Rapid PCR temperature control, detection method, and dual chamber self-calibrating PCR system

Abstract

The application provides a rapid PCR temperature control method, a detection method and a double-chamber self-calibration PCR system, and relates to the technical field of biochemical detection. The temperature control method comprises: a calibration stage, in which a light source heating module synchronously provides heating light beams to PCR reaction chambers and a temperature reference chamber with the same structure, at least one temperature cycle is experienced, a reference liquid temperature measured by a temperature sensor is taken as a liquid temperature reference value in the chamber, an outside surface temperature of the PCR reaction chamber is collected, and a mapping relationship between the outside surface temperature and the liquid temperature in the chamber is established; and an amplification stage, in which the PCR reaction chamber is provided with the heating light beams, the liquid temperature in the chamber after calibration is calculated based on the mapping relationship from the outside surface temperature, and the light source power is adjusted according to the deviation of the liquid temperature in the chamber from a target temperature. The application corrects infrared temperature measurement deviation caused by pipe wall thermal resistance, compensates for infrared low-frequency sampling defects with high-frequency liquid temperature, eliminates temperature control hysteresis and overshoot, and only needs about 11 minutes for 40 cycles, so that extremely fast and accurate temperature control and high-sensitivity nucleic acid detection are realized.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, specifically to a rapid PCR temperature control method, a detection method, and a dual-chamber self-calibrating PCR system. More particularly, it relates to a photothermal PCR method and its temperature calibration system. Background Technology

[0002] Nucleic acid-based diagnostic methods (polymerase chain reaction (PCR)) have long been the gold standard for providing early detection results of infections, exhibiting excellent specificity, accuracy, and sensitivity. They have become indispensable methods for diagnosing cancer, viral and bacterial infections, food safety testing, and environmental monitoring. However, traditional PCR technology requires expensive enzyme reagents, typically takes 1-2 hours for detection, and necessitates costly equipment such as Peltier gels to achieve thermal cycling at different temperatures. Therefore, it is necessary to develop cost-effective and scalable technologies to meet the current needs of biomedical research.

[0003] Traditional PCR technology completes nucleic acid amplification and signal detection by thermal cycling of nucleic acid molecules between high temperature (~95℃) and low temperature (~60℃). Therefore, PCR instruments are equipped with metal blocks, Peltiers, etc. for heating and cooling. The heating and cooling rate of commercial PCR instruments is 2~5℃ / s, which is relatively slow, resulting in a long PCR amplification and detection time.

[0004] Rapid PCR systems utilizing plasmonic metal nanoparticles offer significant advantages in PCR detection time and cost. Nanoparticles absorb light and efficiently convert it into heat energy through plasmon photoexcitation or electron-phonon and phonon-phonon coupling, generating a rapid and localized heating effect around the nanoparticles. Utilizing photothermal conversion, i.e., the plasmonic photothermal effect, the thermal cycling time of the entire PCR detection process (the core process of PCR) can be reduced from one hour to just a few minutes. Using nanoparticles as heaters, light of a specific resonant wavelength irradiates the nanoparticles, which convert the light into heat and rapidly and uniformly transfer it throughout the amplification system. The PCR temperature for nucleic acid amplification is controlled by periodically adjusting the power of the light source.

[0005] Compared to traditional PCR instruments, rapid PCR offers advantages such as small size, fast heating rate, low energy consumption, and ease of integration, making it ideal for the rapid detection needs of point-of-care testing (POCT) scenarios. Currently, temperature control in rapid PCR systems primarily relies on real-time temperature monitoring using infrared thermal imagers. Its working principle is as follows: an infrared camera acquires images of the surface thermal radiation of the PCR reaction tubes; the surface temperature of the tube wall is calculated based on the blackbody radiation law; and then the temperature control system adjusts the light source power to achieve the denaturation, annealing, and extension temperatures required for the PCR cycle.

[0006] The existing technology has the following shortcomings: 1. Traditional PCR is limited by the heating and cooling rates of metal heat-conducting blocks or Peltiers, and the detection time can be as long as 1 to 2 hours.

[0007] 2. Rapid PCR uses infrared thermal imaging temperature sensors, which can only measure the outer wall temperature. There is a significant difference between the liquid temperature and the wall temperature, and there is no standard temperature calibration method for rapid PCR systems. Infrared imaging temperature measurement is greatly affected by ambient temperature, humidity, airflow, and changes in the emissivity of the surface being measured.

[0008] 3. The response time of infrared thermal imaging temperature sensors is >25 milliseconds / time, which is too low. Compared with the response rate of 1 millisecond / time of traditional contact temperature sensors, this will lead to lag and instability in temperature control in the application of real-time temperature measurement in ultra-fast PCR.

[0009] Patent document CN116676179A discloses a PCR instrument based on photothermal conversion, including a heating element and a light source. The heating element has a socket with an opening at the top for inserting or removing reagent tubes, and a photon heat converter at the bottom. The light source is located below the heating element and emits light into the photon heat converter, causing it to generate heat and heat the reagent tubes. Multiple large temperature zones on the heating element are equipped with cooling devices for cooling the reagent tubes. The shortcomings of this patent document are that it does not employ a dual-chamber structure, making it impossible to obtain a reference chamber temperature calibration design, and it does not establish a surface temperature-internal temperature mapping model. Therefore, it cannot solve the problems of liquid temperature and wall temperature deviation and response lag in infrared thermometry, making it difficult to achieve precise temperature control under ultra-fast thermal cycling conditions. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the purpose of this invention is to provide a rapid PCR temperature control and detection method, as well as a dual-chamber self-calibrating PCR system.

[0011] According to the present invention, a rapid PCR temperature control method employs a rapid PCR system comprising a PCR reaction chamber and a temperature reference chamber with identical structures. The rapid PCR system further includes a light source heating module, a temperature sensor inserted into the temperature reference chamber, and an infrared temperature measurement module. The PCR reaction chamber is filled with PCR reaction solution, and the temperature reference chamber is filled with a reference solution of the same volume as the PCR reaction solution. The PCR temperature control method includes a calibration phase and an amplification phase. The calibration phase includes: A1: The heating module of the light source synchronously provides a heating beam to the bottom of the liquid in the two chambers for photothermal heating, so that the two chambers undergo at least one temperature cycle; During the temperature cycling process, the temperature of the reference liquid inside the temperature reference chamber is measured by the temperature sensor as a reference value for the liquid temperature inside the chamber, and the temperature of the outer surface of the PCR reaction chamber is collected by the infrared temperature measurement module. A2: Based on the reference value of the liquid temperature inside the cavity and the surface temperature of the outer side of the PCR reaction cavity, establish a mapping relationship between the surface temperature of the outer side and the liquid temperature inside the cavity; The amplification phase includes: B1: The heating light source module provides a heating beam to the PCR reaction chamber for photothermal heating. During the heating process, the infrared temperature measurement module collects the temperature of the outer surface of the PCR reaction chamber and calculates the calibrated liquid temperature inside the chamber based on the mapping relationship. B2: Adjust the operating power of the light source heating module according to the deviation between the calibrated intracavitary liquid temperature and the target temperature.

[0012] Preferably, the mapping relationship is as follows: T 管内 =f(T 表面 ,dT 管内 / dt); Among them, T 管内 T is the temperature of the liquid inside the cavity, which is the temperature reference. 表面 The infrared thermometer module collects the surface temperature of the outer side of the PCR reaction chamber, dT. 管内 / dt represents the rate of temperature change within the temperature reference cavity, t represents time, and f represents the calibration function obtained through multiple linear regression.

[0013] Preferably, the temperature cycle includes a cooling process after the heating process, and the rapid PCR system further includes a cooling module configured to provide symmetrical cooling airflow to the two chambers; During the heating process of A1 and B1, the heating module of the light source is turned on, and during the cooling process, the heating module of the light source is turned off and the cooling module is started to cool down.

[0014] Preferably, in B2, a PID control algorithm is used to dynamically adjust the PID parameters based on the deviation between the calibrated intracavitary liquid temperature and the target temperature, thereby adjusting the working power of the light source heating module in real time.

[0015] According to a rapid PCR detection method provided by the present invention, based on the rapid PCR temperature control method, a nucleic acid detection stage is further included, wherein the nucleic acid detection stage includes a combination of any one or more of the following methods: 1) During the annealing / extension phase of each temperature cycle, test and collect multiple fluorescence signals for fluorescent qPCR detection; 2) After all thermal cycling procedures are completed, the endpoint fluorescence detection is performed to test the fluorescence signal intensity; 3) After all thermal cycling procedures are completed, the amplification products are sequenced or detected by electrophoresis.

[0016] A dual-chamber self-calibrating PCR system according to the present invention is used to implement the aforementioned rapid PCR temperature control method, comprising: The consumable body contains the PCR reaction chamber and the temperature reference chamber. A sealing cover plate covers and seals the top openings of the two chambers, through which the temperature sensor is inserted into the temperature reference chamber; The light source heating module is located below the consumable body and is configured to simultaneously provide two heating beams of the same energy to the bottom of the liquid in the PCR reaction chamber and the temperature reference chamber. The infrared temperature measurement module is configured to collect the temperature of the outer surface of the PCR reaction chamber. The temperature control module is configured to establish a mapping relationship between the reference liquid temperature measured by the temperature sensor and the outer surface temperature of the PCR reaction chamber collected by the infrared temperature measurement module during the calibration phase; during the amplification phase, it calculates the calibrated intracavitary liquid temperature based on the mapping relationship and the outer surface temperature of the PCR reaction chamber collected by the infrared temperature measurement module; and adjusts the power of the light source heating module according to the deviation between the calibrated intracavitary liquid temperature and the target temperature.

[0017] Preferably, the bottoms of the PCR reaction chamber and the temperature reference chamber are both conical, with the radial dimensions of the bottom of the two chambers contracting to form sharp corners, and the radial dimensions of the two chambers gradually increasing from the bottom to the top opening and then remaining constant.

[0018] Preferably, the PCR reaction solution comprises a nucleic acid template and a nucleic acid amplification reaction solution, the nucleic acid amplification reaction solution contains nano-photothermal materials, and the upper layer of the PCR reaction solution is sealed with an oil seal; the reference solution comprises a nucleic acid amplification reaction solution with the same composition and volume as the nucleic acid amplification reaction solution in the PCR reaction chamber, and enzyme-free water, and the upper layer of the reference solution is sealed with an oil seal.

[0019] Preferably, the light source heating module includes a laser, a beam splitter, and two total reflection mirrors; The laser is located at the middle of the bottom side of the consumable body and emits laser light towards the bottom of the consumable body; the beam splitter is located on the output optical path of the laser and splits the laser light into two beams of the same energy and emits them to the sides respectively; the two total reflection mirrors are located on both sides of the beam splitter and reflect the split beams to the bottom of the PCR reaction chamber and the temperature reference chamber.

[0020] Preferably, it also includes a cooling module and a multi-channel fluorescence detection module; The cooling module includes a fan located between two chambers. The fan's air outlet is aimed at the center of the consumable body, so that the airflow diffuses symmetrically to both sides and sweeps evenly across the outer walls of the two chambers. The multi-channel fluorescence detection module is configured to initiate excitation and data acquisition during each annealing / extension stage, thereby enabling real-time capture of multiple fluorescence signals.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention, by setting up a dual-chamber sealing consumable and using a built-in contact sensor to obtain the actual liquid temperature in the isomorphic reference chamber in real time, corrects the problem of infrared temperature measurement lag and deviation caused by tube wall thermal resistance, thus ensuring the stability of the amplification system.

[0022] 2. This invention eliminates the need for traditional metal heat transfer blocks such as Peltier, and instead uses a centrally located symmetrical fan for cooling and synchronous multi-beam heating, combined with precise PID control. 40 temperature cycles can be completed in just about 11 minutes, and the precise temperature control is significantly superior to traditional PCR equipment.

[0023] 3. This invention combines the advantages of area array monitoring in infrared thermal imaging with the high-frequency response advantages of contact sensors. By dynamically compensating for the low-frequency sampling (>25 milliseconds / time) defects of infrared cameras through high-frequency liquid temperature data, it effectively eliminates control lag and thermal inertia in ultra-fast heating processes above 10℃ / s, eliminates temperature overshoot and system oscillation, and achieves extremely high stability under ultra-fast temperature control.

[0024] 4. Through the precise and lag-free temperature control described above, this invention achieves stable amplification and detection of templates at extremely low concentrations, resulting in nucleic acid amplification with extremely high specificity and sensitivity. Attached Figure Description

[0025] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a plan view illustrating the overall structure of the photothermal PCR system of this invention; Figure 2 This invention primarily demonstrates the heating kinetics curve of the photothermal PCR system; Figure 3 This invention primarily demonstrates the thermal cycling kinetics curve of the photothermal PCR system; Figure 4 This is a comparison diagram that mainly illustrates the performance of photothermal PCR amplification in this invention.

[0026] Figure label: Consumable body 1, PCR reaction chamber 2, temperature reference chamber 3, sealing cover 4, fan 5, temperature sensor 6, laser 7, beam splitter 8, total reflection mirror 9. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0028] This invention provides a dual-chamber self-calibrating PCR system, comprising dual-chamber consumables, a light source heating module, a cooling module, an infrared temperature measurement module, and a temperature control module. The rapid PCR described in this invention refers to PCR technology that achieves rapid temperature cycling through heating, with photothermal PCR as its core implementation method. Specifically, the light source heating module is implemented using a laser 7, a beam splitter 8, and a total reflection mirror 9; the cooling module is implemented using a fan 5; the temperature sensor 6 is implemented using a contact temperature sensor such as a miniature thermocouple or a thermistor; and the infrared temperature measurement module is implemented using an infrared thermal imaging temperature sensor.

[0029] like Figure 1 As shown, the overall structure of the PCR system of the present invention includes: consumable body 1, PCR reaction chamber 2, temperature reference chamber 3, sealing cover plate 4, fan 5, temperature sensor 6, laser 7, beam splitter 8, and total reflection mirror 9.

[0030] The consumable body 1 contains two adjacent chambers: a PCR reaction chamber 2 and a temperature reference chamber 3. Both chambers have identical structures. The PCR reaction chamber 2 and the temperature reference chamber 3 are formed within the consumable body 1, with the sidewalls of the consumable body 1 serving as the sidewalls of the two chambers. The length, width, depth, and radial dimensions of the two chambers are completely identical, ensuring that their thermal response characteristics are the same, thus providing an isomorphic reference for temperature calibration. The two chambers are arranged side-by-side.

[0031] Both the PCR reaction chamber 2 and the temperature reference chamber 3 have conical bottoms. The radial dimensions of the bottom of the two chambers contract to form sharp corners, and the radial dimensions of the two chambers gradually increase from the bottom to the top opening and then remain constant. The conical bottom design allows the light beam to enter the liquid from the bottom sharp corner, which is beneficial for the concentrated absorption and rapid transfer of photothermal energy. A sealing cover 4 is located on top of the consumable body 1, covering and sealing the top openings of the PCR reaction chamber 2 and the temperature reference chamber 3.

[0032] PCR reaction chamber 2 contains PCR reaction solution including nucleic acid template and nucleic acid amplification reaction solution. The nucleic acid amplification reaction solution contains photothermal nanomaterials, including gold nanoparticles, graphene, and silver nanoparticles, which are mixed with enzymes and reaction buffers to prepare the nucleic acid amplification reaction solution. The upper layer of the PCR reaction solution is sealed with an oil seal. Temperature reference chamber 3 contains reference solution, which contains nucleic acid amplification reaction solution with the same composition and volume as the nucleic acid amplification reaction solution in PCR reaction chamber 2, as well as enzyme-free water. The upper layer of the reference solution is also sealed with an oil seal. The volumes of nucleic acid template and enzyme-free water are equal, ensuring that the total volume of the reference solution and the PCR reaction solution is the same. Since the composition of the nucleic acid amplification reaction solution in both chambers is identical, the only difference being that the nucleic acid template in PCR reaction chamber 2 is replaced with enzyme-free water in temperature reference chamber 3, therefore both chambers have the same photothermal response characteristics, providing an isomorphic reference standard for temperature calibration.

[0033] Temperature sensor 6 is inserted into temperature reference chamber 3 through sealing cover plate 4, configured to directly measure the temperature of the reference liquid inside temperature reference chamber 3. Temperature sensor 6 passes vertically through sealing cover plate 4, is inserted into and fixed at the center of the conical bottom of temperature reference chamber 3, and a gap is left between the probe of temperature sensor 6 and the wall of temperature reference chamber 3 to obtain the most representative liquid temperature. In specific implementations, temperature sensor 6 uses a standard contact temperature sensor such as a miniature thermocouple or thermistor, including sensor packaging structure and signal acquisition circuitry.

[0034] The light source heating module is located below the consumable body 1 and is configured to simultaneously provide two heating beams of the same energy to the bottom of the liquid in the PCR reaction chamber 2 and the temperature reference chamber 3, so as to achieve synchronous photothermal excitation of the liquid in the two chambers. In a specific implementation, the light source heating module includes a laser 7, a beam splitter 8, and two total reflection mirrors 9.

[0035] Laser 7 is located at the center of the bottom side of consumable body 1, that is, exactly in the middle of the bottom projection positions of PCR reaction chamber 2 and temperature reference chamber 3, emitting laser light towards the bottom of consumable body 1. Beam splitter 8 is located in the output optical path of laser 7 and below consumable body 1, splitting the laser light into two beams of equal energy that are emitted to opposite sides. Two total reflection mirrors 9 are located on both sides of beam splitter 8, reflecting the split beams to the pointed corners of the conical bottoms of PCR reaction chamber 2 and temperature reference chamber 3.

[0036] The cooling module includes a fan 5 located between the PCR reaction chamber 2 and the temperature reference chamber 3, configured to provide symmetrical cooling airflow to the two chambers. The fan 5 is positioned at the physical midpoint between the PCR reaction chamber 2 and the temperature reference chamber 3, with its outlet aligned with the center of the consumable body 1, allowing the airflow to diffuse symmetrically to both sides and evenly sweep across the outer walls of the PCR reaction chamber 2 and the temperature reference chamber 3, ensuring that the heat dissipation rates of the two chambers are completely consistent during the thermal cycling cooling phase.

[0037] The infrared temperature measurement module is configured to acquire the thermal radiation temperature of the outer surface of the PCR reaction chamber 2. In specific implementation, the infrared temperature measurement module uses an infrared thermal imaging temperature sensor to acquire thermal radiation images of the outer surface of the PCR reaction chamber 2 and calculate the surface temperature of the tube wall according to the blackbody radiation law.

[0038] The temperature control module is configured to establish a mapping relationship between the reference liquid temperature measured by the temperature sensor 6 and the outer surface temperature of the PCR reaction chamber 2 collected by the infrared temperature measurement module during the calibration phase. During the amplification phase, the calibrated liquid temperature inside the chamber is calculated based on the mapping relationship and the outer surface temperature of the PCR reaction chamber 2 collected by the infrared temperature measurement module, and the power of the light source heating module is adjusted according to the deviation between the calibrated liquid temperature inside the chamber and the target temperature.

[0039] Since the infrared temperature measurement module can only collect the surface temperature outside the PCR reaction chamber 2 and cannot directly measure the temperature of the liquid inside the chamber, and because the thermal resistance of the tube wall during heating / cooling causes a lag and deviation between the surface temperature and the liquid temperature inside the tube, it is necessary to establish a mapping relationship between the surface temperature and the liquid temperature inside the chamber. The temperature control method of this invention is divided into a calibration stage and an amplification stage.

[0040] During the calibration phase, a heating beam is simultaneously supplied to the bottom of the liquid in PCR reaction chamber 2 and temperature reference chamber 3 via a light source heating module for photothermal heating, causing both chambers to undergo at least one temperature cycle. During this temperature cycle, the actual temperature of the reference liquid in temperature reference chamber 3, measured by temperature sensor 6, is used as the reference value for the liquid temperature inside the chamber. Based on the isomorphism of the two chambers, this temperature is the approximate temperature of the liquid in PCR reaction chamber 2. Simultaneously, the temperature of the outer surface of PCR reaction chamber 2 is collected by an infrared temperature measurement module as the T-surface temperature, and a calibration function is established through multiple linear regression. T 管内 =f(T 表面 ,dT 管内 / dt) Among them, T 管内 T represents the temperature of the liquid inside temperature reference chamber 3. 表面 The infrared thermometer module collects the surface temperature of the outer side of PCR reaction chamber 2, dT. 管内 / dt represents the rate of temperature change within the temperature reference cavity 3, t represents time, and f represents the calibration function obtained through multiple linear regression.

[0041] During the amplification phase, a heating beam is supplied to the PCR reaction chamber 2 via a light source heating module for photothermal heating. During heating, the outer surface temperature of the PCR reaction chamber 2 is collected by an infrared thermometer module. Based on the aforementioned calibration function, the calibrated internal liquid temperature is calculated. The operating power of the light source heating module is adjusted according to the deviation between the calibrated internal liquid temperature and the target temperature. Once this calibration function is established, the internal liquid temperature can be calculated in real time during the amplification phase simply by collecting the surface temperature using the infrared thermometer module.

[0042] The temperature control module employs a PID control algorithm during the expansion phase. Based on the deviation between the calibrated tube temperature and the target temperature, it dynamically adjusts the PID parameters to regulate the operating power of the light source heating module in real time. The temperature control logic is as follows: during the heating phase, the light source heating module is activated and operates at high power; during the cooling phase, the light source heating module is deactivated and the fan is activated; during the temperature plateau phase, the power is adaptively adjusted to switch between denaturation and annealing / extension states.

[0043] The system also includes a multi-channel fluorescence detection module, configured to initiate excitation and data acquisition during each annealing / extension stage, enabling real-time capture of multiple fluorescence signals.

[0044] The consumable body 1 is fixed on the fixture body, which provides positioning and fixing functions for the consumable body, ensuring that the consumable body 1 maintains a precise relative position with respect to the light path of the light source heating module and the airflow direction of the fan 5.

[0045] This invention also provides a rapid PCR temperature control method, employing the aforementioned dual-chamber self-calibrating PCR system, including a calibration phase and an amplification phase: Calibration phase: A1: The heating beam is simultaneously supplied to the bottom of the liquid in the PCR reaction chamber 2 and the temperature reference chamber 3 through the light source heating module to perform photothermal heating, so that the two chambers undergo at least one temperature cycle; During the temperature cycling process, the temperature of the reference liquid in the temperature reference chamber 3 is measured by the temperature sensor 6 as a reference value for the liquid temperature inside the chamber, while the temperature of the outer surface of the PCR reaction chamber 2 is collected by the infrared temperature measurement module.

[0046] A2: Based on the reference value of the liquid temperature inside the chamber and the temperature of the outer surface of the PCR reaction chamber 2, establish the mapping relationship between the outer surface temperature and the liquid temperature inside the chamber.

[0047] Amplification phase: B1: The heating beam is provided to the PCR reaction chamber 2 through the light source heating module for photothermal heating. During the heating process, the temperature of the outer surface of the PCR reaction chamber 2 is collected by the infrared temperature measurement module, and the calibrated liquid temperature inside the chamber is calculated based on the mapping relationship.

[0048] B2: Adjust the working power of the light source heating module according to the deviation between the calibrated intracavitary liquid temperature and the target temperature.

[0049] During the heating phase, the light source heating module is activated; during the cooling phase, the light source heating module is deactivated and fan 5 is activated for symmetrical cooling. B2 employs a PID control algorithm, dynamically adjusting the PID parameters based on the deviation between the calibrated intracavity liquid temperature and the target temperature to regulate the working power of the light source heating module in real time.

[0050] Using the aforementioned temperature control method, the system achieves 40 temperature cycles in approximately 11 minutes, demonstrating precise temperature control that is significantly superior to traditional PCR equipment. Due to this precise and lag-free temperature control, the system enables stable amplification and detection of extremely low template concentrations, resulting in nucleic acid amplification with exceptionally high specificity and sensitivity.

[0051] This invention also provides a rapid PCR detection method, which, based on the above-mentioned rapid PCR temperature control method, further includes a nucleic acid detection stage, and can select any one or more of the following methods in combination according to experimental requirements: 1. Quantitative real-time PCR (qPCR) detection: During the annealing / extension phase of each temperature cycle, the multi-channel fluorescence detection module is activated, the excitation light source is activated to irradiate the PCR reaction chamber, and the intensity of the corresponding emitted fluorescence signal is collected to achieve quantitative detection of multiple fluorescence signals. 2. Endpoint fluorescence detection: After all thermal cycling procedures are completed, the multi-channel fluorescence detection module is activated, the excitation light source is turned on to irradiate the PCR reaction chamber, and the intensity of the corresponding emitted fluorescence signal is collected to achieve endpoint fluorescence detection. 3. Sequencing or electrophoresis detection: After all thermal cycling procedures are completed, remove the amplification products from the PCR reaction chamber and sequence them using Sanger sequencing or other methods, or perform agarose gel electrophoresis detection.

[0052] To more clearly illustrate the technical solution of this invention, the following embodiments all use the preferred photothermal PCR method of this invention for specific description. This is a typical implementation of rapid PCR. However, the scope of protection of this invention is not limited to photothermal PCR. Any PCR technology that uses the dual-chamber self-calibration principle to achieve rapid temperature control and detection falls within the scope of protection of this invention. The following three embodiments illustrate optional implementation schemes of this invention. All embodiments use the aforementioned PCR system.

[0053] Example 1 The structure of the photothermal PCR system is as follows: Figure 1As shown, a laser with a center wavelength of 808nm and a maximum output power of approximately 2W is installed at the bottom. The main beam is divided into two sub-lasers of equal energy by an optical beam splitter, which illuminate vertically upwards. A dual-chamber sealing consumable, including a PCR reaction chamber and a temperature reference chamber, is installed in the upper fixture, so that the two laser beams are aligned with the bottom of the two chambers respectively.

[0054] Install a centrifugal cooling fan at the symmetrical center of the two chambers, with the air outlet aligned with the center gap of the consumables.

[0055] A non-contact infrared temperature sensor is placed on the side of the consumables in the PCR reaction chamber, with the focal length of the imaging sensor corresponding to the exact center of the PCR reaction chamber. An NTC thermistor temperature sensor is fixed and passes through the center of the silicone cap, and is fixed at the center of the conical bottom of the temperature reference chamber, serving as a contact temperature sensor.

[0056] Example 2 In the system of Example 1, 20 μL of PCR reaction solution containing amplification primers, enzymes, template and gold nanorods (absorption peak near 790 nm) was injected into the PCR reaction chamber; an equal volume of reference blank solution containing the same concentration of gold nanorods was injected into the temperature reference chamber, 40 μL of paraffin oil was added, the chamber was sealed, and the dual chambers were heated for testing.

[0057] The calibration phase begins. During thermal cycling, a contact sensor inside the reference chamber acquires the actual liquid temperature in real time, while an infrared thermometer module collects the surface temperature of the PCR reaction chamber's outer surface. Based on these two sets of data, a mapping relationship is established between the outer surface temperature and the liquid temperature inside the chamber. After calibration, the amplification phase begins. The PID controller calculates the calibrated liquid temperature inside the chamber based on the mapping relationship and calculates the temperature difference between this temperature and the target heating temperature. The output power is adjusted accordingly: during heating, the light source heating module is kept at full load; during cooling, the light source is turned off, and a central fan simultaneously blows air onto both chambers for forced cooling; and when the plateau phase is reached, the power is adaptively fine-tuned.

[0058] Figure 2 The figure mainly illustrates the heating kinetics curves of the PCR system in this invention. The horizontal axis represents time and the vertical axis represents temperature. The figure shows five heating rate curves corresponding to different gold nanorod concentrations (OD). Figure 2The results showed a significant positive correlation between the heating rate and the concentration (OD) of the gold nanorods. When the target denaturation temperature was set to 90-95℃ and the annealing extension temperature to 57℃, the control system performed photoexcitation. The average heating rates for systems with OD values ​​of 0.8, 1.7, 3.3, 6.7, and 10 were 2.95℃ / s, 5.84℃ / s, 8.01℃ / s, 9.45℃ / s, and 10.59℃ / s, respectively. Preferably, when the system OD ≥ 6.7, the system can rapidly heat from 57℃ to 95℃ in approximately 3 seconds, significantly reducing the waiting time for denaturation heating. The rapid PCR based on dual-chamber room temperature control feedback sealed the reaction chamber containing the PCR reaction solution and the temperature reference chamber containing the reference solution within the system. Through this photothermal synergy and precise feedback control, the system stably and rapidly completed 40 temperature cycles without overshoot, with a total time of only approximately 659 seconds (approximately 10.98 minutes).

[0059] Figure 3 The main feature of this invention is the thermocycling kinetics curve of the PCR system. As can be seen, Figure 3 The horizontal axis represents time, and the vertical axis represents temperature. The temperature of the PCR system fluctuates periodically, with the upper and lower limits of temperature remaining relatively stable around 95℃ and 57℃, respectively.

[0060] Example 3: Real-time fluorescence quantitative detection The optimized photothermal system and temperature control model described above were used for actual nucleic acid amplification. At the end of each annealing / extension stage, the integrated multi-channel fluorescence detection module automatically excited and acquired signals to plot real-time amplification curves. The thermal cycling program was set as follows: 95℃ denaturation for 1 second, 57℃ annealing / extension for 10 seconds, for a total of 40 thermal cycles. Using plasmid templates, gradient dilution groups from 4.3 pg / μL to 4.3 fg / μL were set up to verify the amplification performance of the photothermal PCR system. Figure 4 This is a comparison graph of the photothermal PCR amplification performance. The horizontal axis represents the number of cycles, and the vertical axis represents the original fluorescence signal. The amplification results show that the photothermal PCR fluorescence signal rises rapidly after amplification initiation, the exponential phase is steeper than that of conventional PCR, and stable amplification can still be achieved at extremely low concentrations as low as 4.3 fg / μL, confirming that this dual-chamber photothermal system has extremely high amplification specificity and reliability.

[0061] In the description of this application, it should be understood that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0062] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A rapid PCR temperature control method, characterized in that, A rapid PCR system is used, comprising a PCR reaction chamber (2) and a temperature reference chamber (3) with identical structures. The rapid PCR system also includes a light source heating module, a temperature sensor (6) inserted into the temperature reference chamber (3), and an infrared temperature measurement module. The PCR reaction chamber (2) is filled with PCR reaction solution, and the temperature reference chamber (3) is filled with a reference solution of the same volume as the PCR reaction solution. The PCR temperature control method includes a calibration stage and an amplification stage. The calibration phase includes: A1: The heating module of the light source synchronously provides a heating beam to the bottom of the liquid in the two chambers for photothermal heating, so that the two chambers undergo at least one temperature cycle; During the temperature cycling process, the temperature of the reference liquid in the temperature reference chamber (3) is measured by the temperature sensor (6) as a reference value for the liquid temperature in the chamber, and the temperature of the outer surface of the PCR reaction chamber (2) is collected by the infrared temperature measurement module. A2: Based on the reference value of the liquid temperature inside the cavity and the outer surface temperature of the PCR reaction cavity (2), establish a mapping relationship between the outer surface temperature and the liquid temperature inside the cavity; The amplification phase includes: B1: The heating light beam is provided to the PCR reaction chamber (2) through the light source heating module for photothermal heating. During the heating process, the outer surface temperature of the PCR reaction chamber (2) is collected through the infrared temperature measurement module, and the calibrated liquid temperature inside the chamber is calculated based on the mapping relationship. B2: Adjust the operating power of the light source heating module according to the deviation between the calibrated intracavitary liquid temperature and the target temperature.

2. The rapid PCR temperature control method as described in claim 1, characterized in that, The mapping relationship is as follows: T 管内 =f(T 表面 ,dT 管内 / dt); Among them, T 管内 T represents the temperature of the liquid inside the temperature reference chamber (3). 表面 The temperature of the outer surface of the PCR reaction chamber (2) collected by the infrared thermometer module, dT 管内 / dt is the rate of temperature change in the temperature reference cavity (3), t is time, and f is the calibration function obtained by multiple linear regression.

3. The rapid PCR temperature control method as described in claim 1, characterized in that, The temperature cycle includes a cooling process following the heating process, and the rapid PCR system also includes a cooling module configured to provide symmetrical cooling airflow to the two chambers; During the heating process of A1 and B1, the heating module of the light source is turned on, and during the cooling process, the heating module of the light source is turned off and the cooling module is started to cool down.

4. The rapid PCR temperature control method as described in claim 1, characterized in that, In B2, a PID control algorithm is used to dynamically adjust the PID parameters based on the deviation between the calibrated intracavitary liquid temperature and the target temperature, thereby adjusting the working power of the light source heating module in real time.

5. A rapid PCR detection method, characterized in that, The rapid PCR temperature control method according to any one of claims 1 to 4 further includes a nucleic acid detection stage, wherein the nucleic acid detection stage comprises a combination of any one or more of the following methods: 1) During the annealing / extension phase of each temperature cycle, test and collect multiple fluorescence signals for fluorescent qPCR detection; 2) After all thermal cycling procedures are completed, the endpoint fluorescence detection is performed to test the fluorescence signal intensity; 3) After all thermal cycling procedures are completed, the amplification products are sequenced or detected by electrophoresis.

6. A dual-chamber self-calibrating PCR system, characterized in that, A method for implementing the rapid PCR temperature control method according to any one of claims 1 to 4, comprising: The consumable body (1) is provided with the PCR reaction chamber (2) and the temperature reference chamber (3). A sealing cover (4) covers and seals the top openings of the two chambers, through which the temperature sensor (6) is inserted into the temperature reference chamber (3); The light source heating module is located below the consumable body (1) and is configured to simultaneously provide two heating beams of the same energy to the bottom of the liquid in the PCR reaction chamber (2) and the temperature reference chamber (3); The infrared temperature measurement module is configured to collect the temperature of the outer surface of the PCR reaction chamber (2); The temperature control module is configured to establish a mapping relationship between the reference liquid temperature measured by the temperature sensor (6) and the outer surface temperature of the PCR reaction chamber (2) collected by the infrared temperature measurement module during the calibration phase. During the amplification phase, the module calculates the calibrated liquid temperature inside the chamber based on the mapping relationship and the outer surface temperature of the PCR reaction chamber (2) collected by the infrared temperature measurement module, and adjusts the power of the light source heating module according to the deviation between the calibrated liquid temperature inside the chamber and the target temperature.

7. The dual-chamber self-calibrating PCR system as described in claim 6, characterized in that, The bottom of the PCR reaction chamber (2) and the temperature reference chamber (3) are both conical. The radial dimensions of the bottom of the two chambers shrink to form sharp corners. The radial dimensions of the two chambers gradually increase from the bottom to the top opening and then remain unchanged.

8. The dual-chamber self-calibrating PCR system as described in claim 6, characterized in that, The PCR reaction solution contains a nucleic acid template and a nucleic acid amplification reaction solution. The nucleic acid amplification reaction solution contains nano-photothermal materials, and the upper layer of the PCR reaction solution is sealed with oil. The reference solution contains a nucleic acid amplification reaction solution with the same composition and volume as the nucleic acid amplification reaction solution in the PCR reaction chamber (2) and enzyme-free water. The upper layer of the reference solution is sealed with oil.

9. The dual-chamber self-calibrating PCR system as described in claim 6, characterized in that, The light source heating module includes a laser (7), a beam splitter (8), and two total reflection mirrors (9). The laser (7) is located at the middle position on the bottom side of the consumable body (1) and emits laser light towards the bottom of the consumable body (1); the beam splitter (8) is located on the output optical path of the laser (7) and splits the laser light into two beams of the same energy and emits them to the sides respectively; the two total reflection mirrors (9) are located on both sides of the beam splitter (8) and reflect the split beams to the bottom of the PCR reaction chamber (2) and the temperature reference chamber (3).

10. The dual-chamber self-calibrating PCR system as described in claim 6, characterized in that, It also includes a cooling module and a multi-channel fluorescence detection module; The cooling module includes a fan (5) located between two chambers. The air outlet of the fan (5) is aligned with the middle of the consumable body (1), so that the airflow diffuses symmetrically to both sides and sweeps evenly across the outer walls of the two chambers. The multi-channel fluorescence detection module is configured to initiate excitation and data acquisition during each annealing / extension stage, thereby enabling real-time capture of multiple fluorescence signals.

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