Real-time fluorescent PCR instrument suitable for micro-fluidic chip

Abstract

The invention discloses a real-time fluorescent PCR instrument suitable for a micro-fluidic chip, and relates to the technical field of molecular diagnosis and biological detection. The conveying module conveys the chip to a station; the rotating module drives the chip to rotate; the pretreatment auxiliary module punctures a reagent bag of the chip and heats the chip; the amplification temperature control module is used for carrying out heating and cooling circulation on the PCR reaction cavity on the chip; the fluorescence detection module is used for collecting fluorescence signals in the chip PCR reaction cavity; the pressure valve module controls opening and closing of the thin film micro valve on the chip. According to the invention, centrifugal driving, mechanical valve control, semiconductor temperature control, multi-channel optical detection and other technologies are highly integrated in a compact instrument, the physical characteristics and operation requirements of a centrifugal micro-fluidic chip are perfectly matched, full-automatic operation from an original sample to a qPCR quantitative result is really realized, and the operation efficiency is greatly improved. The operation difficulty and personal errors are greatly reduced, the aerosol pollution risk is remarkably reduced, and the detection consistency and reliability are improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and biological detection technology. More specifically, it relates to integrated and automated nucleic acid amplification and detection equipment, particularly a fully automated real-time quantitative PCR (qPCR) analyzer based on centrifugal microfluidic chip technology that integrates sample processing, nucleic acid amplification and multi-wavelength fluorescence detection. Background Technology

[0002] RAP (Recombinase Polymerase Amplification) involves the recombinase forming a complex with primers at a constant temperature of 37–42℃, directly scanning double-stranded DNA and initiating strand displacement synthesis, completing a 10^9-fold amplification within 5–20 minutes. LAMP (Loop-Mediated Isothermal Amplification) uses four (or six) specific primers to recognize six target segments at a constant temperature of 60–65℃, forming a "dumbbell" loop structure through BstDNA polymerase with strand displacement activity, producing 10^12 copies within half an hour, accompanied by magnesium pyrophosphate precipitation, visible turbidity or fluorescence discoloration. With high sensitivity and high specificity, it has been widely used in visual detection and on-site diagnosis in primary laboratories. PCR (polymerase chain reaction) uses a temperature-controlled cycle of denaturation at 95°C, annealing at 55°C, and extension at 72°C. Taq DNA polymerase repeatedly replicates the target sequence, and 30 cycles are enough to amplify a single copy to a detectable level. The technology is mature and the reagent system is complete. It can be seamlessly integrated with fluorescent probes, digital chips, or high-throughput platforms, and it remains the gold standard for nucleic acid diagnosis in scientific research, clinical practice, and forensic medicine.

[0003] Centrifugal microfluidic discs (also known as "Lab-on-a-CD") integrate all conventional experimental procedures (sample addition, metering, mixing, reaction, separation, and detection) onto a single CD-sized plastic disc. They utilize centrifugal force instead of external pumps and valves, achieving liquid step control through speed-time programming. The core structure consists of radially distributed reservoirs, capillary valves, siphon valves, burst valves, and microchannels. As the rotation speed increases or decreases according to a preset curve, the balance between centrifugal force and capillary force / surface tension is sequentially broken, causing the liquid to transfer from the center outwards like dominoes, completing steps such as precise metering, resuspension, washing, nucleic acid amplification, or immunochromatography. Advantages include: no need for external pump tubing, simple structure, low cost, and easy disposable use; centrifugal force naturally and efficiently separates particles / cells / magnetic beads; 24–96 channels can be arranged in parallel for high throughput; natural compatibility with optical readout (fluorescence, colorimetry, scattering); and the entire device can be shrunk into a portable "pocket laboratory."

[0004] Real-time quantitative PCR (qPCR) is the gold standard technology for nucleic acid quantification. However, the traditional qPCR detection process is cumbersome, requiring multiple steps such as nucleic acid extraction, reagent preparation, amplification reaction, and fluorescence detection. It relies on multiple independent instruments and a large amount of manual operation, which is time-consuming, prone to contamination, and requires a high level of professional expertise from operators, making it difficult to apply in non-laboratory environments such as bedside or field settings. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings of existing technologies, such as fragmented qPCR detection processes, low automation, and difficulty in field application, and to provide a highly integrated, easy-to-operate, fully automated real-time fluorescence quantitative PCR analyzer suitable for centrifugal microfluidic chips.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a real-time fluorescence PCR instrument suitable for microfluidic chips, including a base, a delivery module, a rotation module, a pretreatment auxiliary module, an amplification temperature control module, a fluorescence detection module, a pressure valve module, and a main control circuit system;

[0007] The chip delivery module, mounted on the base, automatically delivers the centrifugal microfluidic chip placed at the injection position to the station of the rotating module.

[0008] The rotating module, mounted on the chip delivery module, clamps and drives the centrifugal microfluidic chip to rotate, using centrifugal force to control the flow, mixing and distribution of reagents within the centrifugal microfluidic chip;

[0009] The pretreatment auxiliary module, installed on the base, punctures the reagent capsule on the centrifugal microfluidic chip before centrifugation and heats specific areas of the chip when necessary to assist in sample lysis.

[0010] The amplification temperature control module is installed on the base and cycles the temperature of the PCR reaction chamber on the chip after the centrifugal microfluidic chip stops rotating.

[0011] The fluorescence detection module is located above the centrifugal microfluidic chip and synchronously collects the fluorescence signal in the PCR reaction chamber of the centrifugal microfluidic chip during specific stages of the amplification cycle.

[0012] The pressure valve module controls the opening and closing of the thin-film microvalve on the centrifugal microfluidic chip through mechanical extrusion, thereby regulating the timing of liquid flow.

[0013] The main control circuit system coordinates and controls the collaborative work of each module.

[0014] Preferably, the conveying module includes a first support, a guide rail, a slider, and a first motor. The first motor drives the slider to slide along the guide rail, which is mounted on the base. The rotating module includes a second motor and a flange. The second motor is mounted on the slider, and the flange is mounted on the output shaft of the second motor. The centrifugal microfluidic chip is detachably connected to the flange.

[0015] Preferably, the pretreatment auxiliary module includes a piercing column, a second support, an upper heating plate, a lower heating plate, and a linear motor. The second support is mounted on the base, and the linear motor is fixedly mounted on the second support. The piercing column is connected to the linear motor. The upper and lower heating plates are slidably connected to the second support. When the centrifugal microfluidic chip reaches the working position but before it starts high-speed rotation, the piercing column moves downward under the drive of the linear motor to pierce the corresponding sealed reagent capsule on the centrifugal microfluidic chip and release the reagent. Under the action of the driving mechanism, the upper and lower heating plates clamp the lysis chamber area of ​​the centrifugal microfluidic chip from the upper and lower sides along the slide rail on the second support and heat it to the set temperature.

[0016] Preferably, the expanded temperature control module includes two third motors, a transmission rail, two sets of heat sinks, a fan, a Peltier, a heating aluminum plate, and a third support. The third support is mounted on the base, the transmission rail is vertically mounted on both sides of the third support, the heat sink is slidably connected to the transmission rail, the two third motors drive the two sets of heat sinks to slide up and down along the transmission rail, the Peltier is mounted on the bottom of the upper heat sink, the heating aluminum plate is mounted on the top of the lower heat sink, and a fan is mounted on both sets of heat sinks.

[0017] Preferably, the pressure valve module includes an extrusion component and a support block. The extrusion component is mounted on the upper heat sink, and the support block is mounted on the lower heat sink. The extrusion component moves down and, together with the support block, extrudes the corresponding thin-film valve area on the centrifugal microfluidic chip, so that the microchannel is temporarily closed or opened, thereby controlling the liquid flow direction and step switching.

[0018] Preferably, the extrusion component includes an extrusion seat, a sliding shaft, a spring, and an extrusion column. The extrusion seat is mounted on a heat sink. The extrusion seat array has several cavities. A sliding shaft is slidably connected in each cavity. The spring is sleeved and connected to the outside of the sliding shaft. The top end of the spring abuts against the upper surface of the cavity, and the bottom end abuts against the end face of the sliding shaft. The top end of the sliding shaft extends out of the cavity, and the bottom end is fixedly connected to the extrusion column. The bottom end of the extrusion column extends out of the cavity.

[0019] Preferably, the fluorescence detection module includes a housing and a fluorescence detection channel module. The fluorescence detection channel is installed inside the housing, which is a sealed metal cover. The fluorescence detection channel module has four independent optical detection channels. The detection optical axis of the optical detection channel is perpendicular to the plane of the centrifugal microfluidic chip and is vertically aligned with the center of the four PCR reaction chambers on the chip.

[0020] The beneficial effects of adopting the above technical solution are as follows:

[0021] 1. Modular and fully automated process integration: This invention integrates the entire process of "sample in, result out" (sample loading, transfer, preprocessing, amplification, and detection) of centrifugal microfluidic chip into one device. Through the coordinated control of the main control system, the sequential actions of each module are controlled, realizing the full automation of nucleic acid detection and avoiding the error and contamination risks caused by manual operation.

[0022] 2. Active compression-type high-precision temperature control for microfluidic chips: This invention uses a temperature control component driven by a third motor, which includes a semiconductor Peltier and a thermally conductive aluminum block. During the PCR amplification stage, it actively and tightly compresses the reaction chamber area of ​​the chip. This design ensures efficient heat conduction between the reaction chamber and the hot block, achieving rapid heating and cooling and excellent temperature uniformity.

[0023] 3. Detection throughput and multiplicity: This invention supports single-sample multi-index or multi-sample parallel detection; the 4-wavelength optical system supports multiple detection in each chamber, providing rich information and high efficiency.

[0024] 4. Contamination-proof design and simple operation: In this invention, both samples and reagents are processed within the chip, greatly reducing cross-contamination. Operation is extremely simplified, making it suitable for point-of-care testing (POCT) and non-professional scenarios.

[0025] 5. System Compactness and Consumable Costs: The instrument of this invention has a compact structure. The centrifugal microfluidic chip has a flat structure, which is easy to integrate and injection mold, resulting in low consumable production costs and high yield rates. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the overall three-dimensional structure of the instrument of the present invention;

[0027] Figure 2 This is a schematic diagram of the rotating module of the present invention;

[0028] Figure 3 This is a schematic diagram of the structure of the expanded temperature control module of the present invention;

[0029] Figure 4 This is a schematic diagram of the fluorescence detection module of the present invention (external housing and internal view).

[0030] Figure 5 This is a schematic diagram of the preprocessing auxiliary module of the present invention;

[0031] Figure 6 This is a schematic diagram of the structure of the extrusion part of the present invention;

[0032] In the diagram: 1. Base; 2. Conveying module; 2-1. First support; 2-2. Guide rail; 2-3. Slider; 2-4. First motor; 3. Rotation module; 3-1. Second motor; 3-2. Flange; 4. Pre-treatment auxiliary module; 4-1. Puncture column; 4-2. Second support; 4-3. Upper heating plate; 4-4. Lower heating plate; 5. Amplification temperature control module; 5-1. Third motor; 5-2. Transmission guide rail; 5-3. Heat sink; 5-4. Fan; 5-5. Peltier; 5-6. Heating aluminum sheet; 5-7. Third support; 6. Fluorescence detection module; 6-1. Housing; 6-2. Fluorescence detection channel module; 7. Pressure valve module; 7-1. Extrusion component; 7-101. Extrusion seat; 7-102. Sliding shaft; 7-103. Spring; 7-104. Extrusion column; 7-2. Support block; 8. Centrifugal microfluidic chip. Detailed Implementation

[0033] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0034] This invention highly integrates key technologies such as centrifugation drive, mechanical valve control, semiconductor temperature control, and multi-channel optical detection into a compact instrument, perfectly matching the physical characteristics and operational requirements of centrifugal microfluidic chips. It truly achieves fully automated operation from raw sample to qPCR quantitative results – "sample in, result out." This instrument significantly reduces operational difficulty and human error, substantially reduces the risk of aerosol contamination, and improves the consistency and reliability of detection. It is particularly suitable for applications such as rapid on-site testing, point-of-care diagnostics, and primary healthcare units.

[0035] like Figure 1 As shown, this analyzer adopts a layered structure of "base plate - three-dimensional frame - functional modules". The instrument base 1 serves as the bottom mounting foundation to ensure the entire machine is level.

[0036] The transfer module 2 automatically transfers the centrifugal microfluidic chip 8, placed at the sample injection position, to the station of the rotation module 3. The rotation module 3 clamps and drives the centrifugal microfluidic chip 8 to rotate, using centrifugal force to control the flow, mixing, and distribution of reagents within the chip. The pretreatment auxiliary module 4 punctures the reagent capsules on the centrifugal microfluidic chip 8 before centrifugation and heats specific areas of the chip when necessary to aid sample lysis. The amplification temperature control module 5 performs rapid and precise heating and cooling cycles on the PCR reaction chamber of the chip after the centrifugal microfluidic chip 8 stops rotating. The fluorescence detection module 6 synchronously acquires fluorescence signals within the PCR reaction chamber of the centrifugal microfluidic chip at specific stages of the amplification cycle. The pressure valve module 7 controls the opening and closing of the thin-film microvalve on the centrifugal microfluidic chip 8 through mechanical compression to precisely regulate the timing of liquid flow. The main control circuit system coordinates and controls the collaborative operation of each module.

[0037] like Figure 2 As shown, the conveying module 2 includes a first support 2-1, a guide rail 2-2, a slider 2-3, and a first motor 2-4. The first motor 2-4 drives the slider 2-3 to slide along the guide rail 2-2, which is mounted on the base 1. The conveying module 2 achieves fully automatic and precise positioning of the chip from the operating interface to the core workstation, eliminating the need for manual intervention in chip alignment and placement. This is the first step in achieving process automation and reduces the risk of contamination or positional errors introduced by human operation.

[0038] The rotating module 3 includes a second motor 3-1 and a flange 3-2. The second motor 3-1 is mounted on the slider 2-3, and the flange 3-2 is mounted on the output shaft of the second motor 3-1. The centrifugal microfluidic chip 8 is detachably connected to the flange 3-2. After the chip is transferred to the working position, the second motor 3-1 drives the chip to rotate at different speeds and in different directions according to a preset program. In the sample lysis stage, low-speed alternating forward and reverse rotation can be used to promote mixing; in the liquid transfer stage, continuous rotation at a specific speed is used, and centrifugal force is used to drive the liquid to flow sequentially through different chambers on the chip (such as the lysis chamber, washing chamber, elution chamber, and PCR reaction chamber).

[0039] Rotation module 3 provides the core power for all liquid operations (mixing, transferring, and separating) within the chip. By programmably controlling the rotation speed curve, the fluid operations required for chip design can be accurately reproduced, replacing many traditional manual operations such as pipetting and oscillation.

[0040] like Figure 5As shown, the pretreatment auxiliary module 4 includes a piercing column 4-1, a second support 4-2, an upper heating plate 4-3, a lower heating plate 4-4, and a linear motor. The second support 4-2 is mounted on the base 1. The linear motor is fixedly mounted on the second support 4-2. The piercing column 4-1 is connected to the linear motor, and its position corresponds one-to-one with the pre-packaged reagent capsules on the chip. The upper heating plate 4-3 and the lower heating plate 4-4 are both slidably connected to the second support 4-2. The upper and lower heating plates are embedded with heating films and high-precision thermistors. When the centrifugal microfluidic chip 8 reaches the working position but before it starts high-speed rotation, the piercing column 4-1 moves downward under the drive of the linear motor, piercing the corresponding sealed reagent capsules (such as lysis buffer or washing buffer) on the centrifugal microfluidic chip 8 and releasing the reagents. In the lysis step, the upper heating plate 4-3 and the lower heating plate 4-4, driven by the drive mechanism, clamp the lysis chamber area of ​​the centrifugal microfluidic chip 8 from both sides along the slide rails on the second support 4-2, and heat it to a set temperature (e.g., 60°C) to promote cell lysis or protease digestion. They can also be used to melt the "wax valve" on the chip. The puncture function of this module enables the on-demand release of sealed reagents, ensuring reagent freshness and operational reliability. The auxiliary heating function accelerates the lysis process and shortens the total sample processing time.

[0041] like Figure 3 As shown, the amplification temperature control module 5 includes two third motors 5-1, a transmission rail 5-2, two sets of heat sinks 5-3, a fan 5-4, a Peltier 5-5, a heating aluminum plate 5-6, and a third support 5-7. The third support 5-7 is mounted on the base 1, and the transmission rail 5-2 is vertically mounted on both sides of the third support 5-7. The heat sinks 5-3 are slidably connected to the transmission rails 5-2. The two third motors 5-1 drive the two sets of heat sinks 5-3 to slide up and down along the transmission rails 5-2. The Peltier 5-5 is mounted on the bottom of the upper heat sink 5-3. The heating aluminum plate 5-6 is mounted on the top of the lower heat sink 5-3, and a high-precision platinum resistance temperature sensor is embedded inside it. Fans 5-4 are mounted on both sets of heat sinks 5-3 to dissipate heat from the hot end of the Peltier 5-5. After the chip completes sample preprocessing, nucleic acid elution, and mixing with PCR premixed solution to enter the reaction chamber, the centrifugation drive, i.e., the rotation module, stops rotating. The third motor 5-1 drives the Peltier 5-5, heating aluminum plate 5-6, and third support 5-7 to clamp together via guide rail 5-2, ensuring that the heating aluminum plate 5-7 is in close contact with the PCR reaction chamber area of ​​the chip, thus ensuring good thermal contact. The main control circuit system uses a PID algorithm to control the direction and magnitude of the current in the Peltier 5-5, enabling the heating aluminum plate 5-7 to cycle rapidly and accurately between three temperatures: denaturation (e.g., 95°C), annealing (e.g., 60°C), and extension (e.g., 72°C).

[0042] The pretreatment auxiliary module 4 provides the high-precision, rapidly temperature-controlled environment required for PCR reactions. The extrusion structure ensures efficient heat transfer, which is key to obtaining efficient and uniform amplification.

[0043] like Figure 3 As shown, the pressure valve module 7 includes an extrusion component 7-1 and a support block 7-2. The extrusion component 7-1 is mounted on the upper heat sink 5-3, and the support block 7-2 is mounted on the lower heat sink 5-3. The extrusion component 7-1 moves down and together with the support block 7-2, extrudes the corresponding membrane valve area on the centrifugal microfluidic chip 8, so that the microchannel is temporarily closed or opened, controlling the liquid flow direction and step switching.

[0044] Specifically, such as Figure 6 As shown, the extrusion component 7-1 includes an extrusion seat 7-101, a sliding shaft 7-102, a spring 7-103, and an extrusion post 7-104. The extrusion seat 7-101 is mounted on the heat sink 5-3. The extrusion seat 7-101 array has several cavities. A sliding shaft 7-102 is slidably connected to each cavity. The spring 7-103 is sleeved and connected to the outside of the sliding shaft 7-102. The top end of the spring 7-103 abuts against the upper surface of the cavity, and the bottom end abuts against the end face of the shaft platform of the sliding shaft 7-102. The top end of the sliding shaft 7-102 extends out of the cavity, and the bottom end is fixedly connected to the extrusion post 7-104. The bottom end of the extrusion post 7-104 extends out of the cavity. The number and position of the extrusion posts 7-104 are customized according to the number and layout of the "thin-film valves" on the chip. The extruder 7-1 moves downward, and the extrusion column 7-104 extrudes the corresponding membrane valve area on the chip. At the same time, the bottom support block 7-2 applies uniform pressure to the chip's "membrane valve" from below, causing the chip's microchannels to be temporarily closed or opened, thereby precisely controlling the liquid flow direction and step switching.

[0045] The pressure valve module 7 provides active switching control of the flow path within the chip. Combined with passive centrifugal force drive, it enables precise programming control of complex, multi-step fluid processes, which is key to completing fully automated nucleic acid extraction and purification processes.

[0046] like Figure 4 As shown, the fluorescence detection module 6 includes a housing 6-1 and a fluorescence detection channel module 6-2. The fluorescence detection channel 6-2 is installed inside the housing 6-1. The housing 6-1 is a sealed metal cover, which effectively isolates stray light from the environment and electromagnetic interference generated by other electronic components inside the instrument (such as motors and temperature controllers), providing a stable working environment for high-sensitivity photoelectric detection. The fluorescence detection channel module 6-2 has four independent optical detection channels. The detection optical axis of the optical detection channel is perpendicular to the plane of the centrifugal microfluidic chip in the horizontal direction and is vertically aligned with the center of the four PCR reaction chambers on the chip. This module adopts an orthogonal optical path design to maximize the collection of emitted fluorescence perpendicular to the excitation optical path, thereby significantly reducing background interference of the excitation light and improving the signal-to-noise ratio and detection sensitivity.

[0047] During PCR amplification, once the amplification temperature control module 5 stabilizes the reaction chamber temperature at the fluorescence acquisition temperature, the main control circuit system sends an acquisition command to the fluorescence detection module 6. The four detection channels can operate sequentially or simultaneously according to a preset program: illuminating the corresponding LED, exciting the reaction chamber through the optical path, and acquiring the fluorescence signal. The weak current signal generated by the photoelectric sensor is converted into a voltage signal by the precision preamplifier circuit inside the module, then digitized by the analog-to-digital converter, and transmitted to the main control system for real-time processing, recording, and curve plotting.

[0048] The fluorescence detection module 6 enables real-time, multi-wavelength synchronous acquisition of fluorescence signals from the four reaction chambers. Combined with the four-wavelength optical system, each reaction chamber can simultaneously detect up to four different fluorescently labeled targets, achieving a high degree of detection multiplicity.

[0049] The working process of the instrument of this invention is as follows:

[0050] 1. Sample loading: The user places the centrifugal microfluidic chip 8, which has been mixed with the original sample (such as blood or saliva), onto the carrier tray of the delivery module 2.

[0051] 2. Loading: The transmission module 2 automatically feeds the chip into the working position inside the instrument.

[0052] 3. Reagent Release and Pyrolysis: The puncture column 4-1 of the pressure valve module 7 and the pretreatment auxiliary module 4 operates sequentially to release reagents such as lysis buffer. The upper heating plate 4-3 and the lower heating plate 4-4 heat the pyrolysis zone. The rotation module 3 drives the chip to rotate slowly in both directions to promote mixed pyrolysis.

[0053] 4. Nucleic acid purification: The binding, washing, and elution steps are completed through programmed centrifugation speed control. Finally, the eluted nucleic acid is mixed with the PCR premix in the reaction chamber.

[0054] 5. PCR Amplification and Detection: Rotation module 3 stops rotating, and centrifugation stops. Amplification temperature control module 5 tightly compresses the chip reaction chamber area, initiating rapid temperature cycling. At the end of each cycle, fluorescence detection module 6 collects fluorescence signals from the four reaction chambers.

[0055] 6. Result Output and Unloading: After amplification, the amplification temperature control module 5 is released, and the transfer module 2 returns the chip to the loading / unloading area. The instrument's built-in software automatically analyzes the fluorescence curve, calculates the Ct value, and outputs a test report.

[0056] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A real-time fluorescence PCR instrument suitable for microfluidic chips, characterized in that, It includes a base (1), a transfer module (2), a rotation module (3), a pre-processing auxiliary module (4), an amplification temperature control module (5), a fluorescence detection module (6), a pressure valve module (7), and a main control circuit system; The chip transfer module (2) is installed on the base (1) and automatically transfers the centrifugal microfluidic chip (8) placed at the injection position to the station of the rotating module (3); The rotating module (3) is installed on the chip transfer module (2), clamps and drives the centrifugal microfluidic chip (8) to rotate, and uses centrifugal force to control the flow, mixing and distribution of reagents in the centrifugal microfluidic chip (8); The pretreatment auxiliary module (4) is installed on the base (1) to puncture the reagent capsule on the centrifugal microfluidic chip (8) before centrifugation and to heat a specific area of ​​the chip when necessary to assist in sample lysis. The amplification temperature control module (5) is installed on the base (1). After the centrifugal microfluidic chip (8) stops rotating, it cycles the temperature of the PCR reaction chamber on the chip. The fluorescence detection module (6) is set above the centrifugal microfluidic chip (8) and synchronously collects the fluorescence signal in the PCR reaction chamber of the centrifugal microfluidic chip during a specific stage of the amplification cycle. The pressure valve module (7) controls the opening and closing of the thin film microvalve on the centrifugal microfluidic chip (8) by mechanical extrusion, thereby regulating the liquid flow sequence; The main control circuit system coordinates and controls the collaborative work of each module.

2. The real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 1, characterized in that, The transmission module (2) includes a first support (2-1), a guide rail (2-2), a slider (2-3), and a first motor (2-4). The first motor (2-4) drives the slider (2-3) to slide along the guide rail (2-2). The guide rail (2-2) is mounted on the base (1). The rotation module (3) includes a second motor (3-1) and a flange (3-2). The second motor (3-1) is mounted on the slider (2-3), and the flange (3-2) is mounted on the output shaft of the second motor (3-1). The centrifugal microfluidic chip (8) is detachably connected to the flange (3-2).

3. The real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 1, characterized in that, The pretreatment auxiliary module (4) includes a piercing column (4-1), a second support (4-2), an upper heating plate (4-3), a lower heating plate (4-4), and a linear motor. The second support (4-2) is installed on the base (1), and the linear motor is fixedly installed on the second support (4-2). The piercing column (4-1) is connected to the linear motor. The upper heating plate (4-3) and the lower heating plate (4-4) are slidably connected to the second support (4-2). When the centrifugal microfluidic chip (8) reaches the working position but has not yet started high-speed rotation, the piercing column (4-1) moves downward under the drive of the linear motor, piercing the corresponding sealed reagent capsule on the centrifugal microfluidic chip and releasing the reagent. Under the action of the driving mechanism, the upper heating plate (4-3) and the lower heating plate (4-4) clamp the lysis chamber area of ​​the centrifugal microfluidic chip (8) from the upper and lower sides along the slide rail on the second support (4-2) and heat it to the set temperature.

4. A real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 1, characterized in that, The amplified temperature control module (5) includes two third motors (5-1), a transmission rail (5-2), two sets of heat sinks (5-3), a fan (5-4), a Peltier (5-5), a heating aluminum plate (5-6), and a third support (5-7). The third support (5-7) is mounted on the base (1). The transmission rail (5-2) is vertically mounted on both sides of the third support (5-7). The heat sink (5-3) is slidably connected to the transmission rail (5-2). The two third motors (5-1) drive the two sets of heat sinks (5-3) to slide up and down along the transmission rail (5-2). The Peltier (5-5) is mounted on the bottom of the upper heat sink (5-3), and the heating aluminum plate (5-6) is mounted on the top of the lower heat sink (5-3). A fan (5-4) is mounted on both sets of heat sinks (5-3).

5. A real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 4, characterized in that, The pressure valve module (7) includes an extrusion component (7-1) and a support block (7-2). The extrusion component (7-1) is installed on the upper heat sink (5-3), and the support block (7-2) is installed on the lower heat sink (5-3). The extrusion component (7-1) moves down and together with the support block (7-2) extrudes the corresponding membrane valve area on the centrifugal microfluidic chip (8), so that the microchannel is temporarily closed or opened, controlling the liquid flow direction and step switching.

6. A real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 5, characterized in that, The extrusion component (7-1) includes an extrusion seat (7-101), a sliding shaft (7-102), a spring (7-103), and an extrusion column (7-104). The extrusion seat (701) is mounted on a heat sink (5-3). The extrusion seat (701) array has several cavities. A sliding shaft (7-102) is slidably connected in each cavity. The spring (7-103) is sleeved and connected to the outside of the sliding shaft (7-102). The top end of the spring (7-103) abuts against the upper surface of the cavity, and the bottom end abuts against the end face of the shaft platform of the sliding shaft (7-102). The top end of the sliding shaft (7-102) extends out of the cavity, and the bottom end is fixedly connected to the extrusion column (7-104). The bottom end of the extrusion column (7-104) extends out of the cavity.

7. A real-time fluorescence PCR instrument suitable for microfluidic chips according to claim 1, characterized in that, The fluorescence detection module (6) includes a housing (6-1) and a fluorescence detection channel module (6-2). The fluorescence detection channel (6-2) is installed inside the housing (6-1). The housing (6-1) is a sealed metal cover. The fluorescence detection channel module (6-2) is equipped with four independent optical detection channels. The detection optical axis of the optical detection channel is perpendicular to the plane of the centrifugal microfluidic chip and is vertically aligned with the center of the four PCR reaction chambers on the chip.

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