Self-suction multiple nucleic acid detection micro-fluidic chip

Abstract

The invention discloses a self-suction multiple nucleic acid detection micro-fluidic chip, and belongs to the field of biological detection. The device comprises a supporting layer, a reaction layer, a main channel, a branch channel, a micro reaction chamber, a central sample inlet, a regional sample inlet and a sealing layer. The reaction layer is composed of a plurality of same and independent reaction zones, and each reaction zone sequentially comprises a snake-shaped sample introduction channel, a regional sample introduction hole and a reaction zone from inside to outside from a central sample introduction hole; each reaction area comprises a plurality of micro reaction chambers and branch channels for connecting the micro reaction chambers; and a sealing layer is adhered to the surface of the chip. According to the present invention, the operation is simple, the manufacturing cost is low, the sample, the reagent, the oil phase and the like enter the chip through the self-suction manner, the loading of the sample and the multiple molecular targets is achieved through the liquid automatic directional driving, the characteristics of easy operation, full sealing, no waste liquid and multi-target simultaneous detection are provided, and the multi-target nucleic acid PCR amplification and isothermal amplification detection of the single sample or the multiple samples can be met.

Description

Technical Field

[0001] The invention belongs to the fields of biological, medical and chemical detection, and relates to a self-priming multiple nucleic acid detection microfluidic chip. Background Art

[0002] Nucleic acid testing plays a vital role in modern medicine and public health, particularly in addressing infectious diseases, bacterial infections, food safety monitoring, and personalized medicine. Common nucleic acid testing methods include polymerase chain reaction (PCR), isothermal amplification, next-generation sequencing (NGS), and microarrays. PCR, currently the most widely used nucleic acid testing method, uses specific primers to amplify target DNA fragments and uses fluorescent dyes or probes to monitor the amount of amplified product in real time.

[0003] The polymerase chain reaction (PCR) is widely used in the fields of gene variation, chromosome copy number variation, gene expression, pathogen detection, and infectious disease diagnosis due to its high sensitivity and strong specificity, and has broad application prospects. In addition, isothermal nucleic acid amplification technology (Isothermal Nucleic Acid Amplification Technology) has important applications in pathogen detection. This technology performs nucleic acid amplification at a constant temperature and does not require temperature cycling like PCR, thereby simplifying the operation process, reducing equipment requirements, and improving detection efficiency and portability. For example, recombinase polymerase amplification (RPA) reaction and loop-mediated isothermal amplification reaction (LAMP) have fast reaction speed, high sensitivity, and constant temperature reaction; isothermal amplification reaction is easy to combine with microfluidic chips and is suitable for point-of-care detection technology (POCT) of pathogenic microorganisms, and is used for rapid detection of various pathogens such as respiratory tract and intestinal tract.

[0004] In practical applications, the samples to be tested often contain multiple target nucleic acid sequences. For example, in the field of food safety, food may be contaminated by several foodborne pathogens at the same time; in clinical diagnosis, patients may also be infected with multiple pathogens at the same time. Traditional single-target detection methods require separate testing of each target, which is not only time-consuming but also increases reagent and labor costs. Multiplex nucleic acid detection can complete the screening of multiple targets in a single reaction, significantly reducing detection time, reducing costs, and improving detection efficiency. Therefore, multiplex nucleic acid detection has significant advantages.

[0005] Traditional multiplex PCR achieves the simultaneous amplification of multiple target sequences in a single reaction system, requiring the design of multiple pairs of specific primers. However, interactions or competition may exist between different primers, leading to nonspecific amplification or inefficient amplification of certain target sequences, thereby affecting the accuracy of the test results. At the same time, as the number of test targets increases, the complexity of data analysis also increases. How to accurately distinguish and quantify the amplification products of each target sequence, especially in the case of high background noise, faces huge challenges. As a new type of micro-nano droplet manipulation technology, microfluidic chips have the advantages of miniaturization, closedness, integration and automation; therefore, combining microfluidic technology with nucleic acid amplification technology to achieve rapid multiple nucleic acid detection has become a future development trend. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a self-priming multiple nucleic acid detection microfluidic chip. The chip is easy to manufacture, and samples, reagents and oil phases enter the chip through self-priming. The liquid is automatically driven in a directional manner to realize the loading of samples and multiple molecular targets. It has the characteristics of easy operation, full enclosure, no waste liquid and simultaneous detection of multiple targets, and can meet the multi-target nucleic acid PCR amplification and isothermal amplification detection of single or multiple samples.

[0007] The objective of the present invention is achieved through the following technical solutions: a self-priming multiple nucleic acid detection microfluidic chip, the chip comprising a support layer and a reaction layer, the reaction layer being placed on the support layer, and a sealing layer being attached to the upper surface of the reaction layer; the reaction layer being provided with a central injection hole and a plurality of identical and independent reaction areas, the reaction areas starting from the central injection hole and, from the inside out, being a serpentine injection channel, a regional injection hole, and a reaction area; the reaction area being connected to the central injection hole through a microchannel and being evenly distributed in a radial shape; the reaction area comprising a plurality of microreaction chambers and branch channels, the microreaction chambers being connected through branch channels, and the distances between each microreaction chamber and the corresponding regional injection hole and the central injection hole being equal; the serpentine injection channel being used to provide pressure buffering and prevent liquid backflow, the distance between the serpentine injection channel and the central injection port and the regional injection port being adjustable, the length of the serpentine injection channel being 0.5-30 mm, and the number of bends being 1-20.

[0008] Furthermore, the supporting layer is glass, silicon wafer or metal conductor; the reaction layer is made of a polymer material, and the polymer material includes polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and polycarbonate; the sealing layer is a transparent tape, a glass sheet or an airtight polymer film.

[0009] Furthermore, the support layer and the reaction layer are treated with plasma and then packaged and bonded at room temperature, by hot pressing, injection molding or 3D printing.

[0010] Furthermore, the reaction areas are scalable, with a number of 1-128.

[0011] Furthermore, the number of the micro-reaction chambers is 1-2 n The diameter of the micro-reaction chamber is 0.1-3 mm and the height is 30-500 μm; the micro-reaction chamber is circular, square, diamond and polygonal.

[0012] Furthermore, the micro-reaction chamber is used for continuous sampling and pre-storage of reaction solutions to achieve complex cascade reaction control on a single chip.

[0013] Furthermore, the injection channel of the microfluidic chip has a height of 10-150 microns and a width of 10-300 microns; the distance from the central injection port to the branch channel is equal, and the length from the injection channel to the branch channel is 0.1-30 mm.

[0014] The present invention also provides a method for preparing the self-priming multiple nucleic acid detection microfluidic chip, comprising the following steps:

[0015] According to the structural diagrams of the micro-reaction chambers, serpentine injection channels and branch channels in the chip reaction layer, corresponding graphic masks are designed and manufactured respectively. A chip mold is obtained by processing on a silicon wafer using multi-layer soft lithography technology. Polydimethylsiloxane with a prepolymer and curing agent ratio of 10:1-5:1 is poured on the chip mold, and the mold is cured and demolded. The mold is cut and punched, and the chip is sealed with the support layer after plasma treatment. After high-temperature baking, the chip production is completed. After production is completed, a sealing layer is pasted on the surface of the chip, and the chip is placed in a vacuum pump for vacuum degassing treatment for 30-60 minutes, and then sealed and stored for future use.

[0016] The present invention also provides a detection method based on the self-priming multiple nucleic acid detection microfluidic chip, comprising the following steps:

[0017] S1. Obtain a microfluidic chip, use a needle to puncture the sealing layer on the surface of the central sampling hole and the regional sampling hole, add a specific primer-probe mixture containing a blood, sputum, foodborne or respiratory pathogen to be tested into the corresponding regional sampling hole, and drive the mixture to be evenly dispersed into each microreaction chamber under negative pressure. Repeat this operation and add specific primer-probe mixtures containing other pathogens to be tested into each reaction zone respectively;

[0018] S2. Remove the chip sealing layer and quickly replace it with a new one. Use a needle to pierce the sealing layer of the central injection hole. Add the sample solution containing the nucleic acid of each pathogen to be detected into the central injection hole. Driven by negative pressure, the sample solution is evenly dispersed into each reaction area.

[0019] S3. After the sample solution is injected, a water-immiscible oil phase liquid is added from the central injection hole, and the oil phase liquid is dispersed into the channel to completely separate the reaction solutions in each micro-reaction chamber;

[0020] S4. Prepare polydimethylsiloxane with a ratio of prepolymer to curing agent of 5:1, add platinum catalyst, and use the prepared polydimethylsiloxane to seal the regional injection hole and the central injection hole;

[0021] S5. Place the sealed chip on a heating device for nucleic acid amplification. After the reaction is completed, use an image sensor to collect fluorescent signals to achieve rapid detection of multiple pathogens.

[0022] Furthermore, the detection method includes: mixing a specific primer-probe mixed reagent for detecting the target with a hydrogel or a low-melting-point agarose solution and pre-loading it into the micro-reaction chamber 7 to achieve long-term storage at 4° C., which is suitable for on-site detection.

[0023] The beneficial effects of the present invention are:

[0024] 1. The chip's self-priming liquid-driving method can automatically and sequentially load multiple target molecule primers and probes and samples to be tested. Compared with existing technologies, the liquid loading time on the chip is short and the chip is free from the need for external driving force, thereby realizing the automatic loading of reagents and samples on the chip.

[0025] 2. The chip design has no outlet, which can achieve 100% complete distribution of samples in multiple detection areas. Target primers are independently introduced into independent reaction areas. Compared with existing technologies, there is no sample waste, and cross-contamination of target primers is avoided, which greatly improves the sensitivity and accuracy of detection.

[0026] 3. The chip is adaptable to various nucleic acid amplification reactions and is compatible with variable temperature PCR reactions and isothermal nucleic acid amplification reactions. Compared with existing technologies, it has strong compatibility, a wide range of applications, and is more suitable for point-of-care (POCT) applications.

[0027] 4. The chip is scalable and can detect multiple targets simultaneously from a single sample. Compared with existing technologies, a single chip can simultaneously detect 6, 12, 24, 48, or 96 target molecules, providing rapid, multi-target detection capabilities for blood, respiratory, and intestinal microbial infections.

[0028] 5. The independent loading capability of independent target molecule primers or probes enables pre-loading of multiple target primers or probes in the chip reaction area. Compared with existing technologies, it has lower costs, simpler operation, and is suitable for on-site or timely detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a physical picture of the chip of the present invention;

[0030] Figure 2 Schematic diagram of the chip structure of the present invention;

[0031] Figure 3 This is a schematic diagram of the reaction layer structure of the chip of the present invention;

[0032] Figure 4 This is a graph showing the reaction results of the chip of the present invention used for triple RPA detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus;

[0033] Figure 5 This is a graph showing the reaction results of the chip of the present invention used for triple-PCR detection of influenza A, influenza B, and Mycoplasma pneumoniae;

[0034] In the figure, 1-support layer, 2-reaction layer, 3-sealing layer, 4-central injection hole, 5-regional injection hole, 6-reaction area, 7-micro reaction chamber, 8-serpentine injection channel, 9-branch channel. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field should fall within the scope of protection of the embodiments of the present application.

[0036] In the description of this application, the terms "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention. The terms "connected", "connected", and "set" used in the present invention should be understood in a broad sense. For example, they can be fixed connections or detachable connections; they can be directly connected or indirectly connected through intermediate components; they can be wired electrical connections, radio connections, or wireless communication signal connections. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0037] Example 1

[0038] like Figure 1 、 Figure 2As shown, an embodiment of the present invention provides a self-priming multiple nucleic acid detection microfluidic chip, which includes a support layer 1, a reaction layer 2 and a sealing layer 3. The support layer 1 adopts a glass substrate, and the reaction layer 2 adopts polydimethylsiloxane (PDMS) as a material. The chip mold is processed by multi-layer soft lithography technology, and 10:1 (prepolymer: curing agent) PDMS is poured on the mold, and the mold is cured, demolded, cut and punched. A central injection hole 4 and 6 identical reaction areas are provided on the reaction layer 2, and the reaction areas are, from the inside to the outside, a serpentine injection channel 8, a regional injection hole 5, and a reaction area 6; the reaction area 6 is connected to the central injection hole 4 through a microchannel, and includes several microreaction chambers 7 and branch channels 9.

[0039] The support layer 1 and the reaction layer 2 are bonded at room temperature after being treated with air plasma, and then baked on a hot plate at 85°C for 2 hours; the chip needs to be vacuumed and degassed before use, and a sealing layer 3 is pasted on the surface before being placed in a vacuum pump for vacuuming, and the sealing layer 3 is made of transparent tape.

[0040] like Figure 3 As shown, the central injection hole 4 has a diameter of 0.8 mm and is punched with a 0.8 mm punch; the regional injection hole 5 has a diameter of 0.7 mm and is punched with a 0.7 mm punch; the serpentine injection channel 8 is used to prevent the sample from flowing back to the central injection hole 4; the six reaction areas 6 are evenly distributed with the central injection hole 4 as the center; the microreaction chamber 7 has a diameter of 1.2 mm and a height of 150 μm; the width of the microchannel is 50 μm and the height is 50 μm.

[0041] Example 2

[0042] This example uses the device described in Example 1 and takes RPA detection of common foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella, etc.) as an example to further illustrate the implementation method of the chip of the present invention.

[0043] S1. Chip production: Use CAD to draw the structural diagram of the branch channel 9 and the micro-reaction chamber 7, respectively process the corresponding mask plates, and use multi-layer soft lithography technology to process the chip mold on a 4-inch silicon wafer. Pour PDMS with a 10:1 (prepolymer: curing agent) ratio on the mold, cure and demold, cut and punch, seal it with the support layer 1 after plasma treatment, and bake at 85°C for 2 hours to complete the chip production.

[0044] S2. Stick a layer of transparent tape on the surface of the chip and place it in a vacuum pump for vacuum degassing for 20 minutes.

[0045] S3. Prepare RPA reaction systems for Staphylococcus aureus, Listeria monocytogenes, and Salmonella respectively.

[0046] S4. After the chip is vacuum-evacuated and degassed, the chip is removed, the transparent tape on the surface of the central sampling hole 4 and the regional sampling hole 5 is punctured, and 0.6 μL of a specific primer-probe mixed reagent containing Staphylococcus aureus (the remaining RPA reaction reagents except the Staphylococcus aureus sample and potassium ions) is added to the corresponding regional sampling hole 5. The mixed reagent is evenly dispersed into each micro-reaction chamber 7 under the drive of negative pressure. This operation is repeated to add specific primer-probe mixed reagents containing the pathogens to be tested (Listeria monocytogenes, Salmonella, etc.) to other reaction areas on the chip. At this time, each micro-reaction chamber 7 is not yet completely filled with reagents, and the entire chip is in a negative pressure state, and sampling can be carried out again.

[0047] S5. After the specific primer mixture reagents are dispersed into the corresponding reaction areas 6, the original transparent tape is torn off and quickly replaced with a new layer of transparent tape. The transparent tape on the surface of the central injection hole 4 is punctured, and 1.5 μL of a sample solution containing nucleic acids such as Staphylococcus aureus, Salmonella, and Listeria monocytogenes and potassium ions is added to the central injection hole 4. Under negative pressure, the sample solution is evenly dispersed into each reaction area 6.

[0048] S6. After the sample solution is injected, a water-immiscible oil phase liquid (silicone oil, mineral oil or fluorinated oil, etc.) is immediately added from the central injection hole 4. The oil phase liquid is dispersed into the channel to completely separate the reaction liquids in each micro reaction chamber 7.

[0049] S7. Prepare PDMS and catalyst at a ratio of 5:1 (prepolymer: curing agent), and seal the regional injection hole 5 and the central injection hole 4.

[0050] S8. Place the sealed chip on an in-situ PCR instrument, a regular hot plate, or a water bath, and heat at 40°C for 20-30 minutes to perform nucleic acid amplification.

[0051] S9, after the reaction is completed, the fluorescence signal of the micro reaction chamber 7 is collected using an image sensor. The results are as follows: Figure 4 As shown in Figure 2, the sample contains nucleic acids from Staphylococcus aureus, Listeria monocytogenes, and Salmonella, and significant fluorescence can be detected in the corresponding reaction areas of the chip.

[0052] Example 3

[0053] This example uses the device described in Example 1 and takes PCR detection of common respiratory pathogens (influenza A, influenza B, Mycoplasma pneumoniae, etc.) as an example to further illustrate the implementation method of the chip of the present invention.

[0054] S1. Chip production: Use CAD to draw the structural diagram of the core branch channel 9 and the micro-reaction chamber 7, respectively process the corresponding mask plates, and use multi-layer soft lithography technology to process the chip mold on the 4-inch silicon wafer. Pour PDMS with a ratio of 10:1 (prepolymer: curing agent) on the mold, solidify and demold, cut and punch, seal it with the support layer 1 after plasma treatment, and bake it at 85°C for 2 hours to complete the chip production.

[0055] S2. Paste a layer of transparent tape on the surface of the chip and place it in a vacuum pump for vacuum degassing.

[0056] S3. Prepare PCR reaction systems for influenza A, influenza B, and Mycoplasma pneumoniae respectively.

[0057] S4. After the chip is vacuum-degassed, remove the chip, puncture the transparent tape on the surface of the central sampling hole 4 and the regional sampling hole 5, and add 0.6 μL of a primer-probe mixture reagent containing influenza A virus H1N1 (the remaining PCR reaction reagents except the influenza A virus nucleic acid sample) to the corresponding regional sampling hole 5. Driven by negative pressure, the mixed reagent is evenly dispersed into each micro-reaction chamber 7. Repeat this operation and add a specific primer-probe mixture reagent containing the nucleic acid of the pathogen to be tested (influenza B, Mycoplasma pneumoniae, etc.) to other reaction areas on the chip. At this time, each micro-reaction chamber 7 is not completely filled with reagents, and the entire chip is in a negative pressure state, and sampling can be carried out again.

[0058] S5. After the specific primer mixture reagents are dispersed into the corresponding reaction areas 6, the original transparent tape is torn off and quickly replaced with a new layer of transparent tape. The transparent tape on the surface of the central injection hole 4 is pierced, and 1.5 μL of the sample solution containing influenza A and Mycoplasma pneumoniae nucleic acids is added to the central injection hole 4. Under negative pressure, the sample solution is evenly dispersed into each reaction area 6.

[0059] S6. After the sample solution is injected, water-immiscible oil phase liquid (silicone oil) is immediately added from the central injection hole 4. The oil phase liquid is dispersed into the channel to completely separate the reaction liquids in each micro reaction chamber 7.

[0060] S7. Prepare 5:1 (prepolymer: curing agent) PDMS and catalyst, and seal the regional injection hole 5 and the central injection hole 4.

[0061] S8. Place the oil-sealed chip on an in-situ PCR instrument for nucleic acid amplification.

[0062] S9, after the reaction is completed, the fluorescence signal of the micro reaction chamber 7 is collected using an image sensor. The results are as follows: Figure 5 As shown, the sample contains nucleic acids of influenza A and Mycoplasma pneumoniae, and therefore, significant fluorescence can be detected in the corresponding reaction areas of the chip.

[0063] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only.

[0064] It will be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. A self-priming multiple nucleic acid detection microfluidic chip, characterized in that: The chip comprises a support layer (1) and a reaction layer (2), wherein the reaction layer (2) is placed on the support layer (1), and a sealing layer (3) is attached to the upper surface of the reaction layer (2); the reaction layer (2) is provided with a central injection hole (4) and a plurality of identical and independent reaction areas, wherein the reaction areas are arranged from the central injection hole (4) to the outside in the order of a serpentine injection channel (8), a regional injection hole (5), and a reaction area (6); the reaction area (6) is connected to the central injection hole (4) through a microchannel and is evenly distributed in a radial shape; The reaction zone (6) includes a plurality of micro-reaction chambers (7) and branch channels (9), wherein the micro-reaction chambers (7) are connected via the branch channels (9), and the distances between each micro-reaction chamber (7) and the corresponding regional injection holes (5) and the central injection hole (4) are equal; the serpentine injection channel (8) is used to provide pressure buffering and prevent liquid backflow, and the distance between the serpentine injection channel (8) and the central injection port (4) and the regional injection port (5) can be adjusted. The length of the serpentine injection channel (8) is 0.5-30 mm, and the number of bends is 1-20.

2. A self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The support layer (1) is glass, silicon wafer or metal conductor; the reaction layer (2) is made of high molecular polymer material, and the high molecular polymer material includes polydimethylsiloxane, polymethyl methacrylate, and polycarbonate; the sealing layer (3) is transparent tape, glass sheet or airtight polymer film.

3. A self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The support layer (1) and the reaction layer (2) are packaged and bonded at room temperature, by hot pressing, injection molding, or 3D printing after plasma treatment.

4. A self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The reaction areas are scalable, with a number ranging from 1 to 128.

5. The self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The number of the micro-reaction chambers (7) is 1-2 n The diameter of the particles is 0.1-3 mm and the height is 30-500 μm.

6. A self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The micro-reaction chamber (7) is used for continuous sampling and pre-storing reaction liquid.

7. The self-priming multiple nucleic acid detection microfluidic chip according to claim 1, characterized in that: The injection channel of the microfluidic chip has a height of 10-150 microns and a width of 10-300 microns; the distance from the central injection port to the branch channel is equal, and the length from the injection channel to the branch channel (9) is 0.1-30 mm.

8. A method for preparing a self-priming multiple nucleic acid detection microfluidic chip according to any one of claims 1 to 7, characterized in that: The steps include: According to the structure diagram of the micro-reaction chamber (7), the serpentine injection channel (8) and the branch channel (9) in the chip reaction layer, the corresponding graphic mask plates are designed and manufactured respectively, and the chip mold is processed on the silicon wafer by multi-layer soft lithography technology. Polydimethylsiloxane with a ratio of prepolymer and curing agent of 10:1-5:1 is poured on the chip mold, and the mold is released by curing. The mold is cut and punched, and the chip is sealed with the support layer (1) after plasma treatment. After high-temperature baking, the chip production is completed. After the production is completed, a sealing layer (3) is pasted on the surface of the chip, and the chip is placed in a vacuum pump for vacuum degassing treatment for 30-60 minutes, and then sealed and stored for use.

9. A detection method based on the self-priming multiple nucleic acid detection microfluidic chip according to any one of claims 1 to 7, characterized in that: The steps include: S1. Obtain a microfluidic chip, use a needle to pierce the sealing layer (3) on the surface of the central sampling hole (4) and the regional sampling hole (5), add a specific primer-probe mixed reagent containing a blood, sputum, foodborne or respiratory pathogen to be detected to the corresponding regional sampling hole (5), and the mixed reagent is evenly dispersed into each micro-reaction chamber (7) under the drive of negative pressure. Repeat this operation and add the specific primer-probe mixed reagent containing other pathogens to be detected to each reaction zone (6) respectively; S2. Remove the sealing layer (3) of the chip and quickly replace it with a new sealing layer (3). Use a needle to pierce the sealing layer (3) of the central injection hole (4). Add the sample solution containing the nucleic acid of each pathogen to be detected into the central injection hole (4). Driven by negative pressure, the sample solution is evenly dispersed into each reaction area (6). S3. After the sample solution is injected, a water-immiscible oil phase liquid is added from the central injection hole (4) to disperse the oil phase liquid into the channel, thereby completely separating the reaction liquids in each micro-reaction chamber (7); S4, preparing polydimethylsiloxane with a ratio of prepolymer to curing agent of 5:1, adding a platinum catalyst, and using the prepared polydimethylsiloxane to seal the regional injection hole (5) and the central injection hole (4); S5. Place the sealed chip on a heating device for nucleic acid amplification. After the reaction is completed, use an image sensor to collect fluorescent signals to achieve rapid detection of multiple pathogens.

10. The detection method according to claim 9, characterized in that: The detection method comprises: mixing a specific primer-probe mixed reagent for detecting a target with a hydrogel or a low-melting-point agarose solution and then pre-loading the mixture into a micro-reaction chamber (7) to achieve long-term storage at 4°C.

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