bottom of the reaction chamber 103 under the action of their own weight, thereby ensuring that the PCR amplification module 4 can reliably control the temperature of the micro-droplets in the reaction chamber 103. At the same time, the rotating mechanism 5 can also drive the first working platform 1 to swing back and forth during the amplification process, that is, rotate the aforementioned preset angle (generally ±10°), so that the uniform process of the temperature field accelerated by fluid convection can be improved, the heat transfer efficiency and temperature uniformity in the micro-droplet sample can be improved, and the temperature uniformity requirement of the temperature control module itself can be reduced, thereby improving the efficiency of the amplification link.

[0026] In some embodiments, see Figure 8 As shown, the compact digital PCR instrument also includes a device base plate 201 and a longitudinal movement component (not marked 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 to the device base plate 201 via the longitudinal movement guide rail 2021, and the longitudinal movement motor 2022 can drive the integrated module as a whole to move back and forth in a straight line along the guiding direction of the longitudinal movement guide rail 2021, and 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.

[0027] 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 module (such as the subsequent detection crimping module 7 and the fluorescence detection module 6) in different process flows, simplifying the structural design of the processing module. For example, there is no need for longitudinal displacement control of the processing module, that is, the control is simplified.

[0028] See also 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 crimping module 7 on the second side of the integrated module, wherein the first side and the second side are respectively one of the top side and the bottom side of the first working platform 1. In a specific embodiment, Figure 8 The orientation shown is for reference. The first side is the upper side of the integrated module, and the second side is the lower side of the integrated module. The fluorescence detection module 6 can be controlled to rise and fall to move away from or approach the first working platform 1 to achieve the position correspondence with the fluorescence detection area 104 of the integrated micro-droplet chip 100 and perform fluorescence detection. The detection crimping module 7 can be controlled to rise and fall to move closer to or away from the second working platform 2 to achieve compression docking with the first generation hole 31 and the second generation hole 32 in the droplet generation module 3, so that 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 micro-droplets after the amplification reaction to the fluorescence detection area 104 and enter the sample loading chamber 105 of the integrated micro-droplet chip 100. The fluorescence detection module 6 and the detection crimping module 7 can be controlled to move to correspond to the position of each integrated micro-droplet chip 100.

[0029] In this technical solution, by respectively arranging the fluorescence detection module 6 and the detection crimping module 7 on the upper and lower sides of the integrated module, the PCR instrument of the present invention can integrate the fluorescence detection function of the micro-droplets to achieve functional integration of the instrument.

[0030] See Fig. 9 As shown, in some embodiments, the detection crimping module 7 includes an air cavity 76 and a crimping assembly (not labeled in the figure) connected to the air cavity 76, and the air cavity 76 is used to provide negative pressure for the droplet generation module 3 and provide positive pressure for the detection crimping module 7, wherein 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.

[0031] In this technical solution, the air cavity 76 can serve as a pressure vessel of the instrument and also as an assembly carrier of the crimping assembly, which can further simplify the structural design of the instrument and improve the compactness of the structure of the instrument.

[0032] Continue to see Fig. 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 rise and fall by the first crimping motor 73, the moving block 72 is slidably connected to the air cavity 76 and can be driven to move laterally along the length direction of the first working platform 1 by the second crimping motor 74, 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 the crimping assembly is also provided with corresponding guide rails to guide the smooth lifting and lateral movement of the detection crimping block 71; in a preferred embodiment, the moving block 72 is also provided with a light source 77 to provide a light field for the camera of the fluorescence detection module 6, and the light source 77 is arranged on the moving block 72, so that the relative position of the light source 77 and the detection crimping block 71 is stable, and the two move synchronously, and the control is simplified.

[0033] 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-liquid hole 101 and the chip gas-liquid hole 102 respectively, and the detection oil enters the reaction chamber 103 via the first detection through hole and the second detection through hole. The detection crimping block 71 has a first sealing gasket 75 for forming a seal at the crimping position when the detection crimping block 71 is crimped with the droplet generation module 3.

[0034] See also Figure 3 and Figure 4 As shown, in some embodiments, guide slide bars 121 are vertically arranged at the four corresponding corner areas of the first working platform 1 and the second working platform 2. In a specific embodiment, a sliding connection is formed between the guide slide bar 121 and the first working platform 1 and / or the second working platform 2 through a linear bearing 126. 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 so as to apply a restoring elastic force to separate the first working platform 1 and the second working platform 2. The guide slide bar 121 also includes a downward pressing structure (not marked in the figure). The downward pressing structure can overcome the elastic force of the first return spring 122 under the action of the downward pressure so that the second working platform 2 is switched from the chip placement state to the working state. The aforementioned downward pressure can be specifically achieved by manual downward pressure by an operator.

[0035] In this technical solution, force is applied to the downward pressing structure to overcome the elastic force of the first return spring 122 to achieve the approach of the first working platform 1 and the second working platform 2, thereby achieving the crimping of the droplet generation module 3 and the integrated micro-droplet chip 100. When the downward pressing structure is subjected to reverse force, 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 placement state, and prevent the downward pressing structure from falling under the action of its own weight, thereby facilitating the placement of the chip.

[0036] See Figure 4 As shown, in some embodiments, the pressing structure includes a pressure rod 123 and a roller 124 hinged at both ends of the length of the second working platform 2, and a traction rope 125 (such as a steel wire) is connected between the pressure rod 123 and the first working platform 1, and 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 pressure rod 123 is pressed toward the side of the first working platform 1, the traction rope 125 can pull the first working platform 1 and the second working platform 2 towards each other and be in the working state. Preferably, when the pressure rod 123 is pressed toward the side of the first working platform 1 to the lowest position, 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.

[0037] In this technical solution, the state switching of the second working platform 2 can be driven when the position of the pressure rod 123 changes through the directional guiding function of the roller 124 and the pulling connection function of the rope. The structure is simple, especially after the aforementioned pressure rod 123 is pressed down, because the connection point between the traction rope 125 and the pressure rod 123 is below the center point of the roller 124, the self-locking of the structure is achieved, and there is no need to set up a corresponding locking device separately, which further simplifies the structural design.

[0038] See Figure 5 As shown, the droplet generation module 3 includes 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 (not marked in the figure), the first generation hole 31 and the second generation hole 32 are both formed on the generation block 33, and the droplet generation module 3 also includes 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 through the cover plate sealing gasket 310 when generating micro-droplets and realizing the pressure-connected connection between the air path (not marked in the figure) on the generation cover plate 34 and the chip gas-liquid hole 102.

[0039] In this technical solution, both ends of the generated pressing block 33 are connected to the second working platform 2 via connecting lifting ears, and the structure is reliable and simple.

[0040] In some embodiments, the generation cover plate 34 is hinged to the generation pressing block 33 via a rotating shaft 36, and the generation pressing block 33 has an electromagnet 35. When the electromagnet 35 is energized, the generation cover plate 34 is attracted and rotated around the rotating shaft 36 to be in the covering position. This technical solution realizes the attraction of the generation cover plate 34 through the electromagnet 35, and the structure and control are relatively simple.

[0041] In some embodiments, there are two electromagnets 35, which are respectively located at two ends of the length of the generated pressing block 33, and the two ends of the length of the generated cover plate 34 are respectively provided with a suction block corresponding to the position of each electromagnet 35. The two electromagnets 35 are respectively located at two ends of the length of the generated pressing block 33 to ensure the smooth closure of the generated cover plate 34 and to ensure the reliable sealing of the generated cover plate 34.

[0042] In some embodiments, the rotating shaft 36 is provided with a second return spring 37, which is used to switch the generating cover plate 34 from the closing position to the opening position when the electromagnet 35 is powered off. In this technical solution, the second return spring 37 can be provided to open the generating cover plate 34 by its own elastic force when the electromagnet 35 is powered off. The second return spring 37 can be a torsion spring.

[0043] In some embodiments, a hand-held protrusion (not shown) is provided on one side edge of the generation cover plate 34 away from the rotating shaft 36, so that the operator can apply force to the hand-held protrusion to assist in opening or closing the generation cover plate 34. A cover plate sealing gasket 310 is provided on one side end surface of the generation cover plate 34 facing the generation pressing block 33, which is arranged around each of the generation first hole 31 and the generation second hole 32.

[0044] In some embodiments, the two ends of the length of the generating block 33 can be vertically slidably connected to the second working platform 2 via the connecting ears, and a guide rod 38 is provided between the top surface of the generating block 33 and the bottom surface of the second working platform 2. The third return spring 39 is sheathed on 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 generating block 33. In this technical solution, while the two ends of the generating block 33 can be vertically slidably connected to the second working platform 2 via the connecting ears, the clamping of the third return spring 39 between the second working platform 2 and the generating block 33 can buffer and adjust the horizontal state of the generating block 33, thereby ensuring the crimping sealing of the generating block 33 to the integrated micro-droplet chip 100.

[0045] See Figure 6 As shown, in some embodiments, the PCR amplification module 4 includes a semiconductor heating element 41, a first end of the semiconductor heating element 41 is connected to a heat conductive block 43, the temperature control cavity is formed on the end surface of the heat conductive block 43 facing away from the semiconductor heating element 41, and the second end of the semiconductor heating element 41 is connected to a heat dissipation structure (not marked in the figure), and the aforementioned heat conductive block 43 can evenly transfer the heat of the semiconductor heating element 41 to the temperature control cavity, thereby being able to uniformly control the temperature of the micro-droplets in the reaction chamber 103. The aforementioned semiconductor heating element 41 specifically adopts a Peltier, and according to actual needs, a plurality of Peltiers can be used.

[0046] In some embodiments, the heat dissipation structure includes a first heat dissipation block 44 connected in contact with the second end and a second heat dissipation block 48 connected in contact with the first heat dissipation block 44, and the heat dissipation area of ​​the second heat dissipation block 48 is larger than the heat dissipation area of ​​the first heat dissipation block 44, so as to achieve rapid dissipation of heat from the first end of the semiconductor heating element 41.

[0047] In some embodiments, the heat dissipation structure further includes a third heat dissipation block 47 and a heat pipe 49. The heat pipe 49 may be a heat pipe, which contains a heat-conducting medium and has a high heat transfer efficiency. The first end of the heat pipe 49 is located between the first heat dissipation block 44 and the second heat dissipation block 48, and the second end of the heat pipe 49 is connected to the third heat dissipation block 47 for heat exchange. In this technical solution, the heat at the first heat dissipation block 44 and the second heat dissipation block 48 can be quickly transferred and conducted to the third heat dissipation block 47 through the heat pipe 49 for dissipation. The length of the heat pipe 49 can be adjusted to achieve flexible selection of the location of the third heat dissipation block 47, thereby greatly increasing the heat dissipation area and achieving the purpose of rapid heat dissipation.

[0048] In some embodiments, a heat insulation plate 42 is arranged in a circle at the connection position between the first heat sink 44 and the semiconductor heating element 41 to prevent short-circuiting of cold and hot water and reduce energy consumption; and / or, a temperature probe (not shown in the figure) and a temperature fuse (not shown in the figure) are arranged in the heat conductive block 43, wherein the temperature probe can detect the real-time temperature at the heat conductive block 43, and the temperature fuse can disconnect the power supply of the semiconductor heating element 41 when the real-time temperature of the heat conductive block 43 exceeds a preset value, thereby achieving the purpose of constant temperature control.

[0049] In some embodiments, the chip placement grooves 13 are arranged in sequence and at intervals along the length direction of the first working platform 1, and the droplet generation modules 3 and the PCR amplification modules 4 are arranged one-to-one corresponding to the chip placement grooves 13, thereby improving the processing throughput of the instrument.

[0050] According to an embodiment of the present invention, a control method for the above-mentioned compact digital PCR instrument is also provided, comprising the following steps: Place the integrated micro-droplet chip 100 loaded with the sample liquid into the chip placement groove 13; Controlling the second working platform 2 to switch from a chip picking and placing state to a working state; Controlling the droplet generation module 3 to operate so as to generate micro-droplets in the integrated micro-droplet chip 100, and storing the micro-droplets in the reaction chamber 103 of the integrated micro-droplet chip 100; After the micro-droplets are generated, the first working platform 1 and the second working platform 2 are controlled to synchronously flip 180 degrees, and the PCR amplification module 4 is controlled to operate to amplify the micro-droplets in the reaction chamber 103; After amplification is completed, the detection crimping module 7 and the fluorescence detection module 6 are controlled to move toward the integrated micro-droplet chip 100. After the detection crimping module 7 is docked with the first generation hole 31 and the second generation hole 32 of the droplet generation module 3, the detection crimping module 7 is controlled to operate to drive the amplified micro-droplets from the reaction chamber 103 through the fluorescence detection area 104 of the integrated micro-droplet chip 100 into the sample loading cavity 105 of the integrated micro-droplet chip 100, and the fluorescence detection module 6 is controlled to perform fluorescence detection in the fluorescence detection area 104.

[0051] This technical solution integrates the droplet generation module 3 and the PCR amplification module 4 on the second working platform 2. At the same time, the second working platform 2 has a working state and a chip placement state and can switch between the two states. The first working platform 1 and the second working platform 2 are synchronously flipped by the rotation drive of the rotating mechanism 5. There is no need to use the scheduling mechanism in the prior art to switch or flip the integrated micro-droplet chip 100 between different working modules, so that the structure of the digital PCR instrument is more compact and the instrument volume is smaller, so it has stronger portability, and the manufacturing cost is relatively reduced. In some embodiments, the control method also includes: during the operation of the PCR amplification module 4, controlling the first working platform 1 and the second working platform 2 to swing back and forth synchronously, which can improve the heat transfer efficiency and temperature uniformity in the micro-droplet sample through the uniform process of the temperature field accelerated by fluid convection, and also reduce the temperature uniformity requirements of the temperature control module itself, thereby improving the efficiency of the amplification link; and / or, after the fluorescence detection is completed, controlling the first working platform 1 and the second working platform 2 to rotate 180° in the opposite direction synchronously, and then controlling the second working platform 2 to switch from the working state to the chip placement state.

[0052] See Fig.15 and Fig.16As shown, according to an embodiment of the present invention, a digital PCR method for flow multi-index detection is also provided, which is performed using the above-mentioned digital PCR instrument; the digital PCR method comprises the following steps: The integrated micro-droplet chip 100 containing the sample to be tested is placed in the digital PCR instrument, and the reaction chamber 103 is placed in the temperature control chamber; Controlling the droplet generation module 3 to operate to generate micro-droplets, and 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; The fluorescence detection module 6 is controlled 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 the preset target temperature corresponding to the obtained fluorescence information is synchronously recorded, and it is ensured that during the fluorescence detection process, the reaction chamber 103 is in the temperature control cavity, and the preset target temperature is set according to the gradient adjustment of the detection rule.

[0053] In this technical solution, based on the technical route of flow fluorescence detection, in the detection link, when the droplets pass through the fluorescence detection point, different fluorescences are synchronously excited and detected, and there is no need to switch filters for detection in sequence like the imaging method. Therefore, detection can be performed under the condition of continuous temperature changes, avoiding the waiting time required for temperature equilibrium and the time for switching different fluorescence channels, and compressing the detection time from more than 10 minutes in the existing method to 1 minute, increasing the speed by 10 times, and significantly improving the detection throughput; at the same time, the generated droplets are stored in the reaction chamber 103, and there is no need to form a single layer of tiling, so it is not limited by the chip area, and a 50 microliter system can be loaded to generate more than 100,000 droplets, which is significantly improved compared with the loading volume of less than 25 microliters and the total number of droplets of less than 30,000 in the traditional imaging method. The sensitivity and quantitative range are significantly improved; and more importantly, the PCR amplification module in the present application is always tightly wrapped with the reaction chamber 103 during the outflow of the microdroplets, which can ensure that the microdroplets in the reaction chamber 103 are constantly controlled and adjusted at the preset target temperature according to the detection rule gradient in real time, greatly improving the detection flux and improving the detection efficiency.

[0054] When the integrated micro-droplet chip 100 can be driven to flip up and down 180°, the digital PCR method further includes 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 upward 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, thereby ensuring that the PCR amplification module can fully transfer heat and adjust the temperature of the micro-droplets in the reaction chamber 103.

[0055] The digital PCR method for flow multi-index detection of the present invention is further described below in conjunction with a specific embodiment: The micro-droplet chip containing the sample to be tested (i.e. the aforementioned integrated micro-droplet chip 100, the same below) is placed in the flip module (i.e. the integral component composed of the aforementioned integrated module and the rotating mechanism 5, the same below), as shown in FIG. Fig.11 When the flip module is in the open state, the chip can be placed in or taken out of the flip module.

[0056] Change the flip module into a compacted state, such as Fig.12 In the compressed state, the droplet generation module is airtightly 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 compressed state, the amplification module is in close contact with the micro-droplet chip reaction tube (that is, reaction chamber 103, the same below), and can be used as a temperature control unit in the nucleic acid amplification link to continuously control the temperature of the micro-droplets in the reaction tube to achieve nucleic acid amplification.

[0057] The instrument starts and droplet generation begins. The entire automation process is controlled by the control module to realize all other modules. The droplet generation module can provide a specific pressure. The microdroplet generation oil and the sample in the microdroplet chip enter the chip pipeline respectively under the action of air pressure, and interact in the pipeline to generate microdroplets of uniform size, which are collected in the reaction tube of the microdroplet chip to complete the droplet generation.

[0058] The flip module flips the chip 180°, such as Figure 3 , the generated microdroplets are collected in the heating area of ​​the reaction tube by gravity.

[0059] The amplification module is started to control the temperature of the microdroplets in the reaction tube. In this embodiment, the temperature cycle process is to first perform a pre-denaturation at 95°C for 10 minutes, followed by 40 temperature cycles, with 95°C for 10 seconds and 60°C for 25 seconds in each cycle (ie, the aforementioned detection regular gradient).

[0060] After the amplification is completed, the 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, and completes the fluorescence excitation and detection of each droplet after the droplet queue passes the fluorescence detection point. At the same time, the amplification module controls the temperature of the droplets to be tested in real time, and can change the temperature continuously or in steps. The detected droplet fluorescence signal is converted into a numerical value and recorded by the control module, and the droplet temperature during detection is also recorded synchronously. The detected droplet fluorescence signal and temperature information are plotted as a one-dimensional fluorescence scatter plot, such as Fig.17 , quantitative information of different templates can be obtained for different temperature changes, and the theoretical fluorescence change curve is as follows Fig.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, thereby calculating the copy number of all target templates in the original sample and realizing digital PCR multi-index detection.

[0061] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed.

[0062] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present invention, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. 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; The fluorescence detection module (6) is controlled 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 simultaneously record a preset target temperature corresponding to the obtained fluorescence information, and ensure 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.

2. The digital PCR method according to claim 1, characterized in that: 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.

3. The digital PCR method according to claim 2, 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.

4. The digital PCR method according to claim 3, 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.

5. The digital PCR method according to claim 4, 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).

6. The digital PCR method according to claim 3, 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.

7. The digital PCR method according to claim 6, 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).

8. The digital PCR method according to claim 7, 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.

9. The digital PCR method according to claim 8, 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).

10. According to the digital PCR method of claim 3, 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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