Micro-fluidic chip, real-time fluorescent PCR (polymerase chain reaction) detection system and detection method

Abstract

The invention discloses a micro-fluidic chip, a real-time fluorescent PCR (polymerase chain reaction) detection system and a detection method. The system comprises a disposable micro-fluidic chip and a detection instrument main body. The micro-fluidic chip is formed by bonding an ITO glass upper-layer substrate and a PDMS lower-layer channel layer, a closed triangular annular reaction channel is formed, and a high-density DNA probe array is fixed on the inner surface of the channel. The detection instrument main body is provided with a vertical chip bearing table, a heating module and a lensless imaging optical module. During working, a sample and a PCR reagent are injected into the chip, three-temperature-zone thermal convective circulation is established through direct heating of the ITO layer, PCR amplification and fluorescence product in-situ hybridization are completed, and a lens-free imaging system captures fluorescence signals in real time and performs quantitative analysis. The kit has the advantages of ultrahigh flux, rapidness, simplicity, convenience, low cost, high integration level, portability and the like, is suitable for multiple fields of clinical diagnosis, public health, food safety, agriculture and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of PCR detection technology, specifically to a microfluidic chip, a real-time fluorescence PCR detection system, and a detection method. Background Technology

[0002] Real-time polymerase chain reaction (PCR) is the gold standard technology in molecular diagnostics, widely used in many key areas such as pathogen detection, genotyping, and cancer diagnosis. However, existing technologies have many limitations that restrict their further development and application.

[0003] On the one hand, traditional real-time PCR instruments have extremely limited throughput, typically only capable of 4-6 detections. This is woefully inadequate when faced with complex testing needs requiring the simultaneous screening of multiple pathogens or gene mutations. Although other high-throughput technologies exist, such as microarrays, their processes are exceptionally cumbersome. Taking microarrays as an example, they require PCR amplification followed by numerous steps including microarray hybridization, washing, and scanning. The entire process is time-consuming and highly susceptible to contamination, severely impacting the accuracy and reliability of the test results.

[0004] On the other hand, traditional PCR instruments are complex and expensive. They rely on sophisticated Peltier elements to achieve rapid temperature cycling, and also require complex optical systems, including excitation sources, filter sets, lenses, photomultiplier tubes or CCDs, for focusing and detecting fluorescence signals. These complex structures and components not only result in large instrument sizes, but also significantly increase manufacturing and maintenance costs, limiting their widespread application in resource-constrained scenarios.

[0005] Furthermore, multiplex detection is highly complex. It typically requires intricate primer and probe design and reaction system optimization, placing significant demands on the professional knowledge and skills of the operators. Operators need extensive experience and a solid professional foundation to ensure the smooth operation of the detection process and the accuracy of the results. This, to some extent, limits the widespread adoption and application of real-time fluorescence PCR technology, especially in some primary healthcare institutions or point-of-care testing (POCT) scenarios, where a shortage of qualified operators may become a significant factor hindering the technology's application.

[0006] In summary, existing technologies have many shortcomings in terms of throughput, equipment complexity, cost, and ease of operation, making it difficult to meet the ever-increasing demand for complex detection. Therefore, there is an urgent need in this field for a novel molecular diagnostic platform that integrates ultra-high throughput detection, rapid response, and low-cost portability to overcome the limitations of existing technologies and promote the further development and application of molecular diagnostic technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a microfluidic chip, a real-time fluorescence PCR detection system, and a detection method to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A real-time fluorescence PCR detection system includes: a disposable microfluidic chip comprising: an upper substrate made of ITO conductive glass, the inner surface of which has three independent serpentine heating circuits distributed along the three sides of a triangle; a lower channel layer made of PDMS, bonded to the lower side of the upper substrate to form a closed triangular annular reaction channel; the channel is defined by an outer triangular boundary and an inner concentric triangular island structure, the island structure area accounting for 15%-40% of the total channel area; and a high-density DNA probe array fixed to the inner surface of the bottom of the channel, with a probe density ≥100 points / cm². The main body of the testing instrument includes: a vertical chip carrier with precision electrodes, and the electrodes making elastic contact with the heating circuit pins; The heating module independently controls the current of three heating circuits through a programmable power supply, forming a 95±2℃ denaturation zone, a 60±2℃ annealing zone, and a 72±2℃ extension zone within the channel, driving thermal convection circulation. The lensless imaging module includes a side-emitting LED excitation source, a bottom-emitting filter, and a CMOS image sensor, with the sensor's field of view covering the entire probe array.

[0009] Furthermore, the triangular annular channel has a width of 10 mm, a depth of 500 ± 20 μm, and a total channel length of 5-20 cm; the minimum distance between the island structure and the outer boundary is 1.5-3 times the channel width.

[0010] Furthermore, the power ratio of the three circuits controlled by the heating module (203) is as follows: the power ratio of the circuit in the top edge deformation zone is 40%-50%; the power ratio of the circuit in the bottom edge annealing zone is 20%-30%; and the power ratio of the circuit in the bottom edge extension zone is 25%-35%.

[0011] Furthermore, in the lensless imaging module: the CMOS sensor pixel size is ≤4μm, and the distance Z between the imaging surface and the bottom surface of the PDMS channel is ≤2mm; the excitation light source is a 470nm LED array, which is tilted at an angle of 30°-60° to the horizontal direction to illuminate the probe array.

[0012] A microfluidic chip for a real-time fluorescence PCR detection system, wherein the probe array region surface of the PDMS channel layer is treated with oxygen plasma and modified with amino groups, and DNA probes are fixed by covalent bonds; the probe spot diameter is 150±20μm and the center-to-center spacing of the spots is ≥300μm.

[0013] Furthermore, the resistance of the ITO layer on the upper substrate is 5-20Ω / cm, the line width of the serpentine heating circuit is 50-100μm, and the spacing between adjacent circuits is ≥500μm.

[0014] A real-time fluorescence PCR detection method, comprising: Step S1: Inject the mixture containing the nucleic acid sample to be tested and PCR reagents into the reaction channel of the chip; Step S2: By independently controlling the three heating currents, three temperature zones are established in the channel and thermal convection with a flow rate of 0.5-5 mm / s is driven; Step S3: Hybridization of the amplification product with the solid-phase probe occurs in real time in the annealing / extension region; Step S4: Capture fluorescence projection images at a frequency of ≥0.5Hz using a CMOS sensor; Step S5: Reconstruct the time-series curve of probe spot fluorescence intensity based on the deconvolution algorithm.

[0015] Furthermore, in step S2, the thermal convection flow rate is 1-3 mm / s, the entire PCR amplification process takes 10-25 minutes, and the number of targets detected in a single reaction is ≥500.

[0016] Furthermore, in step S4, a differential imaging strategy is adopted: images are acquired at regular intervals during the annealing stage of each round of thermal convection cycle, and background subtraction is performed using images acquired during the extension stage.

[0017] Furthermore, the fluorescence quantification in step S5 adopts the dynamic threshold method: when the signal intensity of the probe point reaches 5-10 times the standard deviation of the background noise, it is judged as a positive signal.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. Ultra-high throughput detection capability. By combining microarray spotting technology and wide-field-of-view lensless imaging technology, it is possible to simultaneously detect thousands of indicators on a single chip, theoretically reaching 3300 detectable dosimetry, far exceeding traditional qPCR technology (usually 4-6 dosimetry). This allows for screening multiple pathogens or gene mutations in a single reaction, greatly improving detection efficiency, and is especially suitable for complex scenarios requiring simultaneous detection of multiple indicators, such as respiratory multiplex pathogen detection and bloodborne infection screening; 2. Rapid and Convenient Detection Process. Utilizing hot convection PCR technology, the reaction is fast, typically completing in just 20-40 minutes, 6-9 times faster than traditional qPCR (60-90 minutes). Furthermore, amplification and detection are completed in one step, eliminating the cumbersome hybridization, washing, and scanning steps of traditional gene chips. Users simply need to "add sample - place card - wait for results," greatly simplifying the operation process, reducing the professional requirements for operators, and improving the convenience and efficiency of detection. 3. Low-cost and highly integrated design. The chip consumables consist only of ITO glass and PDMS, resulting in low cost. The instrument eliminates the expensive Peltier temperature cycling system and all external heating blocks, replacing them with an extremely simple ITO direct heating method and a programmable power supply, significantly reducing the instrument's manufacturing cost, size, and maintenance cost. Furthermore, by directly integrating the heating element onto the disposable chip, the external heating blocks and their complex mechanical alignment structure are eliminated, resulting in an extremely compact overall instrument structure with no complex moving parts, making it easy to achieve miniaturization and portability, ideal for applications such as point-of-care testing (POCT). 4. High reaction efficiency and specificity. The three-temperature zone design corresponds to the three optimal temperature points of PCR denaturation, annealing, and extension. Compared with a two-temperature gradient design, it can more precisely control the reaction conditions of each step, thereby obtaining higher amplification efficiency and specificity. This not only improves the accuracy of detection but also effectively reduces non-specific amplification, ensuring the reliability of detection results, especially when detecting low-abundance targets. 5. Broad Application Prospects. Applicable to various detection scenarios, including but not limited to clinical diagnosis (such as respiratory infection panels, bloodborne infection / septicemia panels, tumor liquid biopsy, etc.), public health and disease control (such as rapid on-site screening, environmental monitoring, etc.), food safety (detection of foodborne pathogens), agriculture and animal husbandry (rapid diagnosis of animal diseases and breeding screening), etc. Its ultra-multiplex detection capabilities and rapid and convenient characteristics enable it to meet the needs of different fields for high-throughput, rapid, and accurate detection, possessing broad application prospects and market potential. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall structure of a real-time fluorescence PCR detection system according to the present invention; Figure 2 This is one of the structural schematic diagrams of a microfluidic chip for a real-time fluorescence PCR detection system according to the present invention; Figure 3 This is one of the structural schematic diagrams of a microfluidic chip for a real-time fluorescence PCR detection system according to the present invention; Figure 4 This is a schematic diagram illustrating the internal three-temperature zone thermal convection and probe hybridization principle of a microfluidic chip for a real-time fluorescence PCR detection system according to the present invention. Figure 5 This is a simulation diagram of the internal thermal convection velocity of a microfluidic chip for a real-time fluorescence PCR detection system according to the present invention. Figure 6 This is a schematic diagram of the internal thermal simulation of a microfluidic chip for a real-time fluorescence PCR detection system according to the present invention.

[0020] In the diagram: 101, upper substrate; 102, serpentine heating circuit; 103, lower channel layer; 104, triangular annular reaction channel; 105, outer triangular boundary; 106, triangular island structure; 107, probe array; 201, chip carrier stage; 202, precision electrode; 203, heating module; 205, excitation light source; 206, emission filter; 207, CMOS / CCD image sensor; 3, ITO transparent heater; 4, microfluidic chip (PDMS layer); 5, pinhole aperture; 52, excitation light. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Please see Figure 1 The present invention provides a technical solution, which mainly includes a disposable microfluidic chip and a detection instrument body, as well as a matching optical module and heating module.

[0023] (I) Microfluidic Chips: Structure: See Figure 2 and Figure 3 A disposable microfluidic chip includes: an upper substrate 101 made of ITO conductive glass, on which three independent serpentine heating circuits 102 are provided on the inner surface, distributed along the three sides of a triangle; a lower channel layer 103 made of PDMS, bonded to the lower side of the upper substrate to form a closed triangular ring-shaped reaction channel 104; the channel is defined by an outer triangular boundary 105 and an inner concentric triangular island structure 106, the area of ​​the island structure accounting for 15%-40% of the total area of ​​the channel; and a high-density DNA probe array fixed on the inner surface of the bottom of the channel, with a probe density ≥100 points / cm².

[0024] Functional area: In the reaction chamber of the chip, a high-density DNA probe array 107 is pre-prepared on the inner surface of PDMS or ITO glass using a spotting instrument. Each probe spot corresponds to a specific detection index.

[0025] Working principle: A mixture containing the test sample and PCR reagents is injected into a triangular annular channel. The chip is placed vertically on the instrument, which is connected to the ITO glass conductive layer via multiple electrodes. The chip is heated by electricity, while heat is dissipated from the other side of the chip using the instrument structure or ambient air. This creates a stable temperature difference field within the reaction chamber, setting it to the three key temperatures required for PCR (e.g., [missing information]). Figure 4The top edge is 95°C for denaturation, creating a gentle temperature gradient from left (60°C) to right (72°C) at the bottom of the chip, allowing the liquid to sequentially pass through the optimal temperature zones for annealing and extension. These three different temperature boundaries generate a stable temperature field and density difference within the channel, driving continuous, stable, and short-circuit-free natural thermal convection. The liquid circulates along a specific trajectory within the annular channel, sequentially passing through the denaturation, annealing, and extension zones, automatically completing the PCR amplification cycle. During the annealing / extension stage, the amplified fluorescently labeled product hybridizes with the corresponding probe on the solid-phase probe array, causing the fluorescence signal at the probe site to increase in real time as amplification progresses. The central island structure effectively prevents the fluid from taking shortcuts, forcing it to circulate completely along the preset "denaturation-annealing-extension" path, improving reaction uniformity and efficiency.

[0026] (II) Main body of the testing instrument: The instrument is equipped with a vertical chip carrier stage 201 for placing and positioning the microfluidic chip. The chip carrier stage 201 integrates precision electrodes 202 that are connected to the chip's ITO layer and are precisely connected to the three sides 105 of the chip's outer triangular channel. The probe array functional area of ​​the chip is directly opposite the lower side of the carrier stage.

[0027] (III) Optical Module (Lens-Free Imaging): This module is located on the side of the chip stage 201, closely aligned with the probe array 107 area of ​​the chip. It consists of an excitation light source 205, an emission filter 206, and a CMOS / CCD image sensor 207. Excitation light 52 (such as a blue LED) illuminates the chip from the side, penetrating the transparent ITO cover and the liquid, exciting the fluorescent groups on the probe array 107. The generated fluorescence signal passes through the microfluidic chip (PDMS layer) 4 and the emission filter behind it, and is directly captured by the CMOS sensor, forming a raw projection image containing signals from all probe points. The computer processes this image using algorithms such as deconvolution to obtain a clear real-time fluorescence image.

[0028] (iv) Heating Module: The core of this module is a precision programmable power supply. Precise voltage / current is applied to the ITO transparent heater 3 of the chip via electrodes on the stage, directly heating the ITO glass using the Joule effect. By precisely controlling the power input and combining it with fixed heat dissipation conditions, a stable temperature gradient required for PCR thermal convection is established within the reaction chamber, stabilizing at the denaturation, annealing, and extension temperatures required for PCR, thereby creating a stable three-temperature zone thermal convection field within the chip.

[0029] The core function of the detection system is to achieve accurate detection of target substances in samples through the combination of optical detection and microfluidic technology. The collaborative process of each component is as follows: Step 1: Sample Loading and Preprocessing The sample to be tested (such as biological fluid, reaction solution, etc.) is introduced into the microchannels inside the microfluidic chip (PDMS layer), and the chip is fixed on the chip carrier stage / platform. The microfluidic chip utilizes the properties of PDMS material to achieve precise sample transport, mixing, or reaction (such as reaction with reagents), providing a uniform and stable detection environment for subsequent detection.

[0030] Step 2: Temperature control adjustment (optional, depending on testing requirements)

[0031] The ITO transparent heater integrated on the microfluidic chip can control the temperature of the sample within the chip according to detection requirements (such as the specific temperature required for enzyme-catalyzed reactions). By adjusting the heater power, the sample is maintained at the optimal temperature required for the reaction or detection, ensuring reaction efficiency or the stability of the target substance.

[0032] Step 3: Excitation light irradiation

[0033] An excitation light source (LED) emits excitation light of a specific wavelength, which is directed onto the region of the microfluidic chip containing the target material. The wavelength of the excitation light must match the optical properties of the target material (e.g., the target material can be excited by light of this wavelength to produce fluorescence) to ensure that the target material can be effectively excited.

[0034] Fluorescence signal generation and filtering

[0035] When irradiated by excitation light, the target substance in the sample (such as a fluorescently labeled molecule to be detected) absorbs energy and releases a fluorescence signal of a specific wavelength. To avoid interference from excitation light and ambient stray light on the fluorescence signal, the fluorescence signal first passes through an emission filter to filter out light of non-target wavelengths (only allowing the target fluorescence wavelength to pass through); then it passes through a pinhole aperture to further remove stray light and signals from non-focal regions, improving the spatial resolution and signal-to-noise ratio of the fluorescence signal.

[0036] Step 4: Signal Acquisition and Analysis

[0037] The filtered and optimized fluorescence signal is received by a CMOS image sensor, which converts the light signal into an electrical signal. The data processing module then transforms this signal into analyzable image or numerical data. By analyzing the intensity, distribution, and other characteristics of the fluorescence signal, qualitative or quantitative detection of the target substance in the sample can be achieved.

[0038] In summary, this system achieves precise sample manipulation through a microfluidic chip, combined with temperature control from an ITO transparent heater, specific excitation from an excitation light source, and signal purification from an optical filter component. Finally, a CMOS image sensor completes signal acquisition, forming a complete detection chain from sample input to detection result output.

[0039] To verify the above effect, a specific example will be used for illustration.

[0040] Example: Design a prototype for detecting multiple respiratory pathogens.

[0041] Calculation of multiple detection capability: Chip functional area dimensions: 1cm × 3cm (10,000 µm × 30,000 µm).

[0042] Assumptions for spotting parameters: Using a non-contact spotting instrument, probe spots with a diameter (D) of 150 µm can be prepared. To ensure that the signals at each spot do not interfere with each other and can be clearly distinguished, the center distance (L) between the spots is set to twice the spot diameter, i.e., L = 300 µm.

[0043] calculate: ① The number of probe points that can be arranged along a 1cm edge is approximately 33 points, which is approximately 10,000 µm / 300 µm / point. ② The number of probe points that can be arranged along a 3cm edge is N_length = 30,000 µm / 300 µm / point = 100 points; ③ Theoretical maximum number of tests = N_width × N_length = 33 × 100 = 300 tests; ④ Conclusion: At 1cm Within a 3cm area, this invention can theoretically easily achieve ultra-multiplex detection of more than 1,000 indicators.

[0044] Optical module selection and resolution analysis: CMOS Module Selection: Choose a consumer-grade or industrial-grade high-resolution monochrome CMOS sensor, such as the Sony IMX415 (8MP, pixel size p=1.45 µm) or a higher-performance research-grade CMOS (e.g., pixel size 1.1 µm - 2.5 µm).

[0045] We selected a monochrome CMOS sensor with a pixel size of p=2.0 µm, whose photosensitive area is sufficient to cover 1 cm. 3cm area.

[0046] Z-distance estimation: ① PDMS channel layer bottom thickness: 200 µm (0.2 mm) ② Emission filter (select a thin absorption filter): ~500 µm (0.5 mm) ③ CMOS protective glass thickness: ~300 µm (0.3 mm) ④ Total Z-distance (Z)≈200+500+300=1000 µm (1 mm).

[0047] Resolution calculation: ① Pixel resolution limit = 2 × p = 2 × 2.0 µm = 4.0 µm; ② Diffraction-limited resolution: δ≈λ / (2 NA_eff); ③ FAM fluorescence wavelength λ≈520 nm (0.52 µm); ④ Effective numerical aperture NA_eff = sin(arctan(D / 2Z)), where D is the width of the imaging region (1cm = 10,000 µm).

[0048] NA_eff = sin(arctan(10000 / (2 × 1000))) = sin(arctan(5)) ≈ 0.98.

[0049] The diffraction-limited resolution is δ≈0.52 µm / (2 × 0.98)≈0.265 µm.

[0050] Compared to traditional PCR:

[0051] Conclusion: Based on Figure 5 Simulation diagram of internal thermal convection velocity of the chip and Figure 6 The internal thermal simulation of the chip shows that the final resolution of the system is determined by the relatively poor pixel-limited resolution, which is approximately 4.0 µm. This resolution is much smaller than the probe spot spacing (300 µm) and probe spot diameter (150 µm) we designed, and is therefore sufficient to clearly distinguish and quantify the fluorescence signal of each probe spot, while having the potential for higher throughput (nearly 10,000 ppm).

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A real-time fluorescence PCR detection system, characterized in that, include: A disposable microfluidic chip includes: an upper substrate (101) made of ITO conductive glass, on the inner surface of which are provided three independent serpentine heating circuits (102), which are distributed along the three sides of a triangle; A lower channel layer (103) made of PDMS is bonded to the lower side of the upper substrate to form a closed triangular ring-shaped reaction channel (104); the channel is defined by an outer triangular boundary (105) and an inner concentric triangular island structure (106), the area of ​​the island structure accounts for 15%-40% of the total area of ​​the channel; a high-density DNA probe array (107) is fixed on the inner surface of the bottom of the channel, with a probe spot density ≥100 spots / cm²; The main body of the testing instrument includes: a vertical chip carrier stage (201) with precision electrodes (202), wherein the electrodes are in elastic contact with the heating circuit pins; The heating module (203) independently controls the current of the three heating circuits through a programmable power supply, forming a 95±2℃ denaturation zone, a 60±2℃ annealing zone, and a 72±2℃ extension zone in the channel, driving thermal convection circulation. The lensless imaging module includes a side-emitting LED excitation source (205), a bottom-emitting filter (206), and a CMOS image sensor (207), with the sensor's field of view covering the entire probe array.

2. The real-time fluorescence PCR detection system according to claim 1, characterized in that: The triangular annular channel (104) has a width of 10 mm, a depth of 500±20 μm, and a total length of 5-20 cm; the minimum distance between the island structure (106) and the outer boundary is 1.5-3 times the width of the channel.

3. The real-time fluorescence PCR detection system according to claim 1, characterized in that: The heating module (203) controls the power ratio of the three circuits as follows: the power ratio of the top edge deformation zone circuit is 40%-50%; the power ratio of the bottom edge annealing zone circuit is 20%-30%; and the power ratio of the bottom edge extension zone circuit is 25%-35%.

4. The real-time fluorescence PCR detection system according to claim 1, characterized in that: In the lensless imaging module (204): the CMOS sensor pixel size is ≤4μm, and the distance Z between the imaging surface and the bottom surface of the PDMS channel is ≤2mm; the excitation light source is a 470nm LED array, which is tilted at an angle of 30°-60° with the horizontal direction to illuminate the probe array.

5. A microfluidic chip for use in any one of the real-time fluorescence PCR detection systems of claims 1-4, characterized in that: The probe array region (107) surface of the PDMS channel layer (103) is treated with oxygen plasma and modified with amino groups, and the DNA probe is fixed by covalent bonds; the probe spot diameter is 150±20μm and the center-to-center spacing of the spots is ≥300μm.

6. The microfluidic chip according to claim 5, characterized in that: The ITO layer resistance of the upper substrate (101) is 5-20Ω / cm, the line width of the serpentine heating circuit (102) is 50-100μm, and the spacing between adjacent circuits is ≥500μm.

7. A real-time fluorescence PCR detection method, characterized in that, include: Step S1: Inject the mixture containing the nucleic acid sample to be tested and PCR reagents into the reaction channel of the chip described in claims 5-6; Step S2: By independently controlling the three heating currents, three temperature zones are established in the channel and thermal convection with a flow rate of 0.5-5 mm / s is driven; Step S3: Hybridization of the amplification product with the solid-phase probe occurs in real time in the annealing / extension region; Step S4: Capture fluorescence projection images at a frequency of ≥0.5Hz using a CMOS sensor; Step S5: Reconstruct the time-series curve of probe spot fluorescence intensity based on the deconvolution algorithm.

8. The real-time fluorescence PCR detection method according to claim 7, characterized in that: In step S2, the thermal convection flow rate is 1-3 mm / s, the entire PCR amplification process takes 10-25 minutes, and the number of targets detected in a single reaction is ≥500.

9. The real-time fluorescence PCR detection method according to claim 7, characterized in that: In step S4, a differential imaging strategy is adopted: images are acquired at regular intervals during the annealing stage of each round of thermal convection cycle, and background subtraction is performed using images acquired during the extension stage.

10. The real-time fluorescence PCR detection method according to claim 7, characterized in that: The fluorescence quantification in step S5 uses the dynamic threshold method: when the signal intensity of the probe point reaches 5-10 times the standard deviation of the background noise, it is judged as a positive signal.

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