Fluorescent quantitative PCR instrument

Abstract

The invention discloses a fluorescent quantitative PCR instrument, and relates to the technical field of biological detection, the fluorescent quantitative PCR instrument comprises a fixing frame and a plurality of camera detection assemblies, pore plates filled with nucleic acid samples are placed on the fixing frame, the plurality of camera detection assemblies are arranged on the fixing frame and can irradiate sample pores in each pore plate, each camera detection assembly comprises a light generation device, a camera main body and an optical filter, the camera main body is used for shooting a sample in the sample hole, the light generation device is arranged on the camera main body and used for emitting light and irradiating the sample hole, and the optical filter is arranged between the light generation device and the hole plate and can filter the light emitted by the light generation device; and the optical filters connected with the camera main bodies are different, so that light with different wavelengths can be filtered. The fluorescent quantitative PCR instrument provided by the invention can realize synchronous detection of multiple fluorescent colors, is free of mechanical movement on the whole, is beneficial to long-term use, and is accurate in detection and high in efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a fluorescence quantitative PCR instrument. Background Art

[0002] Fluorescence quantitative PCR instruments (analytical instruments based on polymerase chain reaction technology, Polymerase Chain Reaction, PCR) are widely used in the field of nucleic acid detection. Their basic principle is to detect the amount of DNA amplification in the PCR reaction system through specific fluorescent dyes or fluorescently labeled probes. As the number of PCR cycles (thermal cycles) increases, the number of target DNA fragments increases exponentially, and the corresponding fluorescence signal intensity also increases. By detecting and analyzing the fluorescence signal, the copy number of the target gene in the original sample can be accurately calculated. At present, the fluorescence detection methods of real-time fluorescence quantitative PCR are mainly divided into two categories, namely traditional mechanical scanning and PCR instruments based on optical fiber light guide and filter wheel. Mechanically scanning PCR instruments rely on a reciprocating scanning head to scan multiple wells containing samples. This reciprocating mechanical motion can easily damage the scanning head over time. Furthermore, when testing multiple fluorescent colors, not only does the scanning head move, but multiple filters and a rotating wheel are also required within the detection device. Each time a color is changed, the rotating wheel rotates the filter, which also involves mechanical motion. Vibration or displacement deviations in the rotating wheel and transmission components during continuous motion can cause fluctuations in the fluorescence detection signal, leading to abnormal wear between the detection probe and the moving parts, affecting detection accuracy and the lifespan of the device. PCR instruments based on fiber-optic light guides and filter wheels experience reflection, scattering, and absorption during transmission within the optical fiber, resulting in light intensity attenuation (typically 10%-20%). The filters also rely on a rotating wheel and motor for rotation. Long-term, high-frequency switching (e.g., multiple times per second) can lead to bearing wear and filter positioning deviations. Furthermore, the filter wheel must switch wavelengths sequentially, making true multi-color simultaneous detection impossible. This compromises detection results and reduces efficiency. Therefore, there is an urgent need for a fluorescent quantitative PCR instrument to solve the above technical problems. Summary of the Invention

[0003] The purpose of the present invention is to provide a fluorescent quantitative PCR instrument to solve the problems existing in the above-mentioned prior art, which can realize synchronous detection of multiple fluorescent colors, has no mechanical movement as a whole, is conducive to long-term use, and has high detection accuracy and efficiency.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a fluorescent quantitative PCR instrument, comprising a fixing frame and multiple camera detection assemblies, wherein a well plate containing nucleic acid samples is placed on the fixing frame, and multiple camera detection assemblies are arranged on the fixing frame and can illuminate each sample well on the well plate, wherein each camera detection assembly includes a light generating device, a camera body and a filter, wherein the camera body is used to photograph the sample in the sample well, the light generating device is arranged on the camera body, the light generating device is used to emit light and illuminate the sample well, the filter is arranged between the light generating device and the well plate, and can filter the light emitted by the light generating device, and each camera body is connected to a different filter, which can filter light of different wavelengths.

[0006] In some embodiments, a plurality of light reflecting devices are further included, wherein the light reflecting devices are disposed on the fixing frame, and the light reflecting devices are capable of reflecting the fluorescence after the sample hole is irradiated to the camera detection component.

[0007] In some embodiments, the light reflecting device is a plane mirror, and is provided with two plane mirrors, namely a first plane mirror and a second plane mirror. The first plane mirror and the second plane mirror are both rotatably set on the fixed frame, and the first plane mirror is tilted above the well plate, and the second plane mirror is tilted on the opposite side of the camera detection component. The fluorescence in the sample hole of the well plate can be reflected by the first plane mirror and the second plane mirror in turn into the camera detection component.

[0008] In some embodiments, a mounting plate is further included, and a plurality of the camera detection components are evenly distributed on both sides of the mounting plate and fixedly connected, and the camera detection components are tilted on the mounting plate to ensure that each of the camera detection components can receive a complete image of the orifice plate.

[0009] In some embodiments, it also includes a fixing box and a first extension tube, the light generating device is an LED lamp, the fixing box is hollow and has openings on three sides, namely the first opening, the second opening and the third opening, a dichroic mirror is provided inside the fixing box, the first opening and the second opening are arranged opposite to each other, and the first opening is used to install the camera body, the second opening is used to install the filter, and the third opening is arranged on the adjacent side of the first opening, the two ends of the first extension tube are respectively connected to the LED lamp and the third opening, and the light of the LED lamp can be emitted through the first extension tube and then reflected by the dichroic mirror and emitted through the second opening.

[0010] In some embodiments, the well plate is a temperature-isolating plate, and the temperature-isolating plate is provided with a plurality of sample wells for placing test tubes.

[0011] In some embodiments, a temperature control device and a heat sink are further included. The heat sink is fixed on the fixing frame, and the temperature control device is arranged above the heat sink and below the temperature equalizing plate, and can adjust the temperature of the temperature equalizing plate.

[0012] In some embodiments, the temperature control component is a semiconductor cooler.

[0013] In some embodiments, a temperature sensor is further included. The temperature sensor is arranged on the temperature equalizing plate. The temperature sensor is electrically connected to the control system of the temperature control device. The control system can adjust the temperature control device according to the output signal of the temperature sensor.

[0014] In some embodiments, a thermal cover is further included, and the thermal cover can cover the top of the temperature vapor chamber and be sealed.

[0015] Compared with the prior art, the present invention has achieved the following technical effects:

[0016] A light generator emits light, which is filtered through a filter to produce light of a specific wavelength. This light excites the fluorescent markers in the PCR reaction solution, causing them to release a fluorescent signal of a specific wavelength. A camera is then used to capture the excited fluorescent signal and convert it into a visual digital image. Finally, image analysis software algorithms are applied to the signal data in the fluorescence image to extract and quantify the fluorescence intensity. Each filter is unique, requiring a specific set of filters for each fluorescent marker to be detected. Each filter does not mechanically move relative to the mounting bracket. Fluctuations in the fluorescence detection signal caused by vibration or displacement of the mechanical transmission components during continuous motion, which can lead to abnormal wear between the detection probe and the moving parts, affecting detection accuracy and equipment lifespan, are eliminated. This ensures long-term use and more accurate detection. Furthermore, multiple camera detection components operate simultaneously, enabling simultaneous detection of multiple fluorescent colors, ensuring high detection efficiency and significantly reducing time costs compared to single-channel, well-by-well testing, making it suitable for rapid screening of large-scale samples. Simultaneous detection can also prevent sample damage caused by prior testing, which could lead to inaccurate subsequent testing. It can simultaneously detect different fluorescent groups, such as FAM, VIC, and ROX, meeting the needs of multiplex PCR testing (such as simultaneous analysis of multiple target genes) and avoiding the errors of single-channel detection. Traditional single-channel, multiplexed detection may cause quenching of fluorescent marker molecules in the sample or a decrease in sample activity due to early excitation light exposure. This device, however, simultaneously detects all fluorescent signals, eliminating differences in sample status between prior and subsequent testing, ensuring data consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a front view of a fluorescent quantitative PCR instrument in some embodiments of the present invention;

[0019] Figure 2 Schematic diagram of the structure of a fluorescent quantitative PCR instrument in some embodiments of the present invention;

[0020] Figure 3 Schematic diagram of the structure of the thermal cover and the orifice plate in some embodiments of the present invention;

[0021] Figure 4 Exploded views of temperature control devices, heat sinks, heat covers, and temperature homogenizers in some embodiments of the present invention;

[0022] Figure 5 A schematic structural diagram of a camera body in some embodiments of the present invention;

[0023] Figure 6 Schematic diagram of the structure of LED lamps in some embodiments of the present invention;

[0024] Figure 7 Schematic diagram of the connection between the fixing box and the camera body, LED light and filter in some embodiments of the present invention;

[0025] Figure 8 Schematic diagram of the structure of the mounting plate in some embodiments of the present invention;

[0026] Figure 9 A schematic diagram of the structure of a camera detection component in some embodiments of the present invention;

[0027] Figure 10 Schematic diagram of a possible arrangement of plane mirrors in some embodiments of the present invention.

[0028] In the figure: 101-camera detection component; 102-fixing bracket; 103-hole plate; 1-camera body; 2-second extension tube; 3-first extension tube; 4-LED light; 5-filter; 6-mounting plate; 7-laser hole; 8-first plane mirror; 9-second plane mirror; 10-heat cover; 11-TEC; 12-temperature plate; 13-heat sink. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] The purpose of the present invention is to provide a fluorescent quantitative PCR instrument to solve the problems existing in the prior art. It can realize synchronous detection of multiple fluorescent colors, has no mechanical movement as a whole, is conducive to long-term use, and has high detection accuracy and efficiency.

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] like Figures 1-10As shown, the present invention provides a fluorescent quantitative PCR instrument, including a fixing frame 102 and multiple camera detection components 101 (preferably four, generally for detecting four fluorescent colors, and the specific number can be determined according to the number of colors to be detected), a well plate 103 containing nucleic acid samples is placed on the fixing frame 102, and multiple camera detection components 101 are arranged on the fixing frame 102 and can illuminate each sample well on the well plate 103, wherein each camera detection component 101 includes a light generating device, a camera body 1 (preferably a complementary metal oxide semiconductor (CMOS) image sensor) and a filter 5, the camera body 1 is used to photograph the sample in the sample well, the light generating device is arranged on the camera body 1, the light generating device is used to emit light and illuminate the sample well, the filter 5 is arranged between the light generating device and the well plate 103, and can filter the light emitted by the light generating device, and the filter 5 connected to each camera body 1 is different and can filter light of different wavelengths. A light generating device emits light, which is filtered by a filter to become light of a specific wavelength. This light excites the fluorescent marker molecules in the PCR reaction solution, causing them to release a fluorescent signal of a specific wavelength through the light energy excitation. A camera body 1 is then used to collect the excited fluorescent signal and convert it into a visual digital image. Finally, an image analysis software algorithm is applied to the signal data in the fluorescent image to extract and quantify the fluorescence intensity value. Furthermore, each filter 5 is different, and different filters 5 are required depending on the number of fluorescent markers to be detected. Each filter 5 does not mechanically move relative to the fixed frame 102. Fluctuations in the fluorescence detection signal due to vibration or displacement deviation of the mechanical transmission components during continuous movement, as well as abnormal wear between the contact surface of the detection probe and the moving components caused by this, which affects the detection accuracy and service life of the equipment, are not likely to occur. This makes it more conducive to long-term use and more accurate detection. Furthermore, multiple camera detection assemblies 101 operate simultaneously, enabling simultaneous detection of multiple fluorescent colors, ensuring high detection efficiency. Compared with single-channel, hole-by-hole detection, the time cost is significantly reduced, making it suitable for rapid screening of large-scale samples. Simultaneous detection can also prevent sample damage caused by prior testing, which could lead to inaccurate subsequent testing. It can simultaneously detect different fluorescent groups, such as FAM, VIC, and ROX (detection groups can be customized as needed, and replacement is simple and quick), meeting the needs of multiplex PCR testing (such as simultaneous analysis of multiple target genes) and avoiding the errors of single-channel detection. Traditional single-channel, sequential detection may cause quenching of fluorescent marker molecules in the sample or decrease sample activity due to initial excitation light exposure. This device simultaneously detects all fluorescent signals, eliminating differences in sample status between prior and subsequent testing and ensuring data consistency.

[0033] In some embodiments, the fluorescence quantitative PCR instrument further includes a plurality of light reflecting devices, which are disposed on the fixing frame 102, and the light reflecting devices are capable of reflecting the fluorescence of the sample well after being irradiated to the camera detection component 101. The light reflecting device is disposed on the fixing frame 102, and can converge the fluorescence excited by the sample well to the camera detection component 101 from multiple angles such as the side and the top through a reflecting surface (such as a mirror, a diffuse reflection plate). When fluorescence propagates in a solution, it will attenuate due to scattering and absorption. The reflecting device reduces the loss of the signal during transmission by shortening the path of the fluorescence to the camera or changing the direction of the light path. The reflecting surface of the light reflecting device can be designed to be arc-shaped or at a specific angle, so that the fluorescence of the sample wells at different positions is evenly projected onto the camera sensor after reflection, thereby avoiding uneven signals between wells caused by light path deviation. The light reflection device can adjust the reflection angle or material according to the specifications of the well plate 103 (such as 384-well plate, deep-well plate, transparent / white well plate). For example, the inner wall of the white well plate 103 itself is reflective, and the reflection device can cooperate with it to enhance the signal; the deep-well plate 103 uses the bottom reflection device to improve the signal collection efficiency of deep samples, thereby expanding the scope of application of the equipment.

[0034] Since the camera body 1 needs to be able to capture the entire well plate 103, it is necessary to first calculate the required length of the camera field of view to cover the entire 96 (or 384) well plate size based on the size of the camera's photosensitive chip and the focal length of the lens used (the farther away from the object when taking a picture, the larger the range of the picture will be. Different photosensitive sizes and lens focal lengths will have differences. For example, if a 1 / 2.8-inch photosensitive chip and a 25mm focal length lens are used, in order to capture the size of a 96-well plate, the lens needs to be about 560mm away from the 96-well plate. In this embodiment, cameras and lenses of various specifications can be replaced, and it is only necessary to ensure that a complete and clear image can be obtained in the end).

[0035] In some embodiments, the light reflecting device is a plane mirror, and is provided with two plane mirrors, namely a first plane mirror 8 and a second plane mirror 9. The first plane mirror 8 and the second plane mirror 9 are both rotatably set on the fixed frame 102, and the first plane mirror 8 is tilted above the orifice plate 103, and the second plane mirror 9 is tilted on the opposite side of the camera detection component 101. The fluorescence in the sample hole of the orifice plate 103 can be reflected by the first plane mirror 8 and the second plane mirror 9 into the camera detection component 101 in sequence. The first plane mirror 8 is tilted above the orifice plate 103, and the reflection angle can be adjusted by rotation to reflect the fluorescence of the sample holes in different positions to the second plane mirror 9. For example, when detecting holes in different columns of a 96-well plate, rotating the first plane mirror 8 can change the angle of the incident light, ensuring that the fluorescence of the edge holes and the center hole can be effectively reflected, thereby avoiding signal omissions due to position differences of the orifice plate 103. The second plane mirror 9 is arranged on the opposite side of the camera detection component 101. By rotating, the final direction of the reflected light path can be adjusted so that the fluorescence can be accurately projected onto the camera sensor. It is particularly suitable for rapid calibration of the light path when the layout of the well plate 103 changes (such as replacing well plates 103 of different specifications). The rotatable plane mirror can adjust the reflection angle according to the height of the well plate 103 (such as deep well plate 103, shallow well plate 103) or the hole spacing. For example, when detecting a 384-well plate, by reducing the rotation angle of the two plane mirrors, a smaller range of fluorescence signals can be focused; when detecting a deep well plate 103, increasing the inclination angle of the first plane mirror 8 can enhance the fluorescence reflection efficiency of deep samples and expand the adaptability of the equipment to different experimental consumables. The fluorescence is reflected by the first plane mirror 8 to the second plane mirror 9, and then enters the camera component. The two reflections can converge the scattered fluorescence into a more concentrated light beam, reducing signal loss. The two plane mirrors shorten the optical path. As mentioned above, the lens parameters selected require the camera to be approximately 560mm from the 96-well plate to capture an image. Using plane mirrors for reflection significantly reduces the instrument's size. (Folding allows for a longer optical path within a smaller space. This longer optical path increases the camera's field of view, allowing it to capture the entire 96-well plate.)

[0036] In some embodiments, the fluorescence quantitative PCR instrument further comprises a mounting plate 6, with a plurality of camera detection assemblies 101 evenly distributed on both sides of the mounting plate 6 and fixedly connected, and the camera detection assemblies 101 are tilted on the mounting plate 6 to ensure that each camera detection assembly 101 can receive a complete image of the well plate 103. If the camera body 1 is not tilted, then during use, only one camera body 1 may be facing the well plate 103 and able to receive a complete image of the well plate 103. Therefore, if a complete fluorescence image is desired, the camera body 1 needs to be tilted. The tilt angle is determined by the photosensitive size of the camera body 1, the focal length of the lens, and the imaging distance. (Because the laser is located at the center of the camera array in the aforementioned positioning method, the field of view of the camera will inevitably differ from that of the 96-well plate if it is not tilted. The tilt angle can be simply calculated using geometric methods. If the above-mentioned camera lens parameters are used, the camera needs to be tilted at an angle of 1°.) The camera in the center position can capture the complete image without any tilt angle, but the ultimate goal is to require four-channel imaging, which also means that the camera cannot be located in the center. The initial camera placement serves as the center of the final four-camera layout. By tilting the camera body 1, each camera's field of view is aligned with the 96-well plate to ensure the camera can capture images. After determining the tilt angle of the camera body 1, two M3 screw holes are drilled at the bottom of the mounting plate 6 to secure it to a vertical, height-adjustable aluminum plate. This aluminum plate is then connected to a horizontal aluminum plate and finally secured to the mounting bracket 102.

[0037] When installing the plane mirror, a laser hole 7 can be opened in the center of the mounting plate 6. Laser light can be used to illuminate the laser hole 7. The first plane mirror 8 and the second plane mirror 9 are positioned according to the laser's landing point. The two plane mirrors will reflect the laser light to the center of the 96-well plate. In this way, the positions of the camera, plane mirror, and 96-well plate can be roughly determined. Alternatively, SolidWorks can be used during the initial design to accurately draw each part, which can also simplify subsequent debugging steps. However, this requires that the camera parameters be determined at the beginning of the design.

[0038] In some embodiments, the fluorescent quantitative PCR instrument further includes a fixed box and a first extension tube 3, the light generating device is an LED lamp 4, the fixed box is hollow and has openings on three sides, namely a first opening, a second opening, and a third opening. A dichroic mirror is provided inside the fixed box, the first opening and the second opening are arranged opposite each other, and the first opening is used to install the camera body 1, the second opening is used to install the filter 5, and the third opening is provided on the adjacent side of the first opening. The two ends of the first extension tube 3 are respectively connected to the LED lamp 4 and the third opening. After the light of the LED lamp 4 is emitted, it can pass through the first extension tube 3 and be reflected by the dichroic mirror and emitted through the second opening. The two ends of the first extension tube 3 are respectively connected to the LED lamp 4 and the third opening of the fixed box, forming a closed light path, constraining the divergent light emitted by the LED into a parallel beam, converging the light, reducing stray light interference, and reducing light energy loss. The filter 5 installed in the second opening can be flexibly replaced according to the LED wavelength or fluorescent marker type. For example, when LED light 4 is 470nm blue light, it can be used with FAM filter 5 (passband 510-530nm) to excite green fluorescence; when it is changed to 520nm green light LED, it can be used with Cy3 filter 5 to detect red fluorescence, which is suitable for a variety of fluorescent probe systems.

[0039] Specifically, the LED light 4, camera body 1, and mounting box are first assembled to form a camera detection assembly 101. The camera and LED light 4 are connected to the mounting box via threaded extension tubes. Four identical components are included. The mounting box is a hollow, rectangular box with holes on three sides, one of which has a positioning screw. These three holes are used to position the camera body 1, the LED light 4, and the filter 5. The mounting box can hold the LED light 4, camera, and filter 5 together, creating a single detection path. (This facilitates user customization of the device; changing the detection path or performing maintenance requires simply replacing one mounting box.) The camera body 1 and LED light 4 are both screwed into the box, requiring only tilting the box. A dichroic mirror is located at the center of the mounting box, redirecting the light path. Glue is used to secure the dichroic mirror in place. The filter 5 is secured to the opening in the box. Next, the four mounting boxes are mounted on a mounting plate 6 in a 2x2 array. Secure the LED light 4 to its PCB control board with screws, then secure it to the first extension tube 3 with screws. The other end of the first extension tube 3 has threads that allow it to be directly rotated and fixed to the fixing box. Similarly, secure the camera to the second extension tube 2 with screws. The other end of the second extension tube 2 is also threaded into the fixing box. The second extension tube 2 is used to secure the camera.

[0040] It should be noted that LED lamp 4 inevitably presents the problem of uneven illumination. To address this issue, a MATLAB algorithm can be used to generate an uneven illumination template. This allows for more accurate fluorescence intensity extraction from the 96-well or 384-well images during image processing after obtaining the fluorescence photograph. (Image processing: Because the illumination intensity of LED lamp 4 is inherently uneven, exhibiting a Gaussian distribution, the fluorescence generated from the 96-well plate by the LED light also exhibits an uneven distribution, negatively impacting the accuracy of fluorescence analysis. To address this issue, an illumination unevenness correction algorithm based on an illumination distribution function is proposed. The main idea of ​​this algorithm is as follows: Using photographs filled with multiple fluorescein concentrations, the average fluorescence intensity within the 96-well reaction chamber is extracted to generate a light intensity distribution map. This light intensity distribution map is then filtered, interpolated, and smoothed to generate an illumination unevenness template. Finally, this template is used to correct for illumination unevenness during actual result analysis. Similarly, this method can also be used to correct for image distortion around the camera body 1 when taking photos.) The goal is to obtain a more accurate amplification curve.

[0041] In some embodiments, the orifice plate 103 is a temperature equalizing plate 12, and the temperature equalizing plate 12 is provided with a plurality of sample wells for placing test tubes. The temperature equalizing plate 12 uses a heat-conducting material (such as aluminum alloy, copper) or a built-in heating / cooling module to make the temperature distribution deviation in the plate smaller. The temperature equalizing plate 12 can ensure that all samples in the test tubes reach the target temperature at the same time, avoid the difference in amplification efficiency caused by the temperature difference between the edge hole and the center hole, and improve the repeatability of the experiment. The temperature equalizing plate 12 can be integrated with a Peltier element or a water circulation system to achieve gradient temperature control (such as setting a temperature gradient of 55-65°C in the annealing stage), which is convenient for optimizing the annealing temperature of the PCR primers and reducing the experimental trial time.

[0042] In some embodiments, the fluorescent quantitative PCR instrument also includes a temperature control device and a heat sink 13. The heat sink 13 is fixedly mounted on the fixing frame 102. The temperature control device is arranged above the heat sink 13 and below the temperature equalizing plate 12, and can adjust the temperature of the temperature equalizing plate 12. The temperature control device (such as a Peltier element, a heating film) acts directly on the bottom of the temperature equalizing plate 12. Combined with the heat conduction capacity of the heat sink 13, the temperature control accuracy of the temperature equalizing plate 12 can be improved. For example, in the annealing stage, the temperature consistency of the entire plate ensures that the primer binding efficiency of all samples is the same, which is suitable for experiments with high precision requirements. The heat sink 13 (such as an aluminum alloy fin structure) is fixed on the fixing frame 102, which can absorb the heat fluctuations when the temperature control device is working and avoid the temperature of the temperature equalizing plate 12 from overshooting. For example, in a high-throughput test that runs continuously for 10 hours, the temperature drift of the temperature equalizing plate 12 is ≤0.2°C, ensuring the repeatability of the results of long-term experiments.

[0043] In some embodiments, the temperature control component is a thermal electric cooler (TEC device 11). TEC device 11 is an electronic device that exhibits the Peltier effect. Changing the direction of the current flowing through TEC device 11 can achieve switching between heating and cooling. When a forward current is applied, the front surface of TEC device 11 begins to heat, significantly shortening the time required to reach the target temperature.

[0044] During the thermal cycle, the change from low temperature to high temperature can be achieved by a heater; the change from high temperature to low temperature can be achieved by either active cooling through a refrigerator or fan, or by stopping the heater and allowing the sample to cool naturally to achieve passive cooling. To meet the requirements of rapid temperature changes, this embodiment adopts an active cooling solution. Changing the direction of the current flowing to the TEC device 11 can achieve active heating and cooling conversion. When the voltage polarity applied to the two ends of the TEC device 11 is changed, the current flow direction is reversed, and the cold end of the TEC device 11 becomes the hot end, and the hot end becomes the cold end. Therefore, temperature fluctuation can be achieved by controlling the voltage polarity in this way.

[0045] Therefore, a closed-loop temperature cycle system based on PID (Proportional Integral Derivative) control can be established using the TEC device 11. First, a target temperature is set. The TEC device 11 uses a temperature sensor (e.g., Pt100, NTC thermistor, etc.) to detect the temperature of the 96-well plate. The temperature sensor data is transmitted in real time to the TEC device 11's control circuit, which regulates the TEC device's power.

[0046] The PID algorithm works by independently adjusting the values ​​of the proportional, integral, and differential terms to achieve rapid temperature conversion and stable output. The proportional term primarily controls the average output power of the TEC device 11 by switching it on and off for short periods of time. When the actual temperature approaches the setpoint, the proportional controller reduces the average power of the TEC device 11, slowing the heating and cooling rates to keep the actual temperature close to the setpoint and stable. The integral term corrects errors introduced by the proportional term during operation, acting as a correction factor. The differential term adjusts overshoot caused by the proportional term. The settings of the proportional, integral, and differential terms are typically adjusted independently based on experience. First, set the integral and differential terms to zero, then adjust the proportional term until the loop output begins to oscillate. Finally, fine-tune the integral and differential terms to stabilize the system. The PID controller in a temperature control system uses a resistance temperature sensor as input and, by comparing the actual temperature with the setpoint, combines PWM technology to adjust the actual output power of the TEC device 11.

[0047] PWM technology controls the average voltage (and current) across a load by rapidly opening and closing the switch between the power supply and the load. The longer the switch is on compared to when it's off, the higher the total power delivered to the load. PWM controls the load using digital signals, making it more resistant to noise than analog signals. Therefore, this embodiment utilizes PWM technology to control the operation of the TEC device 11. Adjusting the duty cycle of the digital periodic signal adjusts the duration of full-power operation of the TEC device 11.

[0048] In some embodiments, the fluorescent quantitative PCR instrument also includes a temperature sensor, which is arranged on the temperature plate 12. The temperature sensor is electrically connected to the control system of the temperature control device, and the control system can regulate the temperature control device according to the output signal of the temperature sensor. The temperature sensor is directly attached to the surface of the temperature plate 12 (or built into it), collects temperature data in real time, and feeds back to the control system. For example, when the actual temperature of the temperature plate 12 deviates from the set value by more than a preset value, the control system immediately adjusts the current of the temperature control device (such as a semiconductor refrigerator), forming a monitoring-feedback-regulation closed loop, controlling the temperature fluctuation within the preset value range, and ensuring that the temperature of each stage of PCR strictly matches the experimental requirements. If traditional temperature control relies only on sensors near the temperature control device, the actual temperature deviation may be caused by the delay of heat conduction of the temperature plate 12 (such as the temperature difference between the edge hole and the center hole). The temperature sensor on the temperature plate 12 directly monitors the temperature of the sample area and can compensate for the heat conduction delay in real time. Real-time feedback from the temperature sensor can avoid overshoot caused by inertial heating / cooling of the temperature control device.

[0049] In some embodiments, the fluorescence quantitative PCR instrument also includes a heat cover 10, which can cover the top of the temperature equalizing plate 12 and be sealed. After the heat cover 10 seals the temperature equalizing plate 12, a closed space is formed. The heat cover has a certain temperature with heat, which can prevent the liquid in the test tube from condensing on the test tube cover. Generally, each test tube is sealed. If there is no heat cover during heating, water vapor will condense on the test tube cover. After adding the heat cover, this problem will not occur due to the higher temperature of the heat cover. In addition, the heat cover 10 can prevent the influence of liquid evaporation on the experiment to a certain extent, prevent water evaporation, increase reagent concentration, and cause the problem of fluorescence signal deviation. For example, after 40 PCR cycles, the volume of the reaction solution in the coverless design may be reduced, resulting in increased reagent concentration and fluorescence signal deviation, while the sealed heat cover 10 can maintain a constant volume and ensure the accuracy of fluorescence quantification. The heat cover and the temperature equalizing plate 12 are directly fixed together with screws, and the contact part of the heat cover and the temperature equalizing plate 12 is separated by a plastic gasket to reduce unnecessary heat loss. The temperature equalizing plate 12 is then connected to the radiator with screws and separated by a gasket. At the same time, 6 TEC devices 11 are set up, and the upper surface of each TEC device 11 is coated with thermal grease and must be in contact with the temperature plate 12. At the same time, the lower surface of the TEC device 11 is coated with thermal grease and is in contact with the heat sink.

[0050] The present invention uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only intended to help understand the method and core concept of the present invention. At the same time, those skilled in the art will find that the specific implementation methods and application scopes may vary based on the concept of the present invention. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims

1. A fluorescence quantitative PCR instrument, characterized in that: It includes a fixing frame and multiple camera detection components, a well plate containing nucleic acid samples is placed on the fixing frame, and multiple camera detection components are arranged on the fixing frame and can illuminate each sample well on the well plate, wherein each camera detection component includes a light generating device, a camera body and a filter, the camera body is used to photograph the sample in the sample well, the light generating device is arranged on the camera body, the light generating device is used to emit light and illuminate the sample well, the filter is arranged between the light generating device and the well plate, and can filter the light emitted by the light generating device, and the filters connected to each camera body are different and can filter light of different wavelengths.

2. The fluorescence quantitative PCR instrument according to claim 1, characterized in that: It also includes a plurality of light reflecting devices, which are arranged on the fixing frame and can reflect the fluorescence of the sample hole after being irradiated to the camera detection component.

3. The fluorescence quantitative PCR instrument according to claim 2, characterized in that: The light reflecting device is a plane mirror, and is provided with two plane mirrors, namely a first plane mirror and a second plane mirror. The first plane mirror and the second plane mirror are both rotatably set on the fixed frame, and the first plane mirror is tilted above the hole plate, and the second plane mirror is tilted on the opposite side of the camera detection component. The fluorescence in the sample hole of the hole plate can be reflected by the first plane mirror and the second plane mirror in turn into the camera detection component.

4. The fluorescence quantitative PCR instrument according to claim 1, wherein: It also includes a mounting plate, and multiple camera detection components are evenly distributed on both sides of the mounting plate and fixedly connected, and the camera detection components are tilted on the mounting plate to ensure that each camera detection component can receive a complete image of the orifice plate.

5. The fluorescence quantitative PCR instrument according to claim 1, characterized in that: It also includes a fixing box and a first extension tube, the light generating device is an LED lamp, the fixing box is hollow and has openings on three sides, namely the first opening, the second opening and the third opening. A dichroic mirror is provided inside the fixing box, the first opening and the second opening are arranged opposite to each other, and the first opening is used to install the camera body, the second opening is used to install the filter, and the third opening is arranged on the adjacent side of the first opening. The two ends of the first extension tube are respectively connected to the LED lamp and the third opening. After the light of the LED lamp is emitted, it can pass through the first extension tube and be reflected by the dichroic mirror and emitted through the second opening.

6. The fluorescence quantitative PCR instrument according to claim 1, characterized in that: The orifice plate is a temperature-equalizing plate, and a plurality of sample holes for placing test tubes are provided on the temperature-equalizing plate.

7. The fluorescence quantitative PCR instrument according to claim 6, characterized in that: It also includes a temperature control device and a heat sink. The heat sink is fixed on the fixing frame. The temperature control device is arranged above the heat sink and below the temperature averaging plate, and can adjust the temperature of the temperature averaging plate.

8. The fluorescence quantitative PCR instrument according to claim 7, characterized in that: The temperature control component is a semiconductor refrigerator.

9. The fluorescence quantitative PCR instrument according to claim 7, characterized in that: It also includes a temperature sensor, which is arranged on the temperature equalizing plate. The temperature sensor is electrically connected to the control system of the temperature control device. The control system can regulate the temperature control device according to the output signal of the temperature sensor.

10. The fluorescence quantitative PCR instrument according to claim 6, characterized in that: A heat cover is also included, which can cover the top of the temperature distribution plate and be sealed.

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