A mini-polymerase chain reaction system for reducing non-specific amplification

Abstract

The application discloses a kind of for reducing non-specific amplification micro polymerase chain reaction system, which includes: a micro reaction cavity;Integrated temperature control module;Active magnetic field intervention module.The application discards the thought that traditional heat start technology passively inhibits enzyme activity, and innovatively introduces high-frequency alternating magnetic field in the early stage of reaction to actively physically intervene;Magnetic field force can effectively destroy the non-specific hybridization product and primer dimer that has been formed and has relatively weak binding force, and has less effect on correct primer-template hybrid that has more firm binding and higher specificity;This "active cleaning" mechanism can directly remove error initiator from the physical level, thereby reducing the background of non-specific amplification at the root, so that the purity of final product and the brightness of target band are qualitatively leaped.

Description

Technical Field

[0001] This invention relates to the field of nonspecific amplification and primer dimer formation technology, and more specifically to a miniature polymerase chain reaction system for reducing nonspecific amplification. Background Technology

[0002] Since its inception, polymerase chain reaction (PCR) technology has become a core technology in molecular biology, clinical diagnostics, forensic medicine, and other fields. However, traditional PCR technology has a significant drawback: non-specific amplification and primer dimer formation. This problem is particularly prominent during reaction system preparation: when the reaction mixture is assembled at room temperature or low temperature, DNA polymerase may still retain some activity, leading to non-specific binding between primers and template DNA, or between primers themselves, and subsequent extension under polymerase catalysis. These non-target products consume primers, nucleotides, and enzymes in the reaction system, severely interfering with the specific amplification of the target fragment, resulting in reduced amplification efficiency, increased background signal, and even false positive or false negative results.

[0003] To address this issue, hot-start PCR technology emerged and has been widely applied. As described in the document, existing hot-start technologies primarily achieve this by inhibiting DNA polymerase activity in the early stages of the reaction. Its main implementation methods include: Chemical modification method: The active site of the enzyme is blocked by covalently binding chemical groups (such as wax beads, aptamers or small molecule inhibitors). The chemical modification is only removed after heating at high temperature for a period of time, and the enzyme activity is restored.

[0004] Antibody modification method: This method utilizes specific antibodies to bind to DNA polymerase, thereby inactivating it. During the initial denaturation high-temperature phase (usually >90°C), the antibody undergoes irreversible denaturation and dissociation, thereby activating enzyme activity.

[0005] While hot-start techniques significantly improve the specificity of PCR, existing technologies still have several limitations. First, both chemical and antibody modifications increase the cost of the PCR reaction. Second, the addition of modifying agents may slightly affect the optimal activity of DNA polymerase. Most importantly, these methods are essentially "passive" inhibition, preventing errors by "disabling" the enzyme, but they cannot actively "correct" or "remove" non-specific primer bindings or primer dimers that have formed during the heating process. Once the enzyme is activated, these non-specific products pre-formed at low temperatures may still be amplified.

[0006] Therefore, there is an urgent need in this field for a novel technology that can more proactively and directly intervene in the nonspecific binding process without relying on expensive chemical modifiers, in order to further optimize the specificity and reliability of PCR reactions. Summary of the Invention

[0007] The purpose of this invention is to provide a miniature polymerase chain reaction system for reducing nonspecific amplification, thereby overcoming the aforementioned shortcomings of the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A miniature polymerase chain reaction system for reducing nonspecific amplification, the system comprising: A miniature reaction chamber for containing a PCR reaction mixture containing DNA template, primers, nucleotides, and DNA polymerase; An integrated temperature control module, thermally coupled to the micro-reaction chamber, is used to perform precise cyclic temperature control on the PCR reaction mixture to achieve the three core temperature stages of DNA denaturation, primer annealing, and strand extension. An active magnetic field intervention module includes at least one magnetic field generator disposed around or below the micro-reaction chamber, the magnetic field generator being configured to generate a high-frequency alternating magnetic field in the range of 100 kHz to 10 MHz and apply the magnetic field to the PCR reaction mixture within the micro-reaction chamber. And a system control unit, which is electrically connected to the integrated temperature control module and the active magnetic field intervention module, and is programmed to control the entire PCR amplification process; The programming logic of the system control unit specifically includes: during the process of the integrated temperature control module reducing the temperature of the PCR reaction mixture from the denaturation high temperature stage to the primer annealing temperature stage, or during the initial predetermined time period of maintaining the annealing temperature stage, activating the active magnetic field intervention module to apply the high-frequency alternating magnetic field to the reaction mixture with a preset magnetic field frequency and field strength. The preset magnetic field strength ranges from 5 mT to 50 mT.

[0009] Preferably, the magnetic field generator of the active magnetic field intervention module is a miniature planar coil or an electromagnet array, whose geometry and spatial arrangement are optimized to generate a high-frequency alternating magnetic field distribution that is as uniform as possible inside the miniature reaction chamber, ensuring that the entire reaction mixture receives a basically consistent magnetic field intervention effect, and avoiding the problem of excessive or insufficient local intervention due to uneven magnetic field; the manufacturing material of the miniature planar coil or electromagnet array is selected as a metal with high conductivity and good thermal stability. The system control unit is further programmed to precisely modulate the driving current of the magnetic field generator, thereby enabling independent control and real-time fine-tuning of the output magnetic field frequency and field strength. This allows users to input optimal magnetic field intervention parameters through the software interface based on different primer sequence characteristics, reaction system composition, or specific experimental requirements.

[0010] Preferably, the system control unit has multiple programmable modes for activating the active magnetic field intervention module; the first mode is the "cooling process intervention mode", which means that the high-frequency alternating magnetic field is continuously applied throughout the entire cooling range from the high-temperature denaturation stage, such as 94°C to 98°C, until the target annealing temperature, such as 50°C to 65°C is reached. The second mode is the "initial annealing pulse mode", which means that the magnetic field is applied only for the first 10 to 60 seconds after the target annealing temperature is reached. It can be applied continuously or intermittently in a pulsed manner, for example, applying for 2 seconds and then pausing for 1 second. The third mode is the "dynamic tracking mode," in which the system control unit dynamically determines the timing, duration, and intensity of the magnetic field activation based on preset or real-time estimated non-specific binding risk. These different modes provide users with flexible optimization options to adapt to the specific requirements of different PCR amplification procedures.

[0011] Preferably, the integrated temperature control module uses a combination of a thin-film heater and a thermoelectric cooler, or a semiconductor temperature control device based on the Peltier effect, to achieve rapid heating and cooling of the micro-reaction chamber; the system control unit integrates a high-precision temperature sensor to monitor the actual temperature of the reaction mixture in real time and form a closed-loop feedback control to ensure the accuracy and repeatability of temperature control.

[0012] Preferably, the micro-reaction chamber is one or more micro-reaction chambers on a microfluidic chip, with a volume between 1 μL and 50 μL, suitable for amplification of trace samples; the microfluidic chip is made of a material with good light transmittance, low magnetic permeability, and biocompatibility.

[0013] Preferably, the DNA polymerase is a natural, thermostable DNA polymerase that has not undergone chemical or antibody modification.

[0014] A method for reducing nonspecific amplification in PCR reactions using a miniature polymerase chain reaction system, the method comprising the following steps: Step 1: Prepare the PCR reaction mixture and inject it into the micro-reaction chamber; Step 2: The preset PCR amplification program is started through the system control unit, and the integrated temperature control module begins to execute the first denaturation high temperature stage; Step 3: In the subsequent temperature cycle, when the program runs from the denaturation high temperature stage to the primer annealing temperature stage, or during the initial time period of maintaining the annealing temperature stage, the system control unit automatically activates the active magnetic field intervention module according to preset parameters to apply a high-frequency alternating magnetic field of specific frequency and field strength to the reaction mixture. Step 4: After the preset magnetic field intervention time ends, the system control unit shuts down the magnetic field intervention module, and the PCR reaction continues with normal annealing and extension steps, and completes the subsequent cycle. Step 5: After multiple cycles, PCR amplification products with significantly improved specificity are obtained.

[0015] Preferably, the preset magnetic field intervention parameters, including magnetic field frequency, field strength, initial application temperature, duration, and application mode, are predetermined through a series of optimization experiments; The optimization method includes: using primer pairs known to easily produce non-specific amplification or primer dimers, and sample templates with complex backgrounds, performing PCR amplification under different combinations of magnetic field parameters, and then analyzing the band uniformity of the amplification products by agarose gel electrophoresis, or observing the peak time and final fluorescence intensity of the amplification curve by real-time fluorescence PCR, to determine the set of magnetic field parameters that produces the clearest specific bands, the highest amplification efficiency, and the lowest background noise as the optimal parameters under that specific primer and sample conditions; these optimal parameters can be saved as a protocol in the system control unit for direct use in subsequent similar detections.

[0016] Preferably, the system further includes a result detection module, which can be an optical system for real-time fluorescence detection, including an excitation light source, a photodetector, and a filter group, integrated above or below the micro-reaction chamber, for monitoring changes in fluorescence signals during PCR. The system control unit is also configured to adaptively adjust the parameters of the active magnetic field intervention module in subsequent PCR cycles when it is determined based on the real-time fluorescence signal that a certain reaction may have a risk of non-specific amplification. For example, the parameters may be adjusted by appropriately extending the magnetic field intervention time or increasing the field strength in the next cycle, so as to achieve dynamic and intelligent process control and further improve the reliability of complex sample detection.

[0017] Preferably, the control strategy of the system control unit for the active magnetic field intervention module is specifically optimized to synergistically achieve efficient non-specific amplification inhibition and active management of side effects, specifically including the following synergistic control mechanisms: First, the operating parameters of the high-frequency alternating magnetic field are limited to a specific range and a pulsed mode is adopted to balance the intervention effect and side effects. The magnetic field frequency is preferably set between 500 kHz and 2 MHz. The alternating magnetic field within this frequency range can generate a sufficient induced electric field and Lorentz force in the reaction solution to effectively interfere with and disrupt the non-specific hydrogen bond binding and van der Waals force between primers and templates, as well as between primers. At the same time, it avoids the significant increase in dielectric loss and heat generation due to excessively high frequency, or the weak induction effect due to excessively low frequency. The magnetic field strength is preferably set between 5 mT and 20 mT. This field strength range has been verified to be sufficient to physically dissociate non-specific binding products. At the same time, through optimization, it is insufficient to cause breakage damage to the phosphodiester bonds of the DNA strand, and also avoids irreversible denaturation perturbation of the key disulfide bonds and three-dimensional spatial conformation of the Taq enzyme active site.

[0018] Second, the integrated temperature control module includes at least one high-precision temperature sensor that is closely attached to the micro-reaction chamber for real-time monitoring of the actual temperature of the reaction mixture. The system control unit is programmed to read the data from the temperature sensor in real-time during and after the activation of the active magnetic field intervention module.

[0019] Third, the micro-reaction chamber is made of a material with high resistivity, low dielectric loss and non-magnetic properties, such as borosilicate glass, cyclic olefin polymers or specific grades of polydimethylsiloxane. Using such materials can effectively suppress parasitic eddy currents generated on the reaction vessel wall under alternating magnetic fields, thereby reducing additional thermal noise caused by the heating of the vessel itself and reducing unnecessary microcurrent background.

[0020] In the above technical solution, the present invention provides a miniature polymerase chain reaction system for reducing nonspecific amplification. This invention abandons the traditional hot-start technique's passive inhibition of enzyme activity and innovatively introduces a high-frequency alternating magnetic field for active physical intervention in the early stages of the reaction. The magnetic field effectively destroys the already formed, relatively weakly bound nonspecific hybridization products and primer dimers, while having less impact on the more firmly bound and highly specific correct primer-template hybrids. This "active cleanup" mechanism directly removes erroneous starters at the physical level, thereby reducing the background of nonspecific amplification at its source, resulting in a qualitative leap in the purity of the final product and the brightness of the target band. Experiments have shown that, under the same reaction conditions, PCR amplification using the technology of this invention produces significantly lower nonspecific band and primer dimer band intensities compared to traditional hot-start PCR.

[0021] Because the specificity enhancement of this invention does not rely on chemical modification of the DNA polymerase itself, the system is compatible with unmodified natural DNA polymerases. This avoids the use of expensive hot-start enzymes, helping to reduce reagent costs, and is particularly suitable for scenarios requiring large-scale PCR screening. Simultaneously, the system control unit allows users to precisely adjust the frequency, strength, timing, and mode of the magnetic field; this high programmability makes the method extremely flexible. Users can quickly optimize the best magnetic field intervention parameters based on the characteristics of different primer sequences, template complexity, and specific application requirements, achieving "tailor-made" highly specific amplification with greater versatility.

[0022] This invention deeply integrates a magnetic field intervention module with a miniature PCR system. The small volume of the miniature reaction chamber allows for more uniform magnetic field coverage of the entire reaction system, resulting in a more uniform and efficient intervention effect. Simultaneously, the inherent rapid temperature rise and fall characteristics of the miniature system perfectly match the timing of the brief, precise magnetic field pulse intervention in this invention, achieving synergistic effects. This system not only exhibits excellent specificity but also retains the advantages of miniature PCR, such as speed, low consumable costs, ease of integration, and portability. It is highly suitable for cutting-edge applications requiring extremely high sensitivity and specificity, such as rapid on-site detection and single-cell analysis, providing core technological support for the development of next-generation molecular diagnostic equipment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0024] Figure 1 This is a schematic diagram illustrating the steps of an embodiment of a miniature polymerase chain reaction system for reducing nonspecific amplification according to the present invention. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0026] like Figure 1As shown in the embodiment of the present invention, a miniature polymerase chain reaction system for reducing nonspecific amplification is provided. The system includes: a miniature reaction chamber for containing a PCR reaction mixture containing a DNA template, primers, nucleotides, and DNA polymerase; an integrated temperature control module thermally coupled to the miniature reaction chamber for performing precise cyclic temperature control of the PCR reaction mixture to achieve three core temperature stages: DNA denaturation, primer annealing, and chain extension; and an active magnetic field intervention module including at least one magnetic field generator disposed around or below the miniature reaction chamber, the magnetic field generator being configured to generate frequencies between 100 kHz and 10 kHz. The system comprises a high-frequency alternating magnetic field in the MHz range, which is applied to the PCR reaction mixture within a micro-reaction chamber; and a system control unit electrically connected to an integrated temperature control module and an active magnetic field intervention module, which is programmed to control the entire PCR amplification process; wherein the programming logic of the system control unit specifically includes: activating the active magnetic field intervention module during the process of the integrated temperature control module reducing the temperature of the PCR reaction mixture from the denaturation high temperature stage to the primer annealing temperature stage, or during the initial predetermined time period of maintaining the annealing temperature stage, so as to apply a high-frequency alternating magnetic field to the reaction mixture at a preset magnetic field frequency and field strength; In this specific embodiment, the application time of the high-frequency alternating magnetic field is strictly controlled. The purpose is to use the magnetic field force to physically interfere with and destroy the non-specific binding between the primer and the DNA template, as well as the non-specific dimer binding between the primers, which may form during the cooling process or the early stage of annealing. At the same time, it avoids irreversible damage to the natural structure and active site of DNA polymerase or significant impact on the stability of specific binding. The preset magnetic field strength range is between 5 mT and 50 mT; Specifically, in this embodiment, it is ensured that non-specific products bound by non-covalent bonds can be effectively destroyed without causing a significant magnetocaloric effect in the reaction liquid or leading to DNA strand breaks. Through this time-precise and parameter-controllable active intervention of the magnetic field, the system can significantly reduce the generation of non-specific amplification products and primer dimers at the physical level, thereby improving the specificity and efficiency of PCR amplification. It is especially suitable for micro-scale and rapid detection scenarios with low template DNA concentration or complex sample composition.

[0027] Preferably, the magnetic field generator of the active magnetic field intervention module is a miniature planar coil or electromagnet array, whose geometry and spatial arrangement are optimized to generate a high-frequency alternating magnetic field distribution that is as uniform as possible inside the miniature reaction chamber. This ensures that the entire reaction mixture receives a basically consistent magnetic field intervention effect, avoiding problems of excessive or insufficient local intervention due to magnetic field inhomogeneity. The miniature planar coil or electromagnet array is made of metals with high conductivity and good thermal stability, such as copper or aluminum, and may be manufactured using microelectromechanical systems (MEMS) processing technology to achieve high integration with the miniature reaction chamber, reducing the overall system volume and thermal mass, thereby accelerating the response speed of temperature cycling. The system control unit is further programmed to precisely modulate the driving current of the magnetic field generator, thereby achieving independent control and real-time fine-tuning of the output magnetic field frequency and field strength. This allows users to input the optimal magnetic field intervention parameters through the software interface according to different primer sequence characteristics, reaction system composition, or specific experimental requirements. In this specific embodiment, for example, primers rich in GC bases that are easy to form stable secondary structures or non-specific binding can be interfered with by using a relatively high field strength, while primer pairs with high AT base content and relatively weak binding force can be interfered with by using a relatively low field strength to avoid excessive interference with the possible specific binding.

[0028] Preferably, the system control unit has multiple programmable modes for activating the active magnetic field intervention module. The first mode is the "cooling process intervention mode," which involves continuously applying a high-frequency alternating magnetic field throughout the entire cooling range from the denaturation high temperature stage, such as 94°C to 98°C, until the target annealing temperature, such as 50°C to 65°C, is reached. The second mode is the "initial annealing pulse mode," which applies the magnetic field only for the first 10 to 60 seconds after reaching the target annealing temperature. This can be done continuously or intermittently in pulses, such as applying for 2 seconds followed by a 1-second pause. The third mode is the "dynamic tracking mode," in which the system control unit dynamically determines the activation timing, duration, and intensity of the magnetic field based on a preset or real-time estimated non-specific binding risk (e.g., an index calculated based on the temperature drop rate and primer sequence complexity). These different modes provide users with flexible optimization options to adapt to the specific requirements of different PCR amplification programs.

[0029] Preferably, the integrated temperature control module uses a combination of a thin-film heater and a thermoelectric cooler, or a semiconductor temperature control device based on the Peltier effect, to achieve rapid heating and cooling of the micro-reaction chamber; the system control unit integrates a high-precision temperature sensor to monitor the actual temperature of the reaction mixture in real time and form a closed-loop feedback control to ensure the accuracy and repeatability of temperature control; Specifically, in this embodiment, the programming logic of the system control unit also includes a temperature compensation algorithm to compensate for the slight disturbances in the temperature of the reaction mixture that may be caused by the heat generated by the coil resistance or the slight magnetocaloric effect when the active magnetic field intervention module is working. By adjusting the power output of the temperature control module in real time, it is ensured that the reaction mixture can strictly follow the preset temperature curve throughout the PCR cycle, especially during the magnetic field intervention, so as to avoid the temperature deviation introduced by the magnetic field from affecting the quantitative accuracy of the PCR reaction.

[0030] Preferably, the micro-reaction chamber is one or more micro-reaction chambers on a microfluidic chip, with a volume between 1 μL and 50 μL, suitable for amplification of trace samples; the microfluidic chip is made of a material with good light transmittance, low magnetic permeability, and biocompatibility. In this specific embodiment, for example, polydimethylsiloxane, glass, or cyclic olefin polymers are used; the magnetic field generator of the active magnetic field intervention module is correspondingly miniaturized and integrated into the substrate of the microfluidic chip or packaged in a chip fixture near the microreaction chamber, thereby realizing a highly integrated, portable PCR analysis system.

[0031] In this specific embodiment, the DNA polymerase is a naturally occurring, thermostable DNA polymerase that is not chemically or antibody-modified, such as Taq polymerase. Because the present invention actively removes non-specific bindings at a specific time through physical magnetic field intervention, it reduces the dependence on chemical modification of the enzyme to achieve "hot start," thereby allowing the use of lower-cost and potentially more active natural enzymes, while still achieving or even surpassing the effect of traditional hot-start PCR. Alternatively, the DNA polymerase can also be a hot-start enzyme that has undergone chemical or antibody modification. In this case, the magnetic field intervention technology and the hot-start enzyme technology of the present invention produce a synergistic effect, further physically removing residual or newly formed non-specific bindings before the hot-start enzyme is activated (if the magnetic field intervention is performed at low temperature) or after activation, thereby providing dual specificity protection.

[0032] A method for reducing non-specific amplification in PCR reactions using a miniature polymerase chain reaction system includes the following steps: Step 1, preparing a PCR reaction mixture and injecting it into a miniature reaction chamber; Step 2, initiating a preset PCR amplification program via the system control unit, with the integrated temperature control module performing the first denaturation high-temperature stage; Step 3, during subsequent temperature cycles, when the program cools from the denaturation high-temperature stage to the primer annealing temperature stage, or during the initial period of maintaining this annealing temperature stage, the system control unit automatically activates an active magnetic field intervention module according to preset parameters, applying a high-frequency alternating magnetic field of specific frequency and strength to the reaction mixture; Step 4, after the preset magnetic field intervention time, the system control unit shuts off the magnetic field intervention module, and the PCR reaction continues with normal annealing and extension steps, completing subsequent cycles; Step 5, after multiple cycles, PCR amplification products with significantly improved specificity are obtained.

[0033] Preferably, the preset magnetic field intervention parameters, including magnetic field frequency, field strength, application start temperature, duration, and application mode, are determined in advance through a series of optimization experiments. The optimization methods include: using primer pairs known to easily produce non-specific amplification or primer dimers, and sample templates containing complex backgrounds, performing PCR amplification under different combinations of magnetic field parameters, and then analyzing the band uniformity of the amplification products by agarose gel electrophoresis, or observing the peak time and final fluorescence intensity of the amplification curve by real-time fluorescence PCR, to determine the set of magnetic field parameters that can produce the clearest specific band, the highest amplification efficiency, and the lowest background noise as the optimal parameters under the specific primer and sample conditions. Specifically, in this embodiment, these optimal parameters can be saved as a protocol and stored in the system control unit for direct use in subsequent similar detections.

[0034] Preferably, the system further includes a result detection module, which can be an optical system for real-time fluorescence detection, including an excitation light source, a photodetector, and a filter array, integrated above or below the micro-reaction chamber, for monitoring changes in fluorescence signals during PCR. The system control unit is also configured to adaptively adjust the parameters of the active magnetic field intervention module in subsequent PCR cycles when it is determined based on the real-time fluorescence signal that a certain reaction may have a risk of non-specific amplification. For example, the intervention time in the next cycle may be appropriately extended or the field strength may be increased, so as to achieve dynamic and intelligent process control and further improve the reliability of complex sample detection.

[0035] Preferably, the control strategy of the system control unit for the active magnetic field intervention module is specifically optimized to synergistically achieve efficient non-specific amplification inhibition and active management of side effects, specifically including the following synergistic control mechanisms: The operating parameters of the high-frequency alternating magnetic field are limited to a specific range and a pulsed mode is used to balance the intervention effect and side effects. The magnetic field frequency is preferably set between 500 kHz and 2 MHz. The alternating magnetic field in this frequency range can generate a sufficient induced electric field and Lorentz force in the reaction solution to effectively interfere with and disrupt the non-specific hydrogen bonding and van der Waals forces between primers and templates, as well as between primers. At the same time, it avoids the significant increase in dielectric loss and heat generation due to excessively high frequency, or the weak induction effect due to excessively low frequency.

[0036] Specifically, in this embodiment, the magnetic field strength is preferably set between 5 mT and 20 mT. This range has been verified to be sufficient to physically dissociate non-specific binding products, and through optimization, it is insufficient to cause breakage damage to the phosphodiester bonds of the DNA chain, thus avoiding irreversible denaturation perturbations to the critical disulfide bonds and three-dimensional conformation of the Taq enzyme active site. The magnetic field is applied in a pulsed manner. Within each intervention cycle, the duration of the magnetic field is strictly controlled to be a short pulse of 50 to 200 milliseconds, rather than being continuously applied throughout the entire annealing phase. This pulsed working mode can most effectively disrupt newly formed non-specific bindings in the initial stage of annealing, while minimizing the cumulative energy input of the magnetic field to the reaction system, thereby significantly reducing the overall heat load caused by the eddy current effect and reducing the duration of continuous physical perturbation of enzyme molecules and nucleic acid chains by the magnetic field.

[0037] The system integrates a dynamic monitoring and compensation mechanism for the magnetic field-induced thermal effect. The integrated temperature control module includes at least one high-precision temperature sensor closely attached to the miniature reaction chamber for real-time monitoring of the actual temperature of the reaction mixture. The system control unit is programmed to read the temperature sensor data in real-time during and for a brief time window after the active magnetic field intervention module is activated. Specifically, in this embodiment, when the temperature of the reaction mixture exceeds the preset annealing temperature threshold ±0.5°C due to the ohmic heating effect of the magnetic field-induced eddy current (microcurrent), the system control unit immediately adjusts the power output of the integrated temperature control module to dynamically compensate for the temperature, so that the temperature quickly returns to and stabilizes within the set annealing temperature range, thereby eliminating the potential negative impact of local overheating on primer annealing specificity and enzyme activity, and ensuring the accuracy of PCR temperature cycling.

[0038] The system design and material selection aim to minimize irrelevant electromagnetic side effects. The microreactor is made of a non-magnetic material with high resistivity, low dielectric loss, such as borosilicate glass, cyclic olefin polymers, or specific grades of polydimethylsiloxane. The use of such materials can effectively suppress parasitic eddy currents generated on the reaction vessel wall under an alternating magnetic field, thereby reducing additional thermal noise caused by the heating of the vessel itself and reducing unwanted microcurrent background.

[0039] In this specific embodiment, the magnetic field generator of the active magnetic field intervention module preferably uses a coil wound with Litz wire or an optimized planar coil to reduce skin effect loss during high-frequency operation, improve the efficiency of magnetic field generation, and reduce the pressure of coil self-heating on system thermal management.

[0040] By finely setting the magnetic field parameters (frequency, intensity, pulse timing), real-time monitoring and closed-loop compensation of the magnetic field heat generation effect, and targeted selection of materials for key system components, this invention enables the system to efficiently utilize magnetic field physical intervention to improve PCR specificity while minimizing the microcurrent thermal effect, local temperature fluctuations, and interference with Taq enzyme activity that may be caused by varying magnetic fields. This ensures that the PCR reaction has excellent performance with high specificity, high amplification efficiency, and high reproducibility.

[0041] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A miniature polymerase chain reaction system for reducing nonspecific amplification, characterized in that, The system includes: A miniature reaction chamber for containing a PCR reaction mixture containing DNA template, primers, nucleotides, and DNA polymerase; An integrated temperature control module, thermally coupled to the micro-reaction chamber, is used to perform precise cyclic temperature control on the PCR reaction mixture to achieve the three core temperature stages of DNA denaturation, primer annealing, and strand extension. An active magnetic field intervention module includes at least one magnetic field generator disposed around or below the micro-reaction chamber, the magnetic field generator being configured to generate a high-frequency alternating magnetic field in the range of 100 kHz to 10 MHz and apply the magnetic field to the PCR reaction mixture within the micro-reaction chamber. And a system control unit, which is electrically connected to the integrated temperature control module and the active magnetic field intervention module, and is programmed to control the entire PCR amplification process; The programming logic of the system control unit specifically includes: during the process of the integrated temperature control module reducing the temperature of the PCR reaction mixture from the denaturation high temperature stage to the primer annealing temperature stage, or during the initial predetermined time period of maintaining the annealing temperature stage, activating the active magnetic field intervention module to apply the high-frequency alternating magnetic field to the reaction mixture with a preset magnetic field frequency and field strength. The preset magnetic field strength ranges from 5 mT to 50 mT.

2. The micro polymerase chain reaction system according to claim 1, characterized in that, The magnetic field generator of the active magnetic field intervention module is preferably a miniature planar coil or an electromagnet array. Its geometry and spatial arrangement are optimized to generate a high-frequency alternating magnetic field distribution that is as uniform as possible inside the miniature reaction chamber, ensuring that the entire reaction mixture can be subjected to a basically consistent magnetic field intervention effect, and avoiding the problem of excessive or insufficient local intervention caused by uneven magnetic field. The manufacturing material of the miniature planar coil or electromagnet array is selected from metals with high conductivity and good thermal stability. The system control unit is further programmed to precisely modulate the driving current of the magnetic field generator, thereby enabling independent control and real-time fine-tuning of the output magnetic field frequency and field strength. This allows users to input optimal magnetic field intervention parameters through the software interface based on different primer sequence characteristics, reaction system composition, or specific experimental requirements.

3. The micro polymerase chain reaction system according to claim 1, characterized in that, The system control unit has multiple programmable modes for activating the active magnetic field intervention module; the first mode is the "cooling process intervention mode", which means that the high-frequency alternating magnetic field is continuously applied throughout the entire cooling range from the high-temperature denaturation stage, such as 94°C to 98°C, until the target annealing temperature, such as 50°C to 65°C is reached. The second mode is the "initial annealing pulse mode", which means that the magnetic field is applied only for the first 10 to 60 seconds after the target annealing temperature is reached. It can be applied continuously or intermittently in a pulsed manner, for example, applying for 2 seconds and then pausing for 1 second. The third mode is the "dynamic tracking mode," in which the system control unit dynamically determines the timing, duration, and intensity of the magnetic field activation based on preset or real-time estimated non-specific binding risk. These different modes provide users with flexible optimization options to adapt to the specific requirements of different PCR amplification procedures.

4. The micro polymerase chain reaction system according to claim 1, characterized in that, The integrated temperature control module uses a combination of a thin-film heater and a thermoelectric cooler, or a semiconductor temperature control device based on the Peltier effect, to achieve rapid heating and cooling of the micro-reaction chamber. The system control unit integrates a high-precision temperature sensor to monitor the actual temperature of the reaction mixture in real time and form a closed-loop feedback control to ensure the accuracy and repeatability of temperature control.

5. The micro polymerase chain reaction system according to claim 1, characterized in that, The micro-reaction chamber is one or more micro-reaction chambers on a microfluidic chip, with a volume between 1 μL and 50 μL, suitable for amplification of trace samples; the microfluidic chip is made of a material with good light transmittance, low magnetic permeability and biocompatibility.

6. The micro polymerase chain reaction system according to claim 1, characterized in that, The DNA polymerase is a naturally occurring, thermostable DNA polymerase that has not undergone chemical or antibody modification.

7. A method for reducing nonspecific amplification in PCR reactions using a miniature polymerase chain reaction system as described in any one of claims 1 to 6, characterized in that, The method includes the following steps: Step 1: Prepare the PCR reaction mixture and inject it into the micro-reaction chamber; Step 2: The preset PCR amplification program is started through the system control unit, and the integrated temperature control module begins to execute the first denaturation high temperature stage; Step 3: In the subsequent temperature cycle, when the program runs from the denaturation high temperature stage to the primer annealing temperature stage, or during the initial time period of maintaining the annealing temperature stage, the system control unit automatically activates the active magnetic field intervention module according to preset parameters to apply a high-frequency alternating magnetic field of specific frequency and field strength to the reaction mixture. Step 4: After the preset magnetic field intervention time ends, the system control unit shuts down the magnetic field intervention module, and the PCR reaction continues with normal annealing and extension steps, and completes the subsequent cycle. Step 5: After multiple cycles, PCR amplification products with significantly improved specificity are obtained.

8. The method according to claim 7, characterized in that, The preset magnetic field intervention parameters, including magnetic field frequency, field strength, initial application temperature, duration, and application mode, are predetermined through a series of optimization experiments. The optimization method includes: using primer pairs known to easily produce non-specific amplification or primer dimers, and sample templates with complex backgrounds, performing PCR amplification under different combinations of magnetic field parameters, and then analyzing the band uniformity of the amplification products by agarose gel electrophoresis, or observing the peak time and final fluorescence intensity of the amplification curve by real-time fluorescence PCR, to determine the set of magnetic field parameters that produces the clearest specific bands, the highest amplification efficiency, and the lowest background noise as the optimal parameters under that specific primer and sample conditions; these optimal parameters can be saved as a protocol in the system control unit for direct use in subsequent similar detections.

9. The micro polymerase chain reaction system according to claim 1, characterized in that, The system also includes a result detection module, which can be an optical system for real-time fluorescence detection, including an excitation source, a photodetector, and a filter array, integrated above or below the micro-reaction chamber, for monitoring changes in fluorescence signals during PCR. The system control unit is also configured to adaptively adjust the parameters of the active magnetic field intervention module in subsequent PCR cycles when it is determined based on the real-time fluorescence signal that a reaction may have a risk of nonspecific amplification.

10. The micro polymerase chain reaction system according to claim 1, characterized in that, The control strategy of the system control unit for the active magnetic field intervention module has been specifically optimized to synergistically achieve efficient non-specific amplification inhibition and active management of side effects. Specifically, this includes the following synergistic control mechanisms: First, the operating parameters of the high-frequency alternating magnetic field are limited to a specific range and a pulse mode is adopted to balance the intervention effect and side effects. The magnetic field frequency is preferably set between 500 kHz and 2 MHz. Within this frequency range, the alternating magnetic field can generate sufficient induced electric field and Lorentz force in the reaction solution, effectively interfering with and disrupting non-specific hydrogen bond binding and van der Waals forces between primers and templates, and between primers themselves. Simultaneously, it avoids excessively high frequencies leading to a significant increase in dielectric loss and heat generation, or excessively low frequencies leading to a weak induction effect. The magnetic field strength is preferably set between 5 mT and 20 mT. This field strength range has been verified to be sufficient for physically dissociating non-specific binding products. Furthermore, through optimization, it is insufficient to cause breakage damage to the phosphodiester bonds of the DNA strand, and also avoids irreversible denaturation perturbations to the key disulfide bonds and three-dimensional conformation of the Taq enzyme active site. Second, the integrated temperature control module includes at least one high-precision temperature sensor closely attached to the micro-reaction chamber for real-time monitoring of the actual temperature of the reaction mixture. The system control unit is programmed to read the data from the temperature sensor in real-time during and after the activation of the active magnetic field intervention module within a short time window. Third, the micro-reaction chamber is made of a material with high resistivity, low dielectric loss, and non-magnetic properties, such as borosilicate glass, cyclic olefin polymers, or specific grades of polydimethylsiloxane. Using such materials can effectively suppress parasitic eddy currents generated on the reaction vessel wall under an alternating magnetic field, thereby reducing additional thermal noise caused by the heating of the vessel itself and lowering unnecessary microcurrent background.

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