A microfluidic chip and detection method for nucleic acid amplification detection

Abstract

A microfluidic chip and detection method for nucleic acid amplification detection, the chip includes a base layer, a cover layer and a reversing valve, the cover layer is provided with an inlet, a purification and concentration chamber, a waste liquid outlet, a premixing chamber through hole and an outlet through hole, the inlet is connected to the waste liquid outlet and the premixing chamber through hole respectively after passing through the purification and concentration chamber through a flow channel, a porous material embedded with immunomagnetic beads is pre-set in the purification and concentration chamber, the reversing valve is arranged on the outlet side flow channel of the purification and concentration chamber to control the liquid in the purification and concentration chamber to flow to the waste liquid outlet or the premixing chamber through hole, the premixing chamber through hole is used to add reactants to mix with the liquid from the purification and concentration chamber, the base layer is provided with a mixing flow channel and a constant temperature amplification chamber with a transparent area, and the premixing chamber through hole is connected to the constant temperature amplification chamber through the mixing flow channel. The use of this chip can realize the purification, concentration, constant temperature amplification and real-time fluorescence detection of nucleic acids in one step, and obtain one or more target detection results quickly, simply and reliably.

Description

Technical Field

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

[0002] Since Manz and Widmer first proposed the concept of micro-total analysis system (μTAS) in the 1990s, microfluidic technology has developed on the basis of microelectronics, micromechanics, bioengineering and nanotechnology. In the following decades, this field has rapidly developed into one of the most cutting-edge scientific and technological fields in the world. At present, its core technology is microfluidic chips based on microfluidic technology, also known as chip laboratories Lab on chip. Microfluidic chips are widely used in the biomedical field because of their advantages such as low consumption, low cost, high throughput and automated operation. One of the important applications is molecular diagnostic technology based on microfluidic chips.

[0003] 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.

[0004] 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 (SAT) is a new type of nucleic acid detection technology that combines a new generation of nucleic acid constant temperature amplification technology with 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

[0005] The object of the present invention is to overcome at least one of the above disadvantages and to provide a microfluidic chip and a detection method for nucleic acid amplification detection.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A microfluidic chip for nucleic acid amplification detection comprises a base layer, a cover plate layer and a reversing valve, wherein the base layer and the cover plate layer are stacked and pressed together, the cover plate layer is provided with an inlet, a purification and concentration chamber, a waste liquid outlet, a premixing chamber through hole and an outlet through hole, the inlet is respectively connected to the waste liquid outlet and the premixing chamber through hole after passing through the purification and concentration chamber through a flow channel, a porous material embedded with immunomagnetic beads is pre-set in the purification and concentration chamber, the reversing valve is arranged on the flow channel on the outlet side of the purification and concentration chamber to switch and control the liquid in the purification and concentration chamber to flow to the waste liquid outlet or the premixing chamber through hole, the premixing chamber through hole is used to add reactants to mix with the liquid from the purification and concentration chamber, the base layer is provided with a mixing flow channel and a constant temperature amplification chamber for nucleic acid constant temperature amplification and fluorescence detection, the premixing chamber through hole is connected to the constant temperature amplification chamber through the mixing flow channel, the constant temperature amplification chamber has a transparent area, and the constant temperature amplification chamber is connected to the outlet through hole.

[0008] Further:

[0009] A waste liquid pool is arranged on the waste liquid outlet.

[0010] The reversing valve includes a cylindrical reversing valve body, the cover plate layer is provided with a cylindrical through hole that cooperates with the reversing valve body, the base layer is correspondingly provided with a circular cavity that cooperates with the reversing valve body, and the reversing valve body is provided with a three-way flow channel. By rotating the reversing valve body, the purification and concentration chamber can select one of the waste liquid outlet or the premixing chamber through the three-way flow channel.

[0011] The three-way flow channel is T-shaped, and the opening cross section of the flow channel is square.

[0012] There are multiple groups of premixing chamber through holes and the constant temperature amplification chamber, each of the premixing chamber through holes is connected to the constant temperature amplification chamber through a corresponding flow connection, and the reversing valve is connected to each of the premixing chamber through holes through a corresponding branch of a diversion channel.

[0013] The through hole of the premixing chamber is used to add a buffer and a DNA polymerase premixed with specific primers and to apply an external driving pressure.

[0014] The scaffold material forming the porous material is an organic or inorganic material that can be 3D printed, has no adsorption effect on nucleic acids, and can be prepared into a porous structure by freeze-drying technology.

[0015] The porous material is prepared by mixing a printing material that has no adsorption effect on nucleic acid in 1,4-dioxane to form a solution with a concentration of 5%-20%, and then mixing the solution and immunomagnetic beads at a mass ratio of 4-10:1, and then performing low-temperature deposition 3D printing to obtain the porous material. Preferably, the printing material is PLGA, and preferably, the diameter of the immunomagnetic beads is 100nm-5μm.

[0016] The materials of the base layer, the cover plate layer and the valve body of the reversing valve are polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS.

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

[0018] The sample to be tested is perfused from the sample inlet into the purification and concentration chamber, and the waste liquid flows to the waste liquid pool through the reversing valve;

[0019] Injecting washing liquid from the injection port to wash the purification and concentration chamber, and the waste liquid flows to the waste liquid tank;

[0020] Injecting eluent from the injection port and slowly flowing it through the purification and concentration chamber; when the liquid is about to be discharged from the purification and concentration chamber, switching the reversing valve to allow the eluted nucleic acid to flow into the premixing chamber through hole;

[0021] Injecting a buffer solution mixed with primers and DNA polymerase into the premixing chamber through hole, and then immediately driving the fluid in the premixing chamber through hole into the mixing channel, and then into the constant temperature amplification chamber;

[0022] The temperature of the isothermal amplification 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.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides a microfluidic chip and a detection method capable of nucleic acid purification and concentration as well as real-time fluorescent nucleic acid amplification detection. By using the microfluidic chip of the present invention, the automated purification, concentration, constant temperature amplification and real-time fluorescent detection of nucleic acids can be realized on the chip in one step, and one or more target detection results can be obtained quickly, simply and reliably. The microfluidic chip of the present invention can realize automated sample addition, reaction and detection, 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 nucleic acid detection on the chip, the reagent dosage 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 chip can be widely used in the field of rapid diagnosis of nucleic acid molecules, reduce detection costs, simplify personnel operations and thus improve detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the exploded structure of a nucleic acid detection chip integrating nucleic acid purification and concentration and real-time fluorescent nucleic acid isothermal amplification according to an embodiment of the present invention;

[0026] Figure 2 for Figure 1 A schematic diagram of the structure of the substrate layer 1 shown in FIG.

[0027] Figure 3 for Figure 1 The structural schematic diagram of the cover plate layer 2 shown in FIG.

[0028] Figure 4 for Figure 1 The structural schematic diagram of the reversing valve body 3 shown in FIG.

[0029] Figure 5 The assembled structure of a nucleic acid detection chip according to an embodiment of the present invention and its top view;

[0030] Figure 6 Schematic diagram of two switching modes of a reversing valve according to an embodiment of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] 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, the connection can be used for both fixing and circuit / signal communication.

[0033] 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.

[0034] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0035] See also Figures 1 to 6 The present invention provides a microfluidic chip for nucleic acid amplification detection, comprising a base layer 1, a cover layer 2 and a reversing valve, wherein the base layer 1 and the cover layer 2 The layers are stacked and pressed together. The cover plate layer 2 is provided with an inlet 7, a purification and concentration chamber 8, a waste liquid outlet, a premixing chamber through hole 13 and an outlet through hole 14. The inlet 7 is connected to the waste liquid outlet and the premixing chamber through hole 13 respectively after passing through the purification and concentration chamber 8 through a flow channel. A porous material embedded with immunomagnetic beads is pre-installed in the purification and concentration chamber. The reversing valve is arranged on the outlet side flow channel of the purification and concentration chamber 8 to switch and control the liquid in the purification and concentration chamber 8 to flow to the waste liquid outlet or the premixing chamber through hole 13. The premixing chamber through hole 13 is used to add reactants to mix with the liquid from the purification and concentration chamber. The base layer 1 is provided with a mixing flow channel 5 and a constant temperature amplification chamber 6. The premixing chamber through hole 13 is connected to the constant temperature amplification chamber 6 through the mixing flow channel 5. The constant temperature amplification chamber 6 is used for nucleic acid constant temperature amplification and fluorescence detection. The constant temperature amplification chamber 6 has a transparent area for detection. The constant temperature amplification chamber 6 is connected to the outlet through hole 14.

[0036] Among them, the sample inlet can be driven by an external fluid to sequentially introduce the nucleic acid sample to be concentrated, the washing liquid and the elution liquid into the purification and concentration chamber, and the waste liquid flows to the waste liquid pool through the reversing valve, and the eluted nucleic acid solution flows to the next unit by switching the reversing valve.

[0037] The temperature of the isothermal amplification chamber can be maintained at a reaction condition of 37° C. by contacting an external isothermal control plate, and the chip is placed in a fluorescence detection platform to detect changes in fluorescence intensity in real time to reflect the cyclic amplification situation.

[0038] In a preferred embodiment, a waste liquid pool 10 is provided on the waste liquid outlet.

[0039] In a preferred embodiment, the reversing valve includes a cylindrical reversing valve body 3, the cover layer 2 is provided with a cylindrical through hole 11 which cooperates with the reversing valve body 3, the base layer 1 is correspondingly provided with a circular cavity 4 which cooperates with the reversing valve body 3, and a three-way flow channel is opened on the reversing valve body. By rotating the reversing valve body, the purification and concentration chamber 8 can select one of the waste liquid outlet or the premixing chamber through hole 13 through the three-way flow channel.

[0040] In a preferred embodiment, the three-way flow channel is T-shaped, and the opening cross-section of the flow channel is square.

[0041] In a preferred embodiment, there are multiple groups of the premixing chamber through holes 13 and the constant temperature amplification chamber 6, each of the premixing chamber through holes 13 is connected to the constant temperature amplification chamber 6 through a corresponding flow connection, and the reversing valve is connected to each of the premixing chamber through holes 13 through a corresponding branch of the diversion channel 12.

[0042] In a preferred embodiment, the premixing chamber through hole 13 is used to add a buffer and a DNA polymerase premixed with specific primers and to apply an external driving pressure.

[0043] In a preferred embodiment, the scaffold material forming the porous material is an organic or inorganic material that can be 3D printed, has no adsorption effect on nucleic acids, and can be prepared into a porous structure by freeze-drying technology.

[0044] In a preferred embodiment, the porous material is prepared by mixing a printing material that has no adsorption effect on nucleic acids in 1,4-dioxane to form a solution with a concentration of 5%-20%, and then mixing the solution and immunomagnetic beads at a mass ratio of 4-10:1, and then obtaining the porous material by low-temperature deposition 3D printing. Preferably, the printing material is PLGA, and preferably, the diameter of the immunomagnetic beads is 100nm-5μm.

[0045] In a preferred embodiment, the base layer, the cover plate layer and the valve body of the reversing valve are made of polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS.

[0046] The present invention also provides a nucleic acid amplification detection method, which uses the microfluidic chip for nucleic acid amplification detection described in any of the above embodiments to perform nucleic acid amplification detection, comprising the following steps:

[0047] 1) injecting the sample to be tested into the purification and concentration chamber from the sample inlet, and the waste liquid flows to the waste liquid pool through the reversing valve;

[0048] 2) Injecting a washing liquid from the sample inlet to wash the purification and concentration chamber, and the waste liquid flows to a waste liquid tank;

[0049] 3) injecting an eluent mixed with Mg+ from the injection port and slowly flowing it through the purification and concentration chamber; when the liquid is about to be discharged from the purification and concentration chamber, switching the reversing valve to allow the eluted nucleic acid to flow into the premixing chamber through hole;

[0050] 4) injecting a buffer solution mixed with primers and DNA polymerase into the premixing chamber through hole, and then immediately driving the fluid in the premixing chamber through hole into the mixing channel, and then into the constant temperature amplification chamber;

[0051] 5) Control the temperature of the isothermal amplification chamber to 37° C., observe the fluorescence intensity of the reactants under a microscope, and quantitatively analyze the nucleic acid concentration. The nucleic acid detection method is based on real-time fluorescent nucleic acid amplification detection technology.

[0052] Compared with traditional technologies, the embodiments of the present invention combine nucleic acid purification and concentration with real-time fluorescent nucleic acid constant temperature amplification detection technology into the same microfluidic chip, which will greatly reduce personnel operations, shorten detection time, and quickly, easily and reliably obtain one or more target detection results. At the same time, it can also give full play to the advantages of low cost, high throughput, automation and other advantages of microfluidic chip technology itself, meeting the needs of rapid diagnosis in clinical practice.

[0053] The detection device and the detection process of the specific embodiment of the present invention are further described below in conjunction with the accompanying drawings.

[0054] A specific embodiment of the present invention provides a nucleic acid detection chip that integrates nucleic acid purification and concentration and real-time fluorescent nucleic acid isothermal amplification. Figure 1 The figure shows a schematic diagram of the decomposed structure of the nucleic acid detection chip, wherein the chip includes a base layer 1, a cover layer 2, and a reversing valve body 3 embedded in the two layers.

[0055] like Figure 2 As shown, the base layer 1 includes a circular cavity 4 that cooperates with the reversing valve body 3, a plurality of mixing flow channels 5 and a constant temperature amplification chamber 6, and a plurality of flow channels that are interconnected.

[0056] like Figure 3 As shown, the cover layer 2 includes an injection port 7, a purification and concentration chamber 8, a cylindrical through hole 11 that cooperates with the reversing valve body 3, a waste liquid pool 10, a diversion channel 12, a plurality of pre-mixing chamber through holes 13 and a plurality of through holes 14 corresponding to the end flow channel of the constant temperature amplification chamber 6.

[0057] like Figure 4The figure shows the structure of the reversing valve body 3, with a T-shaped channel 15 with a square cross section opened at the lower end, and the communication direction is consistent with the arrow 16 on the rotating head of the valve body.

[0058] The materials of the base layer, cover layer and the valve body of the reversing valve 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 used; for example, the injection molding method is used to pre-process the mold, and then the polypropylene PP material is melted at 220-280°C in a constant temperature barrel, 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 chip assembly, it is necessary to print a PLGA scaffold mixed with immunomagnetic beads with a diameter of 100nm-5μm in the purification and concentration chamber 8 by 3D printing, wherein the PLGA scaffold is prepared by mixing PLGA in 1,4-dioxane to prepare a solution with a concentration of 5%-20%, and then the solution and immunomagnetic beads are mixed at a mass ratio of 4-10:1, and then printed in a low-temperature deposition printer; then the cover layer 2 is placed in a freeze dryer for freeze drying to form a porous scaffold 9 embedded with immunomagnetic beads. Then, the base layer 1 and the cover layer 2 containing the porous support 9 are assembled in a plastic hot pressing machine, and then the reversing valve body 3 is directly embedded in the chip to form a Figure 5 The assembled chip 17 is shown.

[0059] like Figure 6 As shown, the reversing valve is in the initial state 22 to connect the purification and concentration chamber 8 and the waste liquid pool 10 , and the reversing valve body is rotated 390° counterclockwise to the reversing state 23 to connect the purification and concentration chamber 8 and the diversion channel 12 .

[0060] exist Figure 5In the top view 18 of the chip shown, the chip is divided into three units according to function: a nucleic acid purification and concentration unit 19, a reactant mixing unit 20, and a nucleic acid constant temperature amplification and detection unit 21. The nucleic acid purification and concentration unit is pre-embedded with porous materials, and the porous materials are embedded with immunomagnetic beads; the reactant mixing unit includes a plurality of pre-mixing chamber through holes and a mixing flow channel, which can be used for adding and mixing a variety of reactants, and the mixing flow channel is directly connected to the nucleic acid constant temperature amplification and detection unit; the nucleic acid constant temperature amplification and detection unit includes a plurality of constant temperature amplification chambers, involving external equipment such as constant temperature control and fluorescence detection; the units on the chip are connected through flow channels, and the fluid is driven to flow through each unit in sequence through valves integrated on the chip and external drive. The pre-mixing chamber through hole can collect the nucleic acid solution flowing from the nucleic acid purification and concentration unit, and the buffer solution and DNA polymerase pre-mixed with specific primers are added in sequence through a discharge gun, and then the mixture is immediately mixed through the mixing flow channel and enters the next unit; the driving method of the mixed liquid can adopt various methods such as low-speed centrifugation and external pressure drive, and the high-throughput fluid drive method is preferred.

[0061] Preferably, the nucleic acid detection step specifically includes:

[0062] 1) Using an injection gun, 500 μL of the sample to be tested is injected from the injection port 7 of the nucleic acid purification and concentration unit 19 into the purification and concentration chamber 8 at a speed of 200 μL / min, and the waste liquid flows to the waste liquid pool through the reversing valve;

[0063] 2) 100 μL of washing solution is perfused into the nucleic acid purification and concentration unit 19 at a rate of 200 μL / min for washing, and the waste liquid flows into the waste liquid pool;

[0064] 3) 50 μL of eluent mixed with Mg+ is perfused into the nucleic acid purification and concentration unit 19 at a speed of 50 μL / min, and the eluent is allowed to slowly flow through the purification and concentration chamber 8; when the residual washing liquid is about to be discharged, the reversing valve is turned to the reversing state 23, and the eluted nucleic acid flows into the reactant mixing unit 20, and flows to each pre-mixing chamber through hole 13 through the diversion channel 12; the reversing valve is reset to the initial state 22;

[0065] 4) Use a discharge gun to quickly inject the buffer solution mixed with primers and DNA polymerase into each premixing chamber through hole 13 of the reactant mixing unit, and then use the discharge gun to apply pressure above each premixing chamber through hole 13, so that the fluid quickly flows into the mixing channel 5 and enters the constant temperature amplification chamber 6 in the nucleic acid constant temperature amplification and detection unit 21;

[0066] 5) The temperature of the nucleic acid isothermal amplification and detection unit 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.

[0067] The present invention combines real-time fluorescent nucleic acid isothermal amplification and detection technology with a microfluidic chip, and also integrates the nucleic acid purification and concentration process on the chip, which can quickly, easily and reliably obtain one or more target detection results, greatly simplifying the tedious personnel operations, and can fully utilize the advantages of microfluidic chip technology such as low cost, low reagent consumption, high throughput, and automated process, while giving full play to the advantages of fluorescent nucleic acid isothermal amplification and detection technology such as fast, simple, stable, and reliable, to meet the needs of rapid detection in clinical and epidemic detection.

[0068] The background section of the present invention may include background information about the problem or environment of the present invention, but does not necessarily describe the prior art. Therefore, the inclusion of content 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 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. A microfluidic chip for nucleic acid amplification detection, characterized in that: The invention comprises a base layer, a cover plate layer and a reversing valve, wherein the base layer and the cover plate layer are a long strip-shaped structure which are laminated and pressed together, the cover plate layer is provided with an injection port, a purification and concentration chamber, a waste liquid outlet, a premixing chamber through hole and an outlet through hole, the injection port and the waste liquid outlet are respectively arranged on both sides of the width direction of the long strip-shaped structure, the purification and concentration chamber extends along the width direction of the long strip-shaped structure, and is connected to one side of the premixing chamber through hole through a channel extending along the length direction of the long strip-shaped structure, the injection port is respectively connected to the waste liquid outlet and the premixing chamber through hole after passing through the purification and concentration chamber through a flow channel, a porous material embedded with immunomagnetic beads is pre-installed in the purification and concentration chamber, and the porous material is a printing material which has no adsorption effect on nucleic acid mixed in 1,4 A solution with a concentration of 5%-20% is prepared in dioxane, and then the solution and the immunomagnetic beads are mixed at a mass ratio of 4-10:1, and then a porous material is obtained by low-temperature deposition 3D printing, wherein the scaffold material forming the porous material is an organic or inorganic material that can be 3D printed, has no adsorption effect on nucleic acids, and can be prepared into a porous structure by freeze-drying technology, and the reversing valve is arranged on the channel extending along the length direction of the long strip plate-shaped structure to switch and control the liquid in the purification and concentration chamber to flow to the waste liquid outlet or the pre-mixing chamber through hole, and the pre-mixing chamber through hole is used to add reactants to mix with the liquid from the purification The liquid in the concentration chamber is mixed, and a mixing flow channel extending continuously along the length direction of the long strip plate-shaped structure and a constant temperature amplification chamber for nucleic acid constant temperature amplification and fluorescence detection are arranged on the base layer on the other side of the pre-mixing chamber through hole. The outlet through hole is arranged at a position connected to the extended far end of the constant temperature amplification chamber on the cover layer, thereby forming a distribution in which the channel, the reversing valve, the mixing flow channel, the pre-mixing chamber through hole, the constant temperature amplification chamber and the outlet through hole are unidirectionally extended in series along the length direction on the long strip plate-shaped structure, and the constant temperature amplification chamber has a transparent area, and the constant temperature amplification chamber is connected to the outlet through hole.

2. The microfluidic chip for nucleic acid amplification detection according to claim 1, characterized in that: A waste liquid pool is arranged on the waste liquid outlet.

3. The microfluidic chip for nucleic acid amplification detection according to claim 1 or 2, characterized in that: The reversing valve includes a cylindrical reversing valve body, the cover plate layer is provided with a cylindrical through hole that cooperates with the reversing valve body, the base layer is correspondingly provided with a circular cavity that cooperates with the reversing valve body, and the reversing valve body is provided with a three-way flow channel. By rotating the reversing valve body, the purification and concentration chamber can select one of the waste liquid outlet or the premixing chamber through the three-way flow channel.

4. The microfluidic chip for nucleic acid amplification detection according to claim 3, characterized in that: The three-way flow channel is T-shaped, and the opening cross section of the flow channel is square.

5. The microfluidic chip for nucleic acid amplification detection according to any one of claims 1 to 2, characterized in that: There are multiple groups of premixing chamber through holes and the constant temperature amplification chamber, each of the premixing chamber through holes is connected to the constant temperature amplification chamber through a corresponding flow connection, and the reversing valve is connected to each of the premixing chamber through holes through a corresponding branch of a diversion channel.

6. The microfluidic chip for nucleic acid amplification detection according to any one of claims 1 to 2, characterized in that: The through hole of the premixing chamber is used to add a buffer and a DNA polymerase premixed with specific primers and to apply an external driving pressure.

7. The microfluidic chip for nucleic acid amplification detection according to any one of claims 1 to 2, characterized in that: The printing material is PLGA, and the diameter of the immunomagnetic beads is 100nm-5μm.

8. The microfluidic chip for nucleic acid amplification detection according to any one of claims 1 to 2, characterized in that: The materials of the base layer, the cover plate layer and the valve body of the reversing valve are polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC) or ABS.

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