Combined fluorescent quantitative PCR (Polymerase Chain Reaction) device and use method thereof

Abstract

The invention discloses a combined fluorescent quantitative PCR device and a use method thereof, and relates to the technical field of microfluidics in biomolecular detection.The combined fluorescent quantitative PCR device comprises a detection substrate, a PCR plate and a reaction membrane assembly, the PCR plate is installed on the detection substrate, and a reaction tank structure is formed at the top of the PCR plate; the reaction membrane assembly covers the PCR plate; the reaction membrane assembly comprises a fixed membrane and a flexible membrane, the flexible membrane is arranged between the fixed membrane and the reaction tank structure, and a reaction gap for a to-be-detected sample to flow is formed between the fixed membrane and the flexible membrane; wherein the flexible film can sink into the reaction tank structure and be attached to the reaction tank structure or protrude out of the tank opening to be separated from the reaction tank structure. By adopting the detachable PCR plate and the reaction membrane assembly, reaction parts are convenient to replace and clean, and cross contamination among samples is avoided.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics in biomolecule detection, and in particular to a combined fluorescent quantitative PCR device and a method for using the same. Background Art

[0003] With the rapid development of biotechnology, polymerase chain reaction (PCR) technology has been widely used in molecular biology and diagnosis due to its high sensitivity and specificity. As an important variant of PCR technology, fluorescent quantitative PCR (qPCR) technology realizes real-time monitoring and quantitative analysis of the PCR amplification process by introducing fluorescent dyes or probes into the reaction system, greatly improving the sensitivity and accuracy of nucleic acid detection.

[0004] At present, qPCR technology is usually operated using dedicated qPCR instruments. These instruments generally include key components such as PCR reaction modules, fluorescence detection systems, and temperature control systems. When conducting qPCR experiments, operators need to add the prepared reaction mixture to the reaction wells of the PCR reaction plate, and then place the reaction plate into the qPCR instrument to achieve amplification and quantitative analysis of the target nucleic acid by setting appropriate temperature cycle programs and fluorescence detection parameters. For the detection of multiple samples, a multi-well PCR reaction plate is usually required, with each sample occupying one or more reaction wells.

[0005] However, existing qPCR technology has some limitations in multi-sample detection. Traditional qPCR equipment usually uses fixed PCR reaction plates or disposable PCR chips. When testing multiple samples, the reaction plates need to be frequently cleaned or replaced to avoid cross-contamination between samples. This not only increases the operational complexity and time cost of the experiment, but also poses a risk of contamination, affecting the reliability of the test results. Summary of the invention

[0007] The main purpose of the present invention is to provide a combined fluorescent quantitative PCR device and a method of using the same, aiming to simplify the operating procedures of PCR experiments, reduce experimental costs, and improve the accuracy and reliability of detection results.

[0008] To achieve the above-mentioned purpose, the combined fluorescent quantitative PCR device proposed in the present invention comprises: Detection substrate; A PCR plate, the PCR plate is detachably mounted on the detection substrate, the top of the PCR plate is concave downward to form a reaction slot structure with a notch facing upward; A reaction membrane assembly, wherein the reaction membrane assembly is detachably mounted on the detection substrate and covered on the PCR plate; the reaction membrane assembly comprises a fixed membrane and a flexible membrane, wherein the flexible membrane is arranged between the fixed membrane and the reaction tank structure, and a reaction gap is provided between the fixed membrane and the flexible membrane for the flow of the sample to be tested; Among them, a vent hole is opened on the bottom wall of the reaction tank structure opposite to the notch, and the vent hole is connected to an external air pump through an air channel in the PCR plate. The air pump is used to suck the gas in the reaction tank structure through the air channel and the vent hole or discharge the gas into the reaction tank structure, and correspondingly makes the flexible membrane concave into the reaction tank structure and adhere to the reaction tank structure or bulge out of the notch to detach from the reaction tank structure.

[0009] In one embodiment, the reaction membrane assembly further comprises a membrane frame and an injection port, wherein the membrane frame is sleeved outside the fixed membrane and the flexible membrane, the membrane frame is detachably mounted on the detection substrate, and a injection port communicating with the reaction gap is disposed on the top of the fixed membrane.

[0010] In one embodiment, a sealing groove is provided on the PCR plate, a sealing ring is provided at a position of the film frame corresponding to the sealing groove, and the sealing ring frame is provided at the periphery of the flexible film.

[0011] In one embodiment, the reaction tank structure includes an injection tank, a reaction flow channel tank and a fluorescence detection tank; the reaction membrane assembly also includes a guide block, and the guide block is arranged on the side of the flexible membrane facing the PCR plate corresponding to the positions of the injection tank, the reaction flow channel tank and the fluorescence detection tank.

[0012] In one embodiment, there are multiple guide blocks, and the multiple guide blocks are arranged at intervals along the extension direction of the reaction tank structure.

[0013] In one embodiment, there are a plurality of ventilation holes, and the plurality of ventilation holes are arranged at intervals along the extension direction of the reaction tank structure. The number of the ventilation holes is consistent with the number of the guide blocks and are arranged in a one-to-one correspondence.

[0014] In one embodiment, the bottom of the PCR plate is protruded downward to form a plurality of heat-conducting blocks, and the heat-conducting blocks are arranged corresponding to the positions of the reaction tank structure.

[0015] In one embodiment, the detection substrate is formed with a water bath, the water bath is arranged corresponding to the position of the heat conductive block, the heat conductive block extends into and is arranged in the water bath, and the water bath is connected to an external water bath system through a water pipe.

[0016] The present invention also provides a method for using a combined fluorescent quantitative PCR device, using the combined fluorescent quantitative PCR device as described above; the method for using the combined fluorescent quantitative PCR device comprises: Installing the PCR plate on the detection substrate, installing the reaction membrane assembly on the detection substrate and covering the top of the PCR plate; Starting the air pump to adjust the gas environment in the reaction tank structure through the air channel and the vent hole, so that the flexible membrane is deformed and recessed into the reaction tank structure, so that the flexible membrane adheres to the inner wall of the reaction tank structure; Injecting the sample to be tested into the reaction gap, and starting the temperature control system to allow the sample to be tested to undergo an amplification reaction within a preset temperature range; During the reaction, the fluorescence signal of the sample to be tested in the reaction gap is monitored in real time by an optical detection system and quantitatively analyzed to obtain a quantitative result of the sample to be tested; After use, the reaction membrane assembly is replaced to prepare for the next round of experiments.

[0017] In one embodiment, the step of starting the air pump, adjusting the gas environment in the reaction tank structure through the air channel and the vent hole, causing the flexible membrane to deform and sink into the reaction tank structure, and causing the flexible membrane to adhere to the inner wall of the reaction tank structure includes: Starting the air pump, and when the flexible membrane begins to sag, gradually increasing the suction pressure of the air pump with a preset pressure gradient; wherein the preset pressure gradient is 10 mbar to 20 mbar; After the flexible film is attached to the inner wall of the reaction tank structure, the air pump is adjusted to maintain the pressure state.

[0018] The technical solution of the present invention uses a detachable PCR plate and reaction membrane assembly, and adjusts the position of the flexible membrane in combination with a pneumatic control system, so that the flexible membrane can be flexibly recessed into the reaction tank or protruded outward according to experimental needs, thereby achieving effective isolation and flow control of the sample to be tested. In addition, since both the PCR plate and the reaction membrane assembly are detachable, they are easy to clean and replace, which significantly reduces the risk of cross-contamination between samples and simplifies experimental operations. Not only does it improve the flexibility and reliability of qPCR detection, but it also avoids complex mechanical operations through pneumatic control, ensuring the stability and reliability of the device during use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 the structures shown in these drawings without paying creative work.

[0021] Figure 1 A schematic diagram of the structure of an embodiment of a combined fluorescent quantitative PCR device provided by the present invention; Figure 2 A schematic diagram of the explosion structure of an embodiment of a combined fluorescent quantitative PCR device provided by the present invention; Figure 3 A schematic diagram of the explosion structure of another embodiment of the combined fluorescent quantitative PCR device provided by the present invention; Figure 4 A schematic diagram of the structure of an embodiment of a PCR plate provided by the present invention; Figure 5 A schematic structural diagram of an embodiment of a reaction membrane assembly provided by the present invention; Figure 6 A schematic diagram of the explosion structure of an embodiment of a reaction membrane assembly provided by the present invention; Figure 7 The present invention provides a schematic flow chart of an embodiment of a method for using the combined fluorescent quantitative PCR device.

[0022] Description of Figure Numbers: 100, detection substrate; 200, PCR plate; 210, reaction tank structure; 201, vent; 202, airway; 203, sealing groove; 211, injection groove; 212, reaction flow channel groove; 213, fluorescence detection groove; 220, heat conduction block; 110, water bath; 120, water pipe; 300, reaction membrane assembly; 310, fixed membrane; 320, flexible membrane; 301, reaction gap; 330, membrane frame; 340, injection port; 400, sealing ring; 350, guide block.

[0023] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0026] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly.

[0027] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0028] At present, qPCR technology is usually operated using dedicated qPCR instruments. These instruments generally include key components such as PCR reaction modules, fluorescence detection systems, and temperature control systems. When conducting qPCR experiments, operators need to add the prepared reaction mixture to the reaction wells of the PCR reaction plate, and then place the reaction plate into the qPCR instrument to achieve amplification and quantitative analysis of the target nucleic acid by setting appropriate temperature cycle programs and fluorescence detection parameters. For the detection of multiple samples, a multi-well PCR reaction plate is usually required, with each sample occupying one or more reaction wells.

[0029] However, existing qPCR technology has some limitations in multi-sample detection. Traditional qPCR equipment usually uses fixed PCR reaction plates or disposable PCR chips. When testing multiple samples, the reaction plates need to be frequently cleaned or replaced to avoid cross-contamination between samples. This not only increases the operational complexity and time cost of the experiment, but also poses a risk of contamination, affecting the reliability of the test results.

[0030] In order to solve this technical problem, the present invention provides a combined fluorescent quantitative PCR device and a method for using the same.

[0031] See also Figures 1 to 6In one embodiment of the present invention, the combined fluorescent quantitative PCR device includes a detection substrate 100, a PCR plate 200 and a reaction membrane assembly 300. The PCR plate 200 is detachably mounted on the detection substrate 100. The top of the PCR plate 200 is recessed downward to form a reaction tank structure 210 with a notch facing upward. The reaction membrane assembly 300 is detachably mounted on the detection substrate 100 and is covered on the PCR plate 200. The reaction membrane assembly 300 includes a fixed membrane 310 and a flexible membrane 320. The flexible membrane 320 is disposed between the fixed membrane 310 and the reaction tank structure 210, and the fixed membrane 310 is provided with a plurality of flexible membranes. A reaction gap 301 is provided between the membrane 310 and the flexible membrane 320 for the flow of the sample to be tested; wherein, a vent hole 201 is provided on the bottom wall of the reaction tank structure 210 opposite to the notch, and the vent hole 201 is connected to an external air pump through an air channel 202 in the PCR plate 200, and the air pump is used to suck the gas in the reaction tank structure 210 through the air channel 202 and the vent hole 201 or discharge the gas into the reaction tank structure 210, and correspondingly makes the flexible membrane 320 concave into the reaction tank structure 210 and adhere to the reaction tank structure 210 or bulge out of the notch to detach from the reaction tank structure 210.

[0032] Specifically, the combined fluorescent quantitative PCR device of the present invention includes a detection substrate 100, a PCR plate 200 and a reaction membrane assembly 300. Among them, the PCR plate 200 is detachably mounted on the detection substrate 100, and the top of the PCR plate 200 is recessed downward to form a reaction tank structure 210 with a notch upward, which is used to accommodate the sample to be tested for PCR reaction. The reaction membrane assembly 300 is detachably mounted on the detection substrate 100 and covered on the PCR plate 200. The reaction membrane assembly 300 includes a fixed film 310 and a flexible film 320, and the fixed film 310 is arranged above the flexible film 320, and the flexible film 320 is arranged between the fixed film 310 and the reaction tank structure 210, and there is a reaction gap 301 for the sample to be tested to flow between the fixed film 310 and the flexible film 320. Therefore, the sample to be tested can undergo a PCR reaction in the reaction gap 301, and the flexible film 320 can contact or separate with the reaction tank structure 210 as needed to achieve sample isolation and flow control.

[0033] More specifically, a vent hole 201 is provided on the bottom wall of the reaction tank structure 210 opposite to the notch, and the vent hole 201 is connected to an external air pump through an air channel 202 in the PCR plate 200. The air pump can suck the gas in the reaction tank structure 210 or discharge the gas into the reaction tank structure 210 through the air channel 202 and the vent hole 201, thereby controlling the deformation of the flexible membrane 320. When the air pump sucks the gas in the reaction tank structure 210, the flexible membrane 320 will be concave into the reaction tank structure 210 and adhere to the inner wall of the reaction tank structure 210 under the action of the air pressure difference. At this time, the sample to be tested is confined in the reaction gap 301 for PCR reaction. When it is necessary to replace the sample or clean it, the air pump can discharge gas into the reaction tank structure 210, so that the flexible membrane 320 protrudes out of the notch under the action of air pressure and detaches from the reaction tank structure 210, which is convenient for taking out and cleaning the sample. This pneumatic control method can flexibly adjust the position of the flexible membrane 320 to achieve sample isolation and flow, thereby avoiding the risk of cross contamination.

[0034] In addition, under the same conditions, the reaction membrane assembly 300 used in the present invention has obvious advantages over the disposable PCR chip in terms of manufacturing process and cost. First, from the perspective of material selection, the reaction membrane assembly 300 is mainly composed of two parts: a fixed membrane 310 and a flexible membrane 320. These two membrane materials can be made of high molecular polymers, such as polypropylene, polyester, etc., which are relatively cheap, widely available, and have excellent processing performance. In contrast, disposable PCR chips usually use silicon-based materials, such as silicon wafers or glass substrates. These materials are relatively expensive, and the processing technology is relatively complex, requiring professional micro-processing equipment and a clean environment, resulting in a significant increase in production costs. Secondly, from the perspective of production technology, the production of the reaction membrane assembly 300 mainly involves conventional operations such as cutting and bonding of membrane materials. These processes can be achieved through existing mature technologies, such as laser cutting, hot pressing bonding, etc., with high production efficiency and a high degree of automation, and no complex micro-nano processing steps are required. The production of disposable PCR chips usually requires the use of micro-processing processes such as photolithography, etching, and bonding. These processes have high requirements for equipment and environment, are complex to operate, and are prone to introduce defects or pollution, resulting in a decrease in yield and an increase in production costs. Furthermore, from the perspective of functional integration, the structure of the reaction membrane assembly 300 is relatively simple, mainly forming a reaction gap 301 between the fixed membrane 310 and the flexible membrane 320, and the processing requirements for the membrane material are relatively low. The disposable PCR chip needs to integrate multiple functional units such as temperature control and fluorescence detection on the silicon substrate, which requires complex microfluidic channel design and surface modification, greatly increasing the difficulty and cost of the manufacturing process. Finally, from the perspective of the economy of batch production, the reaction membrane assembly 300 adopts a roll-to-roll continuous production mode, which can greatly improve production efficiency and reduce unit cost. The disposable PCR chip adopts a batch processing mode of silicon wafers, with relatively low production efficiency and high unit cost, especially in small batch production, which is even more expensive. Therefore, the reaction membrane assembly 300 proposed in the present invention reduces the implementation cost of microfluidic technology in biomolecule detection relative to the disposable PCR chip.

[0035] In the technical solution provided by the present invention, by adopting a detachable PCR plate 200 and a reaction membrane assembly 300, and combining a pneumatic control system to adjust the position of the flexible membrane 320, the flexible membrane 320 can be flexibly recessed into the reaction tank or protruded outward according to experimental needs, thereby achieving effective isolation and flow control of the sample to be tested. In addition, since both the PCR plate 200 and the reaction membrane assembly 300 are detachable, they are easy to clean and replace, which significantly reduces the risk of cross-contamination between samples and simplifies experimental operations. Not only does it improve the flexibility and reliability of qPCR detection, but also avoids complex mechanical operations through pneumatic control, ensuring the stability and reliability of the device during use.

[0036] As an optional embodiment, the reaction membrane assembly 300 can be made of PDSM material, which has good biocompatibility, light transmittance and flexibility, and is suitable for PCR reaction. At the same time, the hardness of the fixed membrane 310 can be greater than that of the flexible membrane 320, so that under the action of air pressure, the flexible membrane 320 is easily deformed while the fixed membrane 310 remains stable, thereby preventing the fixed membrane 310 from sinking and affecting the flow and reaction of the sample to be detected.

[0037] Please continue reading Figure 5 and Figure 6 In an embodiment of the present invention, the reaction membrane assembly 300 further includes a membrane frame 330 and an injection port 340. The membrane frame 330 is sleeved outside the fixed membrane 310 and the flexible membrane 320. The membrane frame 330 is detachably mounted on the detection substrate 100. The top of the fixed membrane 310 is provided with an injection port 340 connected to the reaction gap 301.

[0038] Specifically, the membrane frame 330 serves as a supporting structure of the reaction membrane assembly 300, fixing the fixed membrane 310 and the flexible membrane 320 together to form a stable reaction gap 301. The membrane frame 330 is detachably mounted on the detection substrate 100, which facilitates the replacement and cleaning of the reaction membrane assembly 300 and improves the maintainability of the device. At the same time, the sample inlet 340 is arranged on the top of the fixed membrane 310 and communicates with the reaction gap 301, providing a unique inlet and outlet channel for the sample to be tested, which can effectively prevent sample leakage or contamination and ensure the safety and reliability of the experimental process.

[0039] As an optional embodiment, the injection port 340 adopts a conical structure, with a larger opening at the upper end and a smaller opening at the lower end connected to the reaction gap 301. This facilitates the addition of the sample to be tested, while reducing the connection area between the injection port 340 and the reaction gap 301, further reducing the risk of sample leakage. In addition, a sealing cover can be provided at the upper end of the injection port 340 to seal it after the sample is added, further improving the safety of the experimental process.

[0040] Please continue reading Figure 4 and Figure 5In an embodiment of the present invention, a sealing groove 203 is provided on the PCR plate 200, and a sealing ring 400 is provided at a position of the film frame 330 corresponding to the sealing groove 203, and the sealing ring 400 is framed on the periphery of the flexible film 320. Specifically, the sealing groove 203 is located on the surface of the PCR plate 200, and its shape matches the sealing ring 400 of the film frame 330. By providing the sealing groove 203 on the PCR plate 200 and configuring the sealing ring 400 at a corresponding position on the film frame 330, the sealing effect between the PCR plate 200 and the reaction membrane assembly 300 is enhanced. When the film frame 330 is installed on the PCR plate 200, the sealing ring 400 is just embedded in the sealing groove 203, and an effective sealing barrier is formed through physical matching. It can not only prevent the leakage of samples during the reaction, but also prevent external contaminants from entering the reaction area.

[0041] At the same time, the periphery of the flexible membrane 320 is fixed by the sealing ring 400. When operated by the pneumatic system, the flexible membrane 320 can be recessed downward or convex upward, thereby forming an adaptation or separation with the reaction tank structure 210. When the flexible membrane 320 is recessed to form a sealed state, the sealing ring 400 around it ensures the sealing between the PCR plate 200, effectively preventing cross contamination, and maintaining the stability of the reaction environment.

[0042] Please continue reading Figure 4 In an embodiment of the present invention, the reaction tank structure 210 includes an injection tank 211, a reaction flow channel tank 212 and a fluorescence detection tank 213; the reaction membrane assembly 300 also includes a guide block 350, and the guide block 350 is arranged on the side of the flexible membrane 320 facing the PCR plate 200 corresponding to the positions of the injection tank 211, the reaction flow channel tank 212 and the fluorescence detection tank 213.

[0043] Specifically, when the air pump draws gas from the reaction tank structure 210, the flexible membrane 320 is sunken into the reaction tank structure 210 under the action of the air pressure difference. At this time, the guide block 350 provided on the flexible membrane 320 can accurately correspond to the injection slot 211, the reaction channel slot 212 and the fluorescence detection slot 213, ensuring that the flexible membrane 320 is closely fitted to the inner wall of the reaction tank structure 210 during the sunken process, forming a tank structure adapted to the reaction tank structure 210. The design of the guide block 350 can significantly improve the flatness and consistency of the tank structure formed after the flexible membrane 320 is sunken, avoiding the dead volume or bubble problem caused by the uneven deformation of the flexible membrane 320, thereby improving the efficiency and reliability of the PCR reaction.

[0044] As an optional embodiment, the guide block 350 is made of the same material as the flexible film 320, such as dimethylsiloxane (PDMS), which can not only withstand the high temperature during the PCR process, but also maintain good contact with the flexible film 320, without affecting the PCR reaction, so as to ensure the integrity and flexibility of the guide block 350 and the flexible film 320. At the same time, the height and shape of the guide block 350 match the injection slot 211, the reaction channel slot 212 and the fluorescence detection slot 213 to achieve the best spatial adaptation effect. The selection of this material and structure not only improves the functionality of the guide block 350, but also reduces the manufacturing cost and facilitates mass production.

[0045] Please continue reading Figure 5 and Figure 6 In an embodiment of the present invention, there are multiple guide blocks 350 , and the multiple guide blocks 350 are arranged at intervals along the extension direction of the reaction tank structure 210 .

[0046] Specifically, the setting of the guide block 350 enables the flexible membrane 320 to be accurately recessed into the designated reaction tank structure 210 under pneumatic control. Each guide block 350 corresponds to a specific reaction area, such as the injection tank 211, the reaction flow channel tank 212 or the fluorescence detection tank 213. By adjusting the size of the guide block 350, the size of the tank body formed after the flexible membrane 320 is recessed can be controlled, thereby accurately controlling the volume of the sample to be tested in the reaction tank, directly affecting the efficiency and reaction time of the PCR reaction. For example, a larger guide block 350 can be used to increase the reaction volume, which is suitable for PCR steps that require a long reaction time, while a smaller guide block 350 is suitable for rapid reactions or the processing of a small amount of samples.

[0047] In addition, by adjusting the distance between two adjacent guide blocks 350, the corresponding groove formed by the depression of the flexible membrane 320 can be further flattened to avoid the formation of uneven bubbles or dead corners in the groove, which is crucial to ensure the uniformity and repeatability of the PCR reaction. A uniform reaction environment helps to improve the predictability of the reaction and the reliability of the results.

[0048] Please continue reading Figure 4 In an embodiment of the present invention, there are multiple vent holes 201, which are spaced apart along the extension direction of the reaction tank structure 210. The number of the vent holes 201 is consistent with the number of the guide blocks 350 and are arranged one-to-one.

[0049] Specifically, the position selection of the vent hole 201 is closely related to the layout of the reaction tank structure 210. For example, in the serpentine reaction channel groove 212, the vent hole 201 is usually arranged at the corner of the reaction channel groove 212 and the connection between the reaction channel groove 212 and the injection groove 211 and the fluorescence detection groove 213. Such an arrangement can ensure that the gas pressure is evenly distributed in the entire reaction tank structure 210, avoiding the situation where the local pressure is too high or too low, thereby ensuring that the flexible membrane 320 achieves synchronous and stable deformation in various regions.

[0050] In addition, by arranging the vent holes 201 to correspond to the guide blocks 350 one by one, independent control of each reaction area can be achieved. When the air pump draws gas from a specific vent hole 201, the flexible membrane 320 above the corresponding guide block 350 will preferentially dent, forming a groove body that matches the reaction groove structure 210 in the area. This precise positioning pneumatic control method can significantly improve the controllability and repeatability of the reaction conditions, which is conducive to obtaining high-quality PCR amplification results.

[0051] As an optional embodiment, the diameter and spacing of the vent holes 201 can be adjusted according to the specific requirements of the PCR reaction. A smaller diameter of the vent holes 201 helps to improve the uniformity of the air pressure distribution, while a larger spacing of the vent holes 201 can avoid interference between adjacent areas. By balancing these two parameters, the best design of the vent holes 201 can be found to meet the needs of different PCR experiments.

[0052] Please continue reading Figure 3 In the embodiment of the present invention, the bottom of the PCR plate 200 is convex downward to form a plurality of heat-conducting blocks 220, and the heat-conducting blocks 220 are arranged corresponding to the positions of the reaction groove structure 210. (As an embodiment, the plurality of heat-conducting blocks 220 are arranged corresponding to the positions of the reaction flow channel groove 212) Specifically, PCR reaction has strict requirements for precise temperature control. In traditional PCR equipment, due to the limited thermal conductivity of the reaction plate material, the temperature distribution in the reaction well is uneven, which affects the amplification effect. In this embodiment, by setting a heat-conducting block 220 corresponding to the reaction tank structure 210 at the bottom of the PCR plate 200, heat can be quickly and evenly transferred to the reaction tank structure 210, ensuring that the reaction solution reaches the same target temperature in each area.

[0053] As an optional implementation, the heat conductive block 220 is made of a material with high thermal conductivity, such as copper, aluminum and other metals.

[0054] As another optional embodiment, the number and position of the heat conductive blocks 220 can be designed according to the specific layout of the reaction tank structure 210. For example, a plurality of heat conductive blocks 220 are arranged at positions corresponding to the reaction flow channel groove 212. Since the reaction flow channel groove 212 is the main occurrence area of ​​the PCR amplification reaction, arranging the heat conductive blocks 220 at these positions can maximize their temperature control effect and help obtain high-quality amplification products.

[0055] In addition, the size and shape of the heat conductive block 220 can also be optimized to adapt to different reaction systems and temperature control requirements. Smaller heat conductive blocks 220 can provide more precise temperature regulation, while larger heat conductive blocks 220 are conducive to improving the temperature uniformity of the entire reaction tank structure 210. The connection method between the heat conductive block 220 and the PCR plate 200 can also be selected according to actual conditions, such as using adhesive fixing, screw fastening, etc., to ensure close contact and efficient heat transfer between the heat conductive block 220 and the PCR plate 200.

[0056] Please continue reading Figure 2 and Figure 3 In an embodiment of the present invention, the detection substrate 100 is formed with a water bath 110, and the water bath 110 is arranged corresponding to the position of the heat conductive block 220, and the heat conductive block 220 extends into and is arranged in the water bath 110, and the water bath 110 is connected to the external water bath system through the water pipe 120.

[0057] Specifically, the role of the heat conductive block 220 in the PCR plate 200 is to optimize the temperature uniformity of the PCR reaction by enhancing heat transfer. By extending the heat conductive block 220 into the water bath 110, the temperature adjustment function of the water bath system can be used to achieve accurate temperature control of the heat conductive block 220 and the adjacent PCR reaction area. Water, as a heat transfer medium, has good thermal conductivity and heat capacity, and can quickly and evenly adjust the temperature of the heat conductive block 220 to ensure that the temperature during the PCR reaction accurately reaches the set value.

[0058] Compared with traditional direct heating methods, water bath heating can distribute heat more evenly, avoiding local overheating or uneven heating problems. In addition, through the connection of the external water bath system, a wider range of temperature control can be achieved to meet the needs of different types of PCR experiments, including PCR reactions with long time or special temperature curves.

[0059] Please continue reading Figures 1 to 6 , and see Figure 7 The present invention also provides a method for using a combined fluorescent quantitative PCR device, using the combined fluorescent quantitative PCR device as described above; the method for using the combined fluorescent quantitative PCR device comprises: Step S10, installing the PCR plate 200 on the detection substrate 100, installing the reaction membrane assembly 300 on the detection substrate 100 and covering the top of the PCR plate 200; Step S20, starting the air pump to adjust the gas environment in the reaction tank structure 210 through the air channel 202 and the vent hole 201, so that the flexible membrane 320 is deformed and sunken into the reaction tank structure 210, so that the flexible membrane 320 is attached to the inner wall of the reaction tank structure 210; Step S30, injecting the sample to be tested into the reaction gap 301, and starting the temperature control system to allow the sample to be tested to undergo an amplification reaction within a preset temperature range; Step S40: During the reaction process, the fluorescent signal of the sample to be tested in the reaction gap 301 is monitored in real time by an optical detection system and quantitatively analyzed to obtain a quantitative result of the sample to be tested; Step S50: After use, replace the reaction membrane assembly 300 to prepare for the next round of experiments.

[0060] Specifically, first, in step S10, the PCR plate 200 is installed on the detection substrate 100, and the reaction membrane assembly 300 is installed on the detection substrate 100, so that it covers the top of the PCR plate 200. This step ensures the correct installation position of the PCR plate 200 and the reaction membrane assembly 300, laying a foundation for subsequent operations. The stable connection between the PCR plate 200 and the detection substrate 100 can effectively reduce vibration interference, and the precise alignment of the reaction membrane assembly 300 and the PCR plate 200 ensures the airtightness of the reaction space, thereby providing ideal environmental conditions for the PCR reaction.

[0061] Next, in step S20, the air pump is started to adjust the gas environment in the reaction tank structure 210 through the air channel 202 and the vent hole 201. Under the action of the air pump, the flexible membrane 320 is deformed and recessed into the reaction tank structure 210, and finally adheres to the inner wall of the reaction tank structure 210. This step utilizes the pneumatic control system to achieve precise matching of the flexible membrane 320 and the reaction tank structure 210, forming a closed reaction space, avoiding the risk of sample evaporation and cross contamination. At the same time, pneumatic control can also adjust the reaction volume as needed to meet the needs of different experiments.

[0062] In step S30, the sample to be tested is injected into the reaction gap 301, and the temperature control system (water bath system) is started to allow the sample to be tested to undergo an amplification reaction within a preset temperature range. This step introduces the sample to be tested into the reaction gap 301, and uses precise temperature control to ensure optimal conditions for the PCR reaction. The design of the heat conducting block 220 and the water bath 110 can significantly improve the uniformity and stability of the temperature, effectively avoiding the adverse effects of local overheating or temperature fluctuations on the amplification efficiency.

[0063] In step S40, the fluorescence signal of the sample to be tested in the reaction gap 301 is monitored in real time by the optical detection system, and quantitative analysis is performed to finally obtain the quantitative result of the sample to be tested. This step utilizes real-time fluorescence detection technology, and realizes quantitative analysis of the target nucleic acid by monitoring the changes in the fluorescence signal generated during the PCR reaction. The highly sensitive optical sensor and optimized optical path design can detect trace amounts of target nucleic acid, greatly improving the sensitivity and specificity of the detection.

[0064] Finally, in step S50, after use, the reaction membrane assembly 300 is replaced to prepare for the next round of experiments. The modular design of the reaction membrane assembly 300 allows it to be quickly disassembled and replaced, greatly simplifying the cleaning and maintenance process and improving the efficiency of the device. Replacing the reaction membrane assembly 300 can not only avoid cross contamination, but also select membrane assemblies of different materials or specifications as needed, thereby meeting the detection requirements of different types of samples.

[0065] It should be understood that the method for using the combined fluorescent quantitative PCR device applies the combined fluorescent quantitative PCR device as described above. The specific structure of the combined fluorescent quantitative PCR device refers to the above embodiment. Since the method for using the combined fluorescent quantitative PCR device adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0066] In an embodiment of the present invention, the step of starting the air pump, adjusting the gas environment in the reaction tank structure 210 through the air channel 202 and the vent hole 201, causing the flexible film 320 to deform and sink into the reaction tank structure 210, and causing the flexible film 320 to adhere to the inner wall of the reaction tank structure 210 includes: Step S21, starting the air pump, and when the flexible membrane 320 begins to sag, gradually increasing the suction pressure of the air pump with a preset pressure gradient; wherein the preset pressure gradient is 10 mbar to 20 mbar; Step S22 : after the flexible film 320 is attached to the inner wall of the reaction tank structure 210 , adjusting the air pump to maintain the pressure state.

[0067] Specifically, in step S21, the air pump is started, and when the flexible membrane 320 begins to sag, the suction pressure of the air pump is gradually increased with a preset pressure gradient. The preset pressure gradient is 10 mbar to 20 mbar. This step utilizes progressive pressure control to avoid the risk of excessive deformation or damage of the flexible membrane 320 under instantaneous high pressure. By slowly increasing the suction pressure, it can be ensured that the flexible membrane 320 is stably attached to the inner wall of the reaction tank structure 210 to form a uniform and closed reaction space. At the same time, the appropriate pressure gradient also helps to discharge the residual gas in the reaction tank structure 210, further optimizing the environmental conditions of the PCR reaction.

[0068] Then, in step S22, after the flexible membrane 320 is completely attached to the inner wall of the reaction tank structure 210, the air pump is adjusted to maintain the pressure state. This step ensures that the reaction space is always in a stable negative pressure environment during the entire PCR reaction process to prevent sample evaporation or cross contamination. By maintaining continuous negative pressure, the generation of bubbles can also be effectively suppressed, eliminating the interference of bubbles on fluorescent signal detection, and improving the accuracy of quantitative analysis. In addition, the stable pressure state is also conducive to maintaining a close fit between the flexible membrane 320 and the reaction tank structure 210, and good sealing can be maintained even when the temperature changes.

[0069] More specifically, the preset pressure gradient is a minimum value of 10 mbar. This is suitable for situations where the film material is relatively fragile or thin, to ensure that the flexible film 320 will not be damaged due to excessive pressure. Although this pressure gradient is small, it is sufficient to cause the flexible film 320 to begin to sink into the reaction tank structure 210 and eventually fit the inner wall. The lower pressure gradient reduces the risk of the flexible film 320 rupturing or overstretching.

[0070] The preset pressure gradient is an intermediate value of 15 mbar, which not only ensures the safety of the flexible membrane 320, but also provides sufficient pressure to ensure that the flexible membrane 320 can evenly and completely fit the inner wall of the reaction tank structure 210. Under the premise of ensuring that the flexible membrane 320 is not damaged, sufficient pressure is provided to make the membrane fully unfold and fit the inner wall. The evenly fitted flexible membrane 320 can reduce the formation of air bubbles and improve the efficiency and repeatability of the PCR reaction.

[0071] The preset pressure gradient is a maximum value of 20 mbar, which can quickly achieve the deformation and fitting of the flexible film 320. A higher pressure gradient can make the flexible film 320 dent and fit faster, shortening the time of the entire operation.

[0072] The above description is only an exemplary embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A combined fluorescent quantitative PCR device, characterized in that: include: Detection substrate; A PCR plate, wherein the PCR plate is detachably mounted on the detection substrate, and the top of the PCR plate is concave downward to form a reaction slot structure with a notch facing upward; A reaction membrane assembly, wherein the reaction membrane assembly is detachably mounted on the detection substrate and covered on the PCR plate; the reaction membrane assembly comprises a fixed membrane and a flexible membrane, wherein the flexible membrane is arranged between the fixed membrane and the reaction tank structure, and a reaction gap is provided between the fixed membrane and the flexible membrane for the flow of the sample to be tested; Among them, a vent hole is opened on the bottom wall of the reaction tank structure opposite to the notch, and the vent hole is connected to an external air pump through an air channel in the PCR plate. The air pump is used to suck the gas in the reaction tank structure through the air channel and the vent hole or discharge the gas into the reaction tank structure, and correspondingly makes the flexible membrane concave into the reaction tank structure and adhere to the reaction tank structure or bulge out of the notch to detach from the reaction tank structure.

2. The combined fluorescent quantitative PCR device according to claim 1, characterized in that: The reaction membrane assembly also includes a membrane frame and an injection port. The membrane frame is sleeved outside the fixed membrane and the flexible membrane. The membrane frame is detachably mounted on the detection substrate. The top of the fixed membrane is provided with an injection port connected to the reaction gap.

3. The combined fluorescent quantitative PCR device according to claim 2, characterized in that: The PCR plate is provided with a sealing groove, the film frame is provided with a sealing ring at a position corresponding to the sealing groove, and the sealing ring frame is provided at the periphery of the flexible film.

4. The combined fluorescent quantitative PCR device according to any one of claims 1 to 3, characterized in that: The reaction tank structure includes an injection tank, a reaction flow channel tank and a fluorescence detection tank; the reaction membrane assembly also includes a guide block, and the guide block is arranged on the side of the flexible membrane facing the PCR plate corresponding to the positions of the injection tank, the reaction flow channel tank and the fluorescence detection tank.

5. The combined fluorescent quantitative PCR device according to claim 4, characterized in that: There are multiple guide blocks, and the multiple guide blocks are arranged at intervals along the extension direction of the reaction tank structure.

6. The combined fluorescent quantitative PCR device according to claim 5, characterized in that: There are a plurality of ventilation holes, and the plurality of ventilation holes are arranged at intervals along the extension direction of the reaction tank structure. The number of the ventilation holes is consistent with the number of the guide blocks and are arranged in a one-to-one correspondence.

7. The combined fluorescent quantitative PCR device according to any one of claims 1 to 3, characterized in that: The bottom of the PCR plate is convex downward to form a plurality of heat-conducting blocks, and the heat-conducting blocks are arranged corresponding to the positions of the reaction tank structure.

8. The combined fluorescent quantitative PCR device according to claim 7, characterized in that: The detection substrate is formed with a water bath, the water bath is arranged corresponding to the position of the heat conductive block, the heat conductive block extends into and is arranged in the water bath, and the water bath is communicated with an external water bath system through a water pipe.

9. A method for using a combined fluorescent quantitative PCR device, characterized in that: Using the combined fluorescent quantitative PCR device as described in any one of claims 1 to 8; The method for using the combined fluorescent quantitative PCR device includes: Installing the PCR plate on the detection substrate, installing the reaction membrane assembly on the detection substrate and covering the top of the PCR plate; Starting the air pump to adjust the gas environment in the reaction tank structure through the air channel and the vent hole, so that the flexible membrane is deformed and recessed into the reaction tank structure, so that the flexible membrane adheres to the inner wall of the reaction tank structure; Injecting the sample to be tested into the reaction gap, and starting the temperature control system to allow the sample to be tested to undergo an amplification reaction within a preset temperature range; During the reaction, the fluorescence signal of the sample to be tested in the reaction gap is monitored in real time by an optical detection system and quantitatively analyzed to obtain a quantitative result of the sample to be tested; After use, the reaction membrane assembly is replaced to prepare for the next round of experiments.

10. The method for using the combined fluorescent quantitative PCR device according to claim 9, characterized in that: The step of starting the air pump, adjusting the gas environment in the reaction tank structure through the air channel and the vent hole, causing the flexible film to deform and sink into the reaction tank structure, and causing the flexible film to adhere to the inner wall of the reaction tank structure includes: Starting the air pump, and when the flexible membrane begins to sag, gradually increasing the suction pressure of the air pump with a preset pressure gradient; wherein the preset pressure gradient is 10 mbar to 20 mbar; After the flexible film is attached to the inner wall of the reaction tank structure, the air pump is adjusted to maintain the pressure state.

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