A microfluidic chip and detection method for real-time fluorescent nucleic acid amplification detection

Abstract

A microfluidic chip and detection method for real-time fluorescent nucleic acid amplification detection, the chip includes a laminated base layer and a cover layer, the cover layer is provided with a liquid inlet, the base layer is provided with a waste liquid receiving part, a filter paper chamber, an injection port, a mixing flow channel, a cleaning liquid injection part and a reaction liquid injection part, the injection port is connected to the liquid inlet of the cover layer, the filter paper chamber has a transparent area, the filter paper chamber is provided with a filter paper for adsorbing nucleic acid, the filter paper chamber is connected to the injection port, the waste liquid receiving part and the cleaning liquid injection part, and the reaction liquid injection part is connected through the mixing flow channel, the cleaning liquid injection part is used to inject cleaning liquid into the filter paper chamber, and the reaction liquid injection part is used to provide a reaction liquid containing primers and enzymes, which flows into the filter paper chamber after passing through the mixing flow channel. The use of the present invention can greatly simplify cumbersome manual operations and shorten the time of real-time fluorescent nucleic acid amplification detection.

Description

Technical Field

[0001] The invention relates to nucleic acid amplification detection technology, in particular to a microfluidic chip and a detection method for real-time fluorescent nucleic acid amplification detection. Background Art

[0002] In recent years, a large number of infectious diseases such as African swine fever and Ebola virus have spread around the world, but the detection of the virus requires professional instruments to extract and amplify nucleic acids, and the finished products are expensive and cannot be widely used. Microfluidics is a miniaturized and highly integrated technology with low cost and automated operation, and is widely used in the medical field.

[0003] Microfluidics originated from Manz, Widmer and others who used chips to achieve electrophoretic separation that had been completed in capillaries. After that, microfluidics developed rapidly, and was gradually applied to various fields such as biochemical medical diagnosis, food commodity inspection, etc. from the initial analytical chemistry platform. The most commonly used microfluidics drive systems are active and passive. The active type mainly uses external driving forces such as mechanical pressure, magnetic field, electric field, etc., while the passive type mainly relies on the design of the flow channel to achieve the flow of fluids. The passive type is simpler than the active type and is easier to miniaturize, but the passive drive has the defect of instability.

[0004] Clinically, molecular diagnosis is widely used in the detection of infectious diseases, tumors and genetic diseases. With major breakthroughs in life sciences, biotechnology and micro-processing technology, the related industries of high-tech nucleic acid molecular detection have become a new field of global competition in the future.

[0005] Molecular diagnosis is a sophisticated detection method that includes a variety of amplification technologies. Among them, real-time fluorescent nucleic acid constant temperature amplification and testing technology, referred to as SAT, is a new type of nucleic acid detection technology that combines a new generation of nucleic acid constant temperature amplification technology and real-time fluorescent detection technology. This technology has the advantages of rapid response, high sensitivity, high specificity, low pollution, and stable reaction. However, like conventional nucleic acid detection technology, this technology cannot get rid of the shortcomings of time-consuming sample preparation and cumbersome operation, and does not meet the needs of rapid and low-cost clinical diagnosis. Summary of the invention

[0006] The main purpose of the present invention is to overcome the above-mentioned defects of the prior art and provide a microfluidic chip and a detection method for real-time fluorescent nucleic acid amplification detection, so as to improve the detection speed, simplify the operation, facilitate control, and reduce the detection cost.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A microfluidic chip for real-time fluorescent nucleic acid amplification detection comprises a base layer and a cover layer, wherein the base layer and the cover layer are stacked and pressed together, the cover layer is provided with a liquid inlet, the base layer is provided with a waste liquid receiving part, a filter paper chamber, an injection port, a mixing flow channel, a cleaning liquid injection part and a reaction liquid injection part, the injection port is connected to the liquid inlet, the filter paper chamber has a transparent area, a filter paper for adsorbing nucleic acid for nucleic acid purification and concentration is arranged in the filter paper chamber, the filter paper chamber is connected to the injection port, the waste liquid receiving part and the cleaning liquid injection part, and is connected to the reaction liquid injection part through the mixing flow channel, the sample to be tested enters the filter paper chamber through the injection port, the cleaning liquid injection part is used to inject cleaning liquid into the filter paper chamber, the waste liquid after cleaning flows to the waste liquid receiving part, the reaction liquid injection part is used to provide a reaction liquid containing a primer and an enzyme, which flows into the filter paper chamber after passing through the mixing flow channel, and the filter paper chamber is used for nucleic acid constant temperature amplification and fluorescence detection.

[0009] Further:

[0010] The cleaning liquid injection part includes a pre-buried cleaning liquid chamber, which is connected to the filter paper chamber through a flow channel provided with a soft valve. The pre-buried cleaning liquid chamber releases the pre-buried cleaning liquid under the drive of an external force, breaks through the soft valve to clean the filter paper in the filter paper chamber for impurities.

[0011] The reaction liquid injection part includes a pre-embedded enzyme chamber and a pre-embedded primer chamber, each of which has a reaction liquid inlet, and the outlets of the pre-embedded enzyme chamber and the pre-embedded primer chamber are both connected to the mixing flow channel. The reaction liquid flowing into the pre-embedded enzyme chamber and the pre-embedded primer chamber driven by external force is mixed with the pre-embedded primer and enzyme respectively, and then flows to the filter paper chamber after mixing through the mixing flow channel. Preferably, the pre-embedded enzyme chamber and the pre-embedded primer chamber are symmetrically arranged.

[0012] The reaction liquid injection part also includes a pre-embedded reaction liquid chamber, which is respectively connected to the pre-embedded enzyme chamber and the pre-embedded primer chamber through two flow channels provided with soft valves. The pre-embedded reaction liquid chamber releases the pre-embedded reaction liquid under the drive of external force, breaks through the soft valve and flows into the pre-embedded enzyme chamber and the pre-embedded primer chamber.

[0013] The filter paper is fixed in the filter paper chamber by gluing or mechanical slotting, and the amount of nucleic acid adsorption is controlled by controlling the diameter of the filter paper.

[0014] A plurality of filter paper chambers are arranged in parallel on the base layer.

[0015] The waste liquid receiving part includes a waste liquid outlet or a waste liquid pool, and the waste liquid pool is connected to the waste liquid vent hole arranged on the cover layer.

[0016] The material of the base layer and the cover layer is polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS.

[0017] A real-time fluorescent nucleic acid amplification detection method, using the microfluidic chip to perform real-time fluorescent nucleic acid amplification detection, the method comprises the following steps:

[0018] (1) injecting the sample to be tested into the filter paper chamber from the injection port, and the waste liquid flows to the waste liquid receiving part;

[0019] (2) injecting cleaning liquid into the filter paper chamber from the cleaning liquid injection part, and the waste liquid flows to the waste liquid receiving part;

[0020] (3) injecting a reaction solution mixed with primers and enzymes into the filter paper chamber from the reaction solution injection portion, and the reaction solution flows toward the filter paper, and the filter paper adsorbs the nucleic acid to achieve nucleic acid purification and concentration;

[0021] (4) The temperature of the filter paper chamber was controlled at 37° C., the fluorescence intensity of the reactants was observed under a microscope, and the nucleic acid concentration was quantitatively analyzed.

[0022] Furthermore, the filter paper chamber is maintained at a reaction condition of 37° C. by contacting an external thermostatic control plate.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention realizes the design of nucleic acid purification concentration and real-time fluorescence constant temperature amplification detection on a microfluidic chip, which can greatly simplify cumbersome manual operation and shorten the detection time. By setting the filter paper chamber, the nucleic acid is extracted and concentrated using filter paper, and the cost of using magnetic beads to extract nucleic acid in the past is also simplified. At the same time, the present invention also brings into play the advantages of microfluidic chip technology itself (low cost, high throughput, automation, etc.), meeting the needs of rapid diagnosis in clinical practice. In a preferred embodiment, the present invention uses pre-embedded technology to pre-embed enzyme and primer in two parallel chambers respectively, effectively solving the problem of sufficient mixing of reaction solution with primer and enzyme respectively.

[0025] By using the microfluidic chip of the present invention, nucleic acid can be purified and concentrated automatically and quickly, and automatic sample addition and reaction can be realized. On this basis, nucleic acid isothermal amplification and real-time fluorescence detection with fast and simple operation and flexible control can be realized. The microfluidic chip has a simple structure and is easy to control, and the chip can be processed in batches and at low cost. In the process of detecting nucleic acids on the chip, the amount of reagents can be accurately controlled to reduce reagent consumption, and multiple indicators can be detected at the same time to ensure that the test results are reliable, stable and can be quickly obtained. The present invention realizes the integration of nucleic acid extraction, purification, concentration, amplification and detection, and can be widely used in the rapid detection of nucleic acid molecules. Compared with the prior art, it is more convenient and faster, and the cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the structure of a microfluidic chip for real-time fluorescent nucleic acid amplification detection according to an embodiment of the present invention;

[0027] Figure 2 for Figure 1 The schematic diagram of the structure of the basal layer shown in; DETAILED DESCRIPTION

[0028] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention.

[0029] It should be noted that when a component is referred to as being "fixed to" or "disposed on" another component, it can be directly on the other component or indirectly on the other component. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for both fixing and circuit / signal communication.

[0030] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0031] Figure 1 The present invention is a schematic structural diagram of a microfluidic chip for real-time fluorescent nucleic acid amplification detection according to an embodiment of the present invention. Figure 2 for Figure 1 The schematic diagram of the structure of the substrate layer 1 is shown in FIG. Figure 1 and Figure 2The embodiment of the present invention provides a microfluidic chip for real-time fluorescent nucleic acid amplification detection, comprising a base layer 1 and a cover layer 2, wherein the base layer 1 and the cover layer 2 are stacked and pressed together, the cover layer 2 is provided with a liquid inlet, the base layer 1 is provided with a waste liquid receiving portion, a filter paper chamber 5, an injection port 6, a mixing flow channel 7, a cleaning liquid injection portion and a reaction liquid injection portion, the injection port 6 is connected to the liquid inlet, the filter paper chamber 5 is a constant temperature amplification chamber, which has a transparent area for detection, and the filter paper chamber 5 is provided with a liquid for adsorbing nucleic acids for nucleic acid purification and concentration. The filter paper chamber 5 is connected with the injection port 6, the waste liquid receiving part and the cleaning liquid injection part, and is connected with the reaction liquid injection part through the mixing flow channel 7. The sample to be tested enters the filter paper chamber 5 through the injection port 6. The cleaning liquid injection part is used to inject cleaning liquid into the filter paper chamber 5. The waste liquid after cleaning flows to the waste liquid receiving part. The reaction liquid injection part is used to provide a reaction liquid containing primers and enzymes, which flows into the filter paper chamber 5 after passing through the mixing flow channel 7, and nucleic acid isothermal amplification and fluorescence detection are performed in the filter paper chamber 5.

[0032] By using the microfluidic chip of the embodiment of the present invention, nucleic acid can be purified and concentrated automatically and quickly, and automatic sample addition and reaction can be realized. On this basis, nucleic acid isothermal amplification and real-time fluorescence detection with fast and simple operation and flexible control can be realized. The microfluidic chip has a simple structure and is easy to control. The chip can be processed in batches and at low cost. In the process of detecting nucleic acids on the chip, the amount of reagents can be accurately controlled to reduce reagent consumption. At the same time, multiple indicators can be detected to ensure that the test results are reliable, stable and can be quickly obtained.

[0033] See also Figure 1 and Figure 2 In a preferred embodiment, the cleaning liquid injection part includes a pre-buried cleaning liquid chamber 12, and the pre-buried cleaning liquid chamber 12 is connected to the filter paper chamber 5 through a flow channel provided with a soft valve 13. The pre-buried cleaning liquid chamber 12 releases the pre-buried cleaning liquid under the drive of external force, breaks through the soft valve 13 to clean the impurities of the filter paper in the filter paper chamber 5, and the waste liquid flows into the waste liquid receiving part, such as the waste liquid tank 4.

[0034] See also Figure 1 and Figure 2In a preferred embodiment, the reaction liquid injection part includes a pre-embedded enzyme chamber 8 and a pre-embedded primer chamber 11, each of which has a reaction liquid inlet, and the outlets of the pre-embedded enzyme chamber 8 and the pre-embedded primer chamber 11 are connected to the mixing channel 7. The reaction liquid flowing into the pre-embedded enzyme chamber 8 and the pre-embedded primer chamber 11 driven by an external force is mixed with the pre-embedded primer and enzyme respectively, and then flows to the filter paper chamber 5 after being mixed through the mixing channel 7. Preferably, the pre-embedded enzyme chamber 8 and the pre-embedded primer chamber 11 are symmetrically arranged.

[0035] In the above embodiment, the enzyme and the primer are respectively pre-embedded in the two chambers using the pre-embedded technology, which is conducive to the full mixing of the reaction solution, the primer and the enzyme.

[0036] See also Figure 1 and Figure 2 In a preferred embodiment, the reaction liquid injection part also includes a pre-embedded reaction liquid chamber 14, which is respectively connected to the pre-embedded enzyme chamber 8 and the pre-embedded primer chamber 11 through two flow channels provided with soft valves 9 and 10. The pre-embedded reaction liquid chamber 14 releases the pre-embedded reaction liquid under the drive of external force, breaks through the soft valves 9 and 10 and flows into the pre-embedded enzyme chamber 8 and the pre-embedded primer chamber 11.

[0037] In some embodiments, the filter paper in the filter paper chamber 5 can be fixed in the filter paper chamber 5 by gluing or mechanical slotting, and the amount of nucleic acid adsorption can be controlled by controlling the diameter of the filter paper.

[0038] In some embodiments, a plurality of filter paper chambers 5 may be arranged in parallel on the base layer 1 so that multiple tests can be performed simultaneously.

[0039] In some embodiments, the waste liquid receiving part may include a waste liquid outlet or a waste liquid pool 4, and the waste liquid pool 4 is connected to the waste liquid vent provided on the cover layer. The waste liquid vent connects the waste liquid pool 4 to the atmosphere, so as to better drive the waste liquid to flow into the waste liquid pool.

[0040] In some embodiments, the material of the base layer 1 and the cover layer 2 may be polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS, but is not limited thereto.

[0041] The present invention also provides a real-time fluorescent nucleic acid amplification detection method, using the microfluidic chip to perform real-time fluorescent nucleic acid amplification detection, see Figure 1 and Figure 2 The detection method is based on real-time fluorescent nucleic acid amplification detection technology and includes the following steps:

[0042] (1) injecting the sample to be tested into the filter paper chamber 5 from the sample inlet 6, and the waste liquid flows to the waste liquid receiving part;

[0043] (2) Injecting cleaning liquid into the filter paper chamber 5 from the cleaning liquid injection portion, and the waste liquid flows to the waste liquid receiving portion;

[0044] (3) Injecting a reaction solution mixed with primers and enzymes into the filter paper chamber 5 from the reaction solution injection portion, the reaction solution flows toward the filter paper, and the filter paper adsorbs the nucleic acid to achieve nucleic acid purification and concentration;

[0045] (4) The temperature of the filter paper chamber 5 is controlled to be 37° C., the fluorescence intensity of the reactants is observed under a microscope, and the nucleic acid concentration is quantitatively analyzed.

[0046] In a preferred embodiment, the filter paper chamber 5 is maintained at a reaction condition of 37° C. by contacting an external thermostatic control plate.

[0047] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings.

[0048] The microfluidic chip of the specific embodiment uses filter paper adsorption to purify and concentrate nucleic acids and conduct real-time fluorescent nucleic acid amplification detection. The real-time fluorescent nucleic acid amplification detection scheme based on the microfluidic chip includes a nucleic acid purification and concentration unit, a reactant mixing unit, and a nucleic acid constant temperature amplification detection unit; the nucleic acid purification and concentration unit uses filter paper to adsorb nucleic acids to purify and concentrate nucleic acids; the reactant mixing unit supplies and mixes necessary reactants, and the chip can be driven by external force to inject reactants and allow microfluids to flow in each chamber; the nucleic acid constant temperature amplification detection unit involves nucleic acid amplification and fluorescence detection under constant temperature control.

[0049] The microfluidic chip specifically comprises a base layer 1 and a cover layer 2. The materials of the base layer 1 and the cover layer 2 are not limited to common medical plastics such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC), ABS, etc., and can be formed by various plastic molding methods such as compression thermoplastic molding and injection molding. The base layer 1 and the cover layer 2 are packaged by a pressing process.

[0050] The nucleic acid purification and concentration unit of the microfluidic chip comprises an injection port 6, a filter paper chamber 5 (purification and concentration chamber), a cleaning liquid pool, a waste liquid pool 4 and a flow channel connected to the reactant mixing unit; the injection port 6 can inject the sample into the chip through an external driving system, the fluid flows through the filter paper and the nucleic acid is adsorbed, and the remaining waste liquid flows into the waste liquid pool 4. The nucleic acid purification and concentration unit inlet and the vent holes of the waste liquid pool 4 are distributed on the cover plate layer 2, and the fluid flow channels are distributed on the bottom flow channel plate; the cleaning liquid pool is pre-buried with cleaning liquid, and the release of the cleaning liquid requires external pressure, so that the cleaning liquid breaks through the soft valve and flows to the filter paper to clean impurities, and the waste liquid flows into the waste liquid pool 4.

[0051] The filter paper chamber 5 contains filter paper capable of adsorbing nucleic acid; the filter paper can be fixed in the chamber by gluing or mechanically slotting, and the amount of adsorbed nucleic acid can be controlled by controlling the diameter of the filter paper.

[0052] The reactant mixing unit comprises a plurality of regularly distributed, open premixing chambers and a mixing channel 7, and the mixing channel 7 is directly connected to the nucleic acid constant temperature amplification detection unit; the purified nucleic acid is wound on the filter paper, and the reaction liquid flows out from the chamber where it is located, and then the pre-buried enzyme and primer are mixed in the flow channel, and after being fully mixed, it flows through the filter paper for nucleic acid amplification and detection.

[0053] The reaction liquid mixing unit includes two symmetrical chambers; primers and enzymes are pre-buried in the two chambers respectively. Driven by external force, the reaction liquid flows into the two chambers in equal amounts, mixes with the primers and enzymes respectively, and then mixes through the curved confluence channel, and finally flows to the filter paper chamber 5.

[0054] The nucleic acid constant temperature amplification detection unit may include a plurality of filter paper chambers 5 and a plurality of flow channels, which are distributed on the base layer 1; the temperature of the filter paper chamber 5 is maintained at a reaction condition of 37°C by contacting an external constant temperature control plate. By placing the chip in the fluorescence detection platform, the change of fluorescence intensity can be detected in real time to reflect the cycle amplification situation. An opening is opened in the cover layer 2.

[0055] The nucleic acid detection method of one embodiment includes the following specific steps:

[0056] (1) using an injection gun to inject the sample to be tested from the sample inlet 6 of the nucleic acid purification and concentration unit into the purification and concentration chamber, and the waste liquid flows into the waste liquid pool 4;

[0057] (2) releasing the cleaning liquid in the cleaning liquid chamber 12, and the waste liquid flows to the waste liquid tank 4;

[0058] (3) releasing the reaction solution mixed with Mg+, mixing primers and enzymes, and flowing to the filter paper for nucleic acid amplification and detection;

[0059] (4) The temperature of the nucleic acid isothermal amplification detection unit was controlled at 37° C., the fluorescence intensity of the reactants was observed under a microscope, and the nucleic acid concentration was quantitatively analyzed.

[0060] like Figure 1 The figure shows a schematic diagram of the structure of a nucleic acid detection chip that integrates nucleic acid purification and concentration and real-time fluorescent nucleic acid isothermal amplification provided by a specific embodiment. The chip includes a base layer 1 and a cover layer 2. Figure 2 As shown, the base layer 1 includes a waste liquid pool vent 3, a waste liquid pool 4, a filter paper chamber 5, an injection port 6, a mixing channel 7, a pre-embedded enzyme chamber 8, a pre-embedded enzyme chamber soft valve 9, a pre-embedded primer chamber soft valve 10, a pre-embedded primer chamber 11, a pre-embedded cleaning liquid chamber 12, a pre-embedded cleaning liquid chamber soft valve 13 and a pre-embedded reaction liquid chamber 14.

[0061] The materials of the base layer and the cover layer are common medical plastics, such as polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate PC, ABS, and various plastic molding methods such as compression molding and injection molding are adopted; for example, the injection molding method is adopted to pre-process the mold, and then the polypropylene (PP) material is melted in a constant temperature barrel at 220-280°C, and then the melted PP material is injected into the mold under pressure of 800-140MPa, and then the pressure is maintained and cooled to form. Before the chip is assembled, the filter paper is pasted to chamber 5 in the filter paper chamber, the cleaning liquid is pre-embedded in chamber 12, and the reaction liquid is pre-embedded in chamber 14. The nucleic acid detection steps specifically include:

[0062] (1) using a syringe gun to inject 20 μL of the sample to be tested from the injection port (6) of the nucleic acid purification and concentration unit into the paper-based chamber 5 at a rate of 200 μL / min;

[0063] (2) the squeezing chamber 12, after the cleaning liquid flows through the paper base, it flows to the waste liquid tank 4;

[0064] (3) Extrusion chamber 14, after the reaction liquid flows through the paper base, it flows to the waste liquid tank 4;

[0065] (4) The temperature of the nucleic acid isothermal amplification detection unit is controlled to 37° C., the fluorescence intensity of the reactants is observed under a microscope, and the nucleic acid concentration is quantitatively analyzed.

[0066] The chip can be driven by external force to inject fluid and make the fluid flow in each chamber.

[0067] The present invention can realize the integration of nucleic acid extraction, purification, concentration, amplification and detection, and can be widely applied to the detection of nucleic acid molecules, which is convenient and fast.

[0068] The background section of the present invention may contain background information about the problem or environment of the present invention, but does not necessarily describe the prior art. Therefore, the content included in the background section is not an admission by the applicant that the prior art is available.

[0069] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. An extrusion microfluidic chip for real-time fluorescent nucleic acid amplification detection, characterized in that: The invention comprises a base layer and a cover layer, wherein the base layer and the cover layer are stacked and pressed together to form a double-layer structure, the cover layer is provided with a liquid inlet, the base layer is provided with a waste liquid receiving part, a filter paper chamber, an injection port, a mixing flow channel, a cleaning liquid injection part and a reaction liquid injection part, the injection port is connected to the liquid inlet, the filter paper chamber has a transparent area, a filter paper for adsorbing nucleic acid for nucleic acid purification and concentration is arranged in the filter paper chamber, the filter paper chamber is connected to the injection port, the waste liquid receiving part and the cleaning liquid injection part, and is connected to the reaction liquid injection part through the mixing flow channel, the sample to be tested enters the filter paper chamber through the injection port, the cleaning liquid is injected into the filter paper chamber, and the reaction liquid is injected into the filter paper chamber. The washing liquid injection part is used to inject washing liquid into the filter paper chamber, and the waste liquid after washing flows to the waste liquid receiving part. The reaction liquid injection part is used to provide a reaction liquid containing primers and enzymes, which flows into the filter paper chamber after passing through the mixing flow channel. The filter paper chamber is used for constant temperature amplification and fluorescence detection of nucleic acid; the washing liquid injection part includes a pre-buried washing liquid chamber, and the pre-buried washing liquid chamber is connected to the filter paper chamber through a flow channel provided with a soft valve. When the pre-buried washing liquid chamber is squeezed, the pre-buried washing liquid is released, breaking through the soft valve to clean the filter paper in the filter paper chamber from impurities; the reaction liquid injection part includes a pre-buried reaction liquid chamber, a pre-buried enzyme chamber and The pre-embedded primer chamber, the pre-embedded reaction liquid chamber is connected to the pre-embedded enzyme chamber and the pre-embedded primer chamber respectively through two flow channels provided with soft valves, the pre-embedded reaction liquid chamber releases the pre-embedded reaction liquid when squeezed, breaks through the soft valve and flows into the pre-embedded enzyme chamber and the pre-embedded primer chamber, the pre-embedded enzyme chamber and the pre-embedded primer chamber respectively have a reaction liquid inlet, the outlets of the pre-embedded enzyme chamber and the pre-embedded primer chamber are connected to the mixing flow channel, the reaction liquid flowing into the pre-embedded enzyme chamber and the pre-embedded primer chamber driven by external force is mixed with the pre-embedded primer and enzyme respectively, and then flows to the filter paper chamber after being mixed through the mixing flow channel; the squeezing The pressure-type microfluidic chip is configured to perform the following operations when in use: (1) injecting the sample to be tested into the filter paper chamber from the injection port, and the waste liquid flows to the waste liquid receiving part; (2) squeezing the pre-buried cleaning liquid chamber, injecting the cleaning liquid into the filter paper chamber from the cleaning liquid injection part, and the waste liquid flows to the waste liquid receiving part; (3) squeezing the pre-buried reaction liquid chamber, injecting the reaction liquid mixed with primers and enzymes into the filter paper chamber from the reaction liquid injection part, and flowing to the filter paper, so that the filter paper adsorbs the nucleic acid to achieve nucleic acid purification and concentration; (4) controlling the temperature of the filter paper chamber to 37°C so as to observe the fluorescence intensity of the reactant under a microscope and quantitatively analyze the nucleic acid concentration.

2. The extrusion type microfluidic chip according to claim 1, characterized in that: The pre-embedded enzyme chamber and the pre-embedded primer chamber are symmetrically arranged.

3. The extrusion type microfluidic chip according to any one of claims 1 to 2, characterized in that: The filter paper is fixed in the filter paper chamber by gluing or mechanical slotting, and the amount of nucleic acid adsorption is controlled by controlling the diameter of the filter paper.

4. The extrusion type microfluidic chip according to any one of claims 1 to 2, characterized in that: A plurality of parallel filter paper chambers are arranged on the base layer.

5. The extrusion type microfluidic chip according to any one of claims 1 to 2, characterized in that: The waste liquid receiving part includes a waste liquid outlet or a waste liquid pool, and the waste liquid pool is connected to the waste liquid vent hole arranged on the cover layer.

6. The extrusion type microfluidic chip according to any one of claims 1 to 2, characterized in that: The material of the base layer and the cover layer is polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS.

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