A method for thermal cycling control of a polymerase chain reaction and a thermal cycling device

Abstract

The application discloses a thermal cycle control method and a thermal cycle device for polymerase chain reaction, and comprises a thermal cycle module for performing thermal cycle on a sample, the thermal cycle module comprises a Peltier heating element, a sample tube receiving part comprising N receiving well units connected with one end of the heating element directly or indirectly, wherein N is an integer not less than 2, and a heat dissipation part connected with the other end of the heating element directly or indirectly opposite to the sample tube receiving part; and a temperature control module, the temperature control module comprises at least two groups of control modes for controlling the heating element at at least two different environmental temperatures, so that the sample can be controlled by different control modes suitable for the environmental temperature to perform temperature rising and falling operation. The control method can adapt to the differences of different environmental temperatures and different instruments and equipment, so that the control module can complete the temperature cycle control with high precision and differentiation.

Description

Technical Field

[0001] This invention relates to the field of thermal cycling control of polymerase chain reaction (PCR) in medical devices, and specifically to a method and apparatus for thermal cycling control of PCR. Background Technology

[0002] In vitro diagnostic equipment analyzes the characteristics of biological samples by amplifying the nucleic acids contained within them. In disease control and public safety, it can determine whether a person is infected by a virus by detecting the presence of nucleic acid fragments of invading viruses in respiratory specimens (nasal swabs, throat swabs), blood (whole blood samples), or digestive tract samples (anal swabs, fecal samples). In food safety, it analyzes the characteristics of agricultural products and food to determine their safety. In criminal investigations, forensic or investigative personnel often use this method to verify evidence or supporting materials. The most widely used method in these scenarios is a technique called polymerase chain reaction (PCR), which amplifies nucleic acids in the region to be analyzed in a base sequence-specific manner. In PCR, a reaction mixture containing nucleic acids and reagents used to amplify the nucleic acids is heated to approximately 95°C for denaturation, and then cooled to approximately 60°C for annealing and extension reactions. Amplification can be completed by repeating the temperature variations 30 times (or more, up to 40 times). There are also three-step amplification schemes, with a similar principle (except that annealing and extension have different temperatures, usually with annealing at a lower temperature than extension), which will not be described in detail here. In most cases, fluorescence intensity at different cycle numbers is detected by mixing a fluorescently labeled compound with fluorescence intensity varying according to the amount of PCR product into the reaction mixture and irradiating the mixture with excitation light. The nucleic acid amplification process is dynamically reflected by changes in fluorescence intensity. Combined with optical sensors, a fluorescence quantification curve can be obtained to determine whether the sample solution contains the target nucleic acid sequence and to relative quantify the target nucleic acid sequence.

[0003] The core technical solution involves a temperature cycling device for heating and cooling the sample solution. Currently, the most widely used heating element is the Peltier, which can be used to design heating modules with dual heating and cooling functions. However, during the heating or cooling process of the liquid inside the sample tube, due to the hysteresis effect caused by heat transfer inherent in the system, when the heating element raises the temperature block (or sample tube receiving part) to the target temperature, the liquid inside the sample tube is still in a slow temperature rise process. Under these conditions, cycling according to the theoretically designed time will inevitably lead to insufficient thermal cycling, which may even cause amplification failure. The amplification results at this time will not reflect the actual cycling results. To facilitate the rapid attainment of the target temperature in the reaction mixture, many developers have designed special control methods. For example, when the temperature rises, the temperature control block is raised to a temperature higher than the target temperature of the reaction mixture to achieve overshoot (or overshoot) and solve this problem. Japanese invention patent application JP2001521379A, "Improvement of Thermal Cyclist for PCR," proposes a scheme combining a temperature control module with an optimized overshoot mode. This scheme incorporates symmetrical overshoot processes during both heating and cooling, improving upon the rapid and accurate heating and cooling control required by ideal control methods. However, because the same overshoot is consistently applied, it cannot address the diverse application scenarios of reagents used in various analyses, particularly the specific requirements of nucleic acid amplification enzymes. Furthermore, the actual ambient temperature significantly impacts the temperature control during machine operation. These factors render traditional fixed overshoot mode temperature correction control highly unreliable. Additionally, variations in instrument component manufacturing and assembly processes lead to significant differences in the thermal characteristics of finished instruments. Clearly, traditional overshoot control schemes are no longer suitable for the high-quality, accurate detection demands of modern life.

[0004] To address the need for high-precision amplification and more accurate detection in different scenarios using various PCR instruments, there is an urgent need to develop a more adaptable temperature cycling control scheme. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems by providing a thermal cycling control method for polymerase chain reaction (PCR). This method allows for temperature cycling control using different control modes at different ambient temperatures. Furthermore, it can be combined with immersing a temperature sensor in water or a liquid with similar properties to the sample solution before shipment to obtain the temperature control of the sample tube under ideal cycling conditions at different room temperatures in a real-world scenario. This enables the temperature inside the sample tube to adapt to the characteristics of the temperature, generating a corresponding temperature control mode, which is then stored in the temperature control module. This achieves a method for pre-programmed adaptive temperature control modes for different instruments, fully adapting to different test items, ambient temperatures, and instrument differences, making it suitable for high-precision and accurate temperature cycling control.

[0006] This invention employs two technical solutions. The first aspect proposes a thermal cycling control method for polymerase chain reaction (PCR), comprising a thermal cycling module for performing thermal cycling on a sample. The thermal cycling module includes a Peltier heating element, a sample tube receiving section containing N receiving well units (where N is an integer not less than 2) directly or indirectly connected to one end of the heating element, and a heat dissipation section directly or indirectly connected to the other end of the heating element opposite to the sample tube receiving section. The second aspect is a temperature control module, which includes at least two sets of control modes for controlling the heating element according to the corresponding ambient temperature under at least two different ambient temperatures, thereby enabling the sample to undergo heating and cooling operations in different control modes suitable for the ambient temperature.

[0007] Furthermore, the temperature control module includes a judgment unit, which can determine, based on the current ambient temperature, either a control mode corresponding to a certain temperature or a control mode that is associated with two control modes corresponding to different temperatures, as the final control mode.

[0008] Furthermore, the temperature control mode includes an overshoot mode, and the overshoot mode parameters include at least one of the following: overshoot temperature, overshoot temperature holding time, and the relationship between the overshoot temperature and the steady temperature.

[0009] Furthermore, at least one of the overshoot temperature, the overshoot temperature duration, and the relationship between the overshoot temperature and the stable temperature is different in the at least two ambient temperatures.

[0010] Furthermore, at least one of the overshoot temperature, overshoot temperature holding time, and the relationship between the overshoot temperature and the stable temperature is pre-calibrated and pre-programmed into the temperature control module before the machine leaves the factory.

[0011] Furthermore, it also includes a temperature sensor unit, which works with the control module to perform heating and cooling operations on the sample. The control module can also correct the overshoot temperature based on the running time and the measured value of the temperature sensor, including at least one of the following: overshoot temperature maintenance time, and the relationship between the overshoot temperature and the stable temperature.

[0012] Furthermore, in the relationship between the overshoot temperature and the steady-state temperature, the temperature change within the same time period decreases over time.

[0013] Furthermore, the overshoot temperature is determined according to the safe temperature range jointly determined by the inactivation temperature of the nucleic acid amplification enzyme and the ambient temperature.

[0014] Furthermore, the overshoot temperature maintenance time is negatively correlated with the ambient temperature.

[0015] Secondly, the present invention also discloses a thermal cycling device for realizing polymerase chain reaction, comprising a thermal cycling module for performing thermal cycling on a sample, the thermal cycling module comprising a Peltier heating element, a sample tube receiving section comprising N receiving well units directly or indirectly connected to one end of the heating element, wherein N is an integer not less than 2, and a heat dissipation section directly or indirectly connected to the other end of the heating element opposite to the sample tube receiving section; and a temperature control module comprising at least two sets of control modes for controlling the heating element according to the corresponding ambient temperature under at least two different ambient temperatures, thereby realizing the heating and cooling operation of the sample in different control modes suitable for the ambient temperature.

[0016] Thanks to the adoption of the above technical solutions, the control scheme and the equipment using it can adapt to different ambient temperatures to achieve precise temperature control.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0018] 1. The temperature control mode stored in the control module of this invention is related to the ambient temperature. For different ambient temperatures, it can call the cyclic amplification control scheme corresponding to the nearest ambient temperature to achieve adaptability to the ambient temperature.

[0019] 2. By parameterizing the control mode, the optimal memory ratio of the control mode is achieved. At the same time, the most representative parameters of the control mode include at least one of the following: overshoot temperature, overshoot temperature holding time, and the relationship between overshoot temperature and steady temperature. This realizes the visualization of the control mode and ensures the accuracy of the implementation. Different control modes can be directly reflected by the difference of at least one of the above parameters.

[0020] 3. Before leaving the factory, the actual temperature following characteristics of the liquid in the sample tube during the heating process of the heating block are used to determine at least one of the following: overshoot temperature, overshoot temperature maintenance time, and the relationship between overshoot temperature and stable temperature. This ensures that the machine being calibrated has a precise and personalized control mode, rather than a fixed temperature control mode for each machine. This solves the problem of differences between instruments caused by production and assembly.

[0021] 4. When used with a temperature sensor, the heating element can precisely control the temperature inside the heating block according to the control mode, thereby achieving high-precision and accurate temperature control for the entire system.

[0022] 5. The results obtained from determining at least one of the following parameters—overshoot temperature, overshoot temperature duration, and the relationship between overshoot temperature and stable temperature—differences exist, reflecting the adaptability of the control scheme to environmental temperature. Furthermore, the overshoot temperature is correlated with enzyme inactivation temperature and, combined with environmental temperature, confirms a safe temperature range, ensuring that enzyme activity is not affected under reliable temperature control and guaranteeing the accuracy of system results. The relationship between overshoot temperature and stable temperature is similarly modified based on the measured temperature following characteristics, enabling the system to recover to the final stable temperature more quickly and accurately during overshoot, and allowing the sample solution to reach the stable temperature more precisely and rapidly. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of a system control implementation provided by the present invention;

[0024] Figure 2 This is a schematic diagram of a scheme for determining the final temperature control mode provided by the present invention;

[0025] Figure 3 This is a schematic diagram of different control modes obtained under different ambient temperatures provided by the present invention;

[0026] Figure 4 This is a comparison chart of the temperature control effects of the control scheme of this invention and the control scheme without overshoot;

[0027] Figure 5 This is a schematic diagram illustrating the implementation of the final temperature control mode of the control module output by the present invention.

[0028] Figure 6 It is a schematic diagram of the actual control mode curve selected from multiple control modes;

[0029] Figure 7 This is a schematic diagram of the process implemented using the solution of the present invention under actual ambient temperature control.

[0030] Figure 8This is a schematic diagram of the process of controlling the actual ambient temperature after the control mode is preset before leaving the factory using the solution of the present invention;

[0031] Figure 9 This is a schematic diagram of a thermal module structure that can be designed using the scheme of the present invention;

[0032] Figure 10 This is a schematic diagram of another thermal module structure that can be designed using the scheme of the present invention;

[0033] Figure 11 This is a schematic diagram of another thermal module structure that can be designed using the scheme of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0035] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0036] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0037] Currently, most temperature control schemes used in PCR (polymerase chain reaction) are based on the early discovery of a temperature delay effect, requiring overshooting to allow the sample solution in the temperature block to reach a stable temperature more quickly. However, since the introduction of PCR overshoot control schemes, many manufacturers still use the early "spear-shaped" overshoot control scheme, which uses a basically symmetrical triangle for overshoot control. For certain specific reactions, calculations according to publicly available technical solutions show that "spear-shaped" overshoot control requires an overshoot maximum temperature of close to 10°C. Taking 95°C polymerization amplification as an example, at this type of overshoot temperature, the temperature of a local area in the sample tube can be heated to above 97°C. Such a high sample solution temperature... Temperature fluctuations may reduce the activity of thermosensitive amplification enzymes, thereby decreasing the overall amplification efficiency and directly affecting the detection quality. Furthermore, in this overflush mode, the sample solution temperature may be closer to the boiling point, potentially exacerbating bubble formation or evaporation. Therefore, existing instruments are unsuitable for high-sensitivity enzyme applications, and may even exhibit significant differences in detection results due to variations in bubble formation or evaporation at different temperatures despite using the same overflush scheme. To enable the machine to be applicable to a wider range of detection scenarios while ensuring accurate results, a more adaptive temperature cycling control scheme, or more specifically, a more adaptive overflush control scheme, needs to be developed.

[0038] Figure 1This invention provides a system control implementation principle diagram. The control module 10 includes multiple control modes corresponding to different ambient temperatures, including a first control mode 101 corresponding to a first ambient temperature and a second control mode 102 corresponding to a second ambient temperature. Of course, more sets of control modes can be stored for different ambient temperatures; this is not limited here. For example, in the process of corresponding to ambient temperatures, multiple sets of control modes within the range of 15℃-40℃ are stored. The temperature difference interval between each set of control modes can be 0.5, 1, 1.5, 2, 3℃, etc., or the temperature interval data can be randomly set. There are no restrictions on the location. Taking 5℃ as an example, the range of 15℃-40℃ includes six control modes corresponding to 15, 20, 25, 30, 35, and 40℃. The actual room temperature can be any temperature within the above range. For example, if the room temperature is 26.3℃, the control mode corresponding to 25℃, which is closer, can be selected. Of course, correction parameters can also be set in the control module to correct the control mode corresponding to 25℃ to improve the accuracy of the temperature control mode. For temperatures in between two room temperature scenarios, the user can choose the corresponding control mode or the automatic selection program in the control module can be used to select the corresponding control mode automatically.The control module is electrically connected to the heating unit. The control mode within the control module can be a temperature parameter. After conversion by the calculation unit within the control module, it is transformed into electrical control signals such as voltage and current, which are then output to the heating unit to control the heating output. The heating element 30 can be the most widely used Peltier heating element. The number of heating elements is not limited (it can be 1, 4, 6, etc.). One end of the Peltier element 30 is connected to the sample tube receiving section 20, which has N receiving units (N can be 16, 48, 96, 288, 384, etc.). The sample tube receiving section 20 is heated or cooled by changing the electrical control signal of the Peltier element 30. Heat can be transferred between the two through thermal conduction and / or thermal radiation. The sample receiving section 20 can also be considered a temperature block, in which one or more temperature sensors are arranged to acquire real-time temperature change signals. Combined with adjustments by the control module, precise control of the temperature block temperature can be achieved according to the room temperature control mode of this invention. Of course, to ensure temperature control... To ensure accuracy, multiple temperature sensors can be set within the temperature block. The acquired temperature signals can be further processed within the control module 10 to generate power control signals that can drive and adjust the Peltier element 30, achieving a closed-loop control effect according to the control mode set by the control module. The other end of the Peltier element 30 is directly or indirectly connected to a heat sink 40. The heat sink 40 can include a conventional aluminum heat sink, produced using processes such as tooth cutting. To achieve better heat dissipation, a corresponding cooling fan 50 is generally required. The fan speed adjustment is used to match the heat dissipation requirements of the entire system. Of course, to ensure the intelligence of the system operation, the control module 10 can also arrange autonomous calibration based on machine running time, running status, and other historical parameters. It can adaptively adjust the relevant parameters in the control mode using the temperature detected by the temperature sensor in the sample tube receiving part 20 under the same ambient temperature. This can achieve the effect of efficient and accurate temperature control of the system at all times. Of course, the fan speed control in different control modes can also be different.

[0039] Figure 2This is a schematic diagram of a scheme provided by the present invention, which measures the temperature following characteristics of a sample liquid by measuring a temperature block according to a fixed mode before leaving the factory to determine the final temperature control mode. Using the scheme provided by the present invention, sampling calibration can be performed on each machine or some machines in each batch before leaving the factory. When the temperature following characteristics of the machines in the batch are good, the number of calibration machines can be reduced adaptively. Of course, calibration and storage can also be performed on different batches of machines during the production process. Using the gradually accumulated historical calibration curve library, the closer curve parameters are selected from the historical calibration curve library at a certain temperature as the basic parameters used for the current calibration equipment, thereby placing the curves at different temperatures into the control module. Of course, the implementation method is not limited here, but the optimal method is to calibrate each machine according to the method of the present invention. The calibration method can involve using the same microplate as the one actually used, adding a solution with the same or similar thermal properties as the sample solution, or simply water, according to the sample volume required in actual use. Temperature sensors are placed in at least some of the wells within the container to obtain the thermal following characteristics of the solution within the microplate when the temperature changes in the temperature block. (The number of temperature sensors can be set to 2, 3, 4, 5, etc., according to actual needs. The data obtained by each temperature sensor can be processed according to a certain rule stored in the control module to obtain the comprehensive temperature following parameters.) See attached. Figure 2 The sensor shown, in conjunction with the control module, operates a trapezoidal temperature control scheme according to the temperature rise requirements during the temperature cycle, such as controlling the temperature from room temperature to the denaturation temperature of 95°C. The temperature sensor, which is in direct contact with the solution inside the orifice, can directly obtain the following temperature characteristics of the solution within the orifice. The obtained following temperature can be in the form of a discrete data table, or it can be fitted into a continuous function form within the control module. Here, the following temperature is expressed as having a primary relationship with time: T1 = g(t). Based on this, at least some parameter characteristics of the control mode can be generated. For example, the control can include at least one of the following parameter characteristics: overshoot temperature, overshoot temperature maintenance time, and the relationship between the overshoot temperature and the stable temperature. The relationship between the overshoot temperature and the stable temperature can be described as follows:

[0040] T(t) = h(T) 超 T 稳 ,T1) (1),

[0041] Where T1 represents the temperature change characteristics of the sample tube solution obtained directly from the temperature sensor before shipment and under the trapezoidal ideal temperature control mode. This directly incorporates the differential characteristics of each instrument into the overshoot control algorithm. When the final control mode includes all parameters such as overshoot temperature, overshoot temperature holding time, and the relationship between overshoot temperature and steady-state temperature, the control mode can be described as follows:

[0042] S(t)=f(T超 T 稳 ,T(t)) (2);

[0043] Of course, the overshoot control function establishment process of the entire control mode is described here in the form of function S(t). The steps for constructing the control mode with discrete data such as lists are similar and will not be elaborated here. In the process of establishing the entire control mode, the differences in the following characteristics of different machines under the same ambient temperature and the same heating or cooling process are taken into account. Therefore, the constructed control mode can adapt to the differences in different components and production processes. Moreover, the following characteristics of the temperature block during the heating or cooling of the liquid in the sample tube are used to construct the relationship between the overshoot temperature and the steady temperature in the control mode, which can adjust more accurately and quickly. Once the sample solution reaches the required stable temperature, the heat transfer characteristics of the temperature block in the differential system can be correlated with the ability to predict the temperature transfer characteristics from the specific temperature block to the sample solution in advance. This allows for the rational allocation of the overshoot temperature maintenance time in overshoot control and the provision of a more accurate prediction adjustment scheme, ensuring the liquid quickly reaches a stable state after overshoot. Furthermore, since the relationship between the overshoot temperature and the stable temperature in the overshoot control of this invention is directly based on the following relationship, it eliminates the need for complex fuzzy control schemes to fit the differences in temperature across different instruments. Therefore, the entire control module design is simpler, and the final solution S... F In other words, it is the integral value of the straight segment of the relationship between the overshoot temperature and the steady temperature. In other words, the determination of energy only requires a simple calculation of the accumulated result of the follower function of water or similar characteristic samples, without the need for complex component design such as integrators.

[0044] Combination Figure 3 and Figure 4 To elaborate further, Figure 3 The diagram illustrates the temperature tracking characteristic curves of the solution in the sample tube as the hot block rises from different ambient temperatures to a preset temperature. For example, C102, C202, and C302 correspond to ambient temperatures of 20℃, 25℃, and 30℃, respectively, as the hot block rises to the desired stable temperature (e.g., the denaturation temperature of 95℃). The corresponding temperature tracking curves in the sample tube are S101, S201, and S301. This means that even the same instrument can exhibit significant differences in temperature tracking characteristics under different ambient temperatures. Therefore, it is essential to set different control modes for different temperatures within the control module. This allows the instrument to adapt to different ambient temperatures, ensuring its adaptability to variations in ambient temperature. Figure 2 The approach can obtain different control modes corresponding to different temperatures within the control module, combined with... Figure 4To further illustrate the overshoot mode control mechanism of the present invention, when there is no overshoot, the temperature following curve of the sample solution is S. D Depend on Figure 4 It can be seen that without an overshoot scheme, reaching the set temperature would take a long time, which could lead to insufficient testing in this stage. The cumulative effect of this insufficient testing over dozens of tests could be amplified, potentially causing false negatives or other detection failures. The present invention also employs an overshoot control mode, where the highest temperature maintained by the overshoot is denoted as T. 超 The denaturation temperature can be determined within a safe temperature range jointly defined by the inactivation temperature of the nucleic acid amplification enzyme and the ambient temperature. This avoids the possibility of the sample solution temperature rising too high, which could damage the activity of highly sensitive enzymes, as is possible in "spear-shaped" overflush schemes. This effect is also cumulative; after 30 or 40 cycles, it can cause serious consequences and may lead to false negatives. The overshoot temperature needs to be maintained for a period of time to ensure that the sample solution can quickly reach or even exceed the stable temperature T under the entire overflush design. 稳 The overshoot temperature maintenance time is t. 维持 Its overshoot temperature is negatively correlated with ambient temperature; that is, the higher the ambient temperature, the shorter the required overshoot temperature holding time. This is mainly because the system dissipates heat more slowly at higher ambient temperatures. Therefore, a shorter holding time in the control mode ensures that the entire control reaches a stable temperature quickly. The relationship between the overshoot temperature and the stable temperature in the overshoot control mode is S. F like Figure 4 As shown, to ensure the adaptability of this changing relationship to different equipment and different ambient temperatures, the following measures can be taken: Figure 2 The method shown is used to determine this relationship; the specific process and principle will not be elaborated here. Using the overshoot mode control scheme of this invention, the final actual temperature curve S can be obtained. T Because the control scheme of this invention enables the actual temperature control to reach the stable temperature T more quickly and stably. 稳 This refers to the target temperature. The diagram is for illustrative purposes only and does not represent the actual time scale. This allows us to obtain N different control modes corresponding to the same target control temperature under different ambient temperatures. N can be set to different values ​​according to different needs, thus obtaining at least two sets of control modes corresponding to at least two different ambient temperatures, which are then stored in the control module.

[0045] Figure 5This is a schematic diagram illustrating the implementation of the final temperature control mode output by the control module of the present invention. The control module stores temperature cycle control modes corresponding to different ambient temperatures and also includes a judgment unit. The judgment unit can determine, based on the current ambient temperature, either a control mode corresponding to a certain temperature or a control mode associated with two control modes corresponding to different temperatures as the final control mode. For example, the judgment unit includes a comparator. The comparator can first compare the current ambient temperature with different ambient temperatures corresponding to the control modes and select the ambient temperature of the closest control mode as a reference. Then, a threshold s can be set. If the absolute value of the difference between the current temperature and the selected temperature is less than the set threshold s, the control mode corresponding to the selected temperature is used as the final control mode. When the absolute value of the difference is greater than the set threshold s, the two sets of control modes corresponding to the selected temperature and the other temperature closest to the selected temperature are used as the basis. The final control mode can be obtained by using a weighting factor to obtain a new mode that integrates the two modes as the final control mode (of course, the control module can also store frequently used operating states to generate new control modes corresponding to different ambient temperatures, thus generating self-learning type autonomous operating states). Of course, other methods can also be used to integrate the two control modes, which is not limited here.

[0046] Figure 6 This is a schematic diagram of the final temperature control mode of the temperature block output by the control module of the present invention. The temperature sensor and the control module work together to realize the heating and cooling operation of the sample. The control module can also correct the overshoot temperature based on the running time and the measured value of the temperature sensor, including at least one of the following: the overshoot temperature maintenance time, the relationship between the overshoot temperature and the stable temperature, so as to realize that the temperature inside the temperature block runs according to the temperature control curve preset by the control module. Figure 6 In the temperature cycle, three overshoot control schemes (M01, M02, and M03) obtained using the present invention are set. Of course, the parameters of the three overshoot modes can be different and can be determined based on the parameters obtained from actual calibration. Alternatively, they can be set only during partial temperature rises or falls. Or, in the non-denaturation stage, a traditional spear-shaped overshoot control scheme can be used, that is, M02 and / or M01 can be set as spear-shaped or trapezoidal overshoot, while M03 can be set as the irregular-shaped overshoot scheme of the present invention. This is not limited here. However, to improve the system's versatility and more accurate temperature control characteristics, setting overshoot control schemes in all three regions of the temperature cycle as shown in the figure is a better solution. Figure 6 This can correspond to overshoot control at a specific ambient temperature, and the control curves for other different ambient temperatures can be similar. Figure 6The solution has at least some differences in the overshoot stage, namely M01, M02 and M03 in the figure. The process of generating control curves at different temperatures will not be described here. The relationship between the overshoot temperature and the stable temperature during heating and cooling can be derived from the temperature rise relationship obtained by factory test at the corresponding ambient temperature. The control module stores control curves at at least two different ambient temperatures, so that the method of the present invention has stronger environmental adaptability.

[0047] Figure 7 The control module of this invention implements the following steps: In actual use, the instrument system's control module includes different control modes corresponding to ambient temperature. The instrument can determine the ambient temperature based on its own temperature sensor measurements or through user input. Then, it selects the control mode closest to the ambient temperature to complete the control mode selection step S11. After the instrument selects the control mode, it can coordinate with the temperature sensor in the temperature block to execute the heating / cooling operation step S12. According to the determined control mode, the basic parameters of the control mode can include at least one of the following: overshoot temperature, overshoot temperature maintenance time, and the relationship between overshoot temperature and stable temperature. Using the control mode determined by the above parameters, the sample solution is heated / cooled to achieve different target temperatures. After implementing the overshoot mode, the temperature maintenance step S13 needs to be executed. At this time, due to the completion of the overshoot control mode, the final power control signal is maintained, ensuring that the system heating and heat dissipation reach a balanced state, maintaining the stable temperatures corresponding to denaturation, annealing, and extension. More cycles (such as 30, 35, 40, etc.) are executed to achieve temperature cycling of the instrument system, further completing the cyclic amplification of the entire sample solution.

[0048] Figure 8 This is a further detailed description of the control implementation steps of the control module of the present invention, and... Figure 7 Similar steps will not be repeated here, but it is important to note that... Figure 8 The control modes corresponding to different ambient temperatures are pre-calibrated and pre-programmed into the instrument system before leaving the factory. In other words, the control modes of the system control module are pre-programmed using the scheme of this invention. The pre-programming step is S21, in which the temperature following characteristics of the sample tubes of different machines are measured using water (or other liquids with similar thermal properties) before leaving the factory, and the corresponding control modes are generated and pre-programmed into the control module based on different ambient temperatures. The specific method can be referred to the steps given in the previous embodiments, and will not be repeated here.

[0049] Figure 9This is a schematic diagram of a system temperature cycling module driven by the scheme of the present invention. The sample tube receiving part 20 includes 48 well units for receiving 48 sample tubes containing PCR reaction solution. To ensure rapid and uniform heat transfer, its lower part includes a heat-conducting element 82 and four sets of Peltier heating elements 83. To ensure better heat conduction with the heat dissipation part 40, a heat-conducting element 82 may also be included between the Peltier element 30 and the heat dissipation part 40. Using the overshoot mode control of the present invention, this temperature cycling module can simultaneously perform more precise temperature control for 48 nucleic acid detection samples. A method including... Figure 9 The thermal circulation device of the thermal circulation module.

[0050] Figure 10 This is a schematic diagram of another system temperature cycling module driven by the scheme of the present invention. The sample tube receiving part 20 includes only 16 well units, and is similarly connected to the Peltier heating element 93 through the heat-conducting element 82. Here, a single Peltier element 30 design can be adopted. The heat dissipation part 40 is connected to the Peltier element 30 through the heat-conducting element 82. Using the overshoot mode control of the present invention, this temperature cycling module can perform more precise temperature control for 16 nucleic acid detection samples simultaneously. A method including... Figure 10 The thermal circulation device of the thermal circulation module.

[0051] Figure 11 This is a schematic diagram of another system temperature cycling module driven by the scheme of the present invention. The sample tube receiving part 20 includes 96 well units, which are similarly connected to the Peltier heating element 103 through the heat-conducting element 82. Here, a design of 6 Peltier elements 30 can be used. The heat dissipation part 40 is connected to the Peltier element 30 through the heat-conducting element 82. Using the overshoot mode control of the present invention, this temperature cycling module can perform more precise temperature control for 96 nucleic acid detection samples simultaneously. A method including... Figure 11 The thermal circulation device of the thermal circulation module.

[0052] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the present method and its core ideas. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0053] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0054] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A thermal cycling device for polymerase chain reaction, characterized in that, The device includes a thermal cycling module for performing thermal cycling on a sample, the thermal cycling module including a Peltier heating element, a sample tube receiving section including N receiving well units directly or indirectly connected to one end of the heating element, where N is an integer not less than 2, and a heat dissipation section directly or indirectly connected to the other end of the heating element opposite to the sample tube receiving section. A temperature control module includes at least two sets of control modes for controlling the heating element according to the corresponding ambient temperature under at least two different ambient temperatures, thereby enabling the sample to perform heating and cooling operations in different control modes suitable for the ambient temperature; the control mode includes an overshoot mode, and the overshoot mode parameters include at least one of overshoot temperature, overshoot temperature duration, and the relationship between overshoot temperature and stable temperature; at least one of the overshoot temperature, overshoot temperature duration, and the relationship between overshoot temperature and stable temperature is different in at least two ambient temperatures; The temperature control module includes a judgment unit, which can determine the final temperature control mode based on the current ambient temperature, either using a control mode corresponding to a certain temperature or a control mode that is associated with two control modes corresponding to different temperatures. At least one of the overshoot temperature, overshoot temperature duration, and the relationship between the overshoot temperature and the steady temperature is pre-calibrated and pre-programmed into the temperature control module before the machine leaves the factory. It also includes a temperature sensor unit, which works with the control module to perform heating and cooling operations on the sample. The control module can also correct the overshoot temperature based on the running time and the measured value of the temperature sensor, including at least one of the following: overshoot temperature maintenance time and the relationship between the overshoot temperature and the stable temperature.

2. The thermal cycling apparatus for polymerase chain reaction as described in claim 1, characterized in that, In the relationship between the overshoot temperature and the steady-state temperature, the temperature change within the same time period decreases with time.

3. The thermal cycling apparatus for polymerase chain reaction as described in claim 1, characterized in that, The overshoot temperature is determined according to the safe temperature range determined by the inactivation temperature of the nucleic acid amplification enzyme and the ambient temperature.

4. The thermal cycling apparatus for polymerase chain reaction as described in claim 1, characterized in that, The overshoot temperature duration is negatively correlated with the ambient temperature.

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