A fully enclosed microfluidic chip for nucleic acid amplification detection

Abstract

The invention discloses a fully enclosed microfluidic chip for nucleic acid amplification detection, comprising a reaction layer, a first adhesive layer, an elastic film, a second adhesive layer and a sample loading layer which are sequentially attached from bottom to top, wherein a pressure cover is snapped on the top of the sample loading layer; a sample inlet, a reaction pool, a gas filter pool, an inflow channel and an outflow channel are provided in the middle of the top surface of the reaction layer; a free-form notch is provided on the first adhesive layer; an air inlet through hole and a control channel connected with the air inlet through hole are also provided on the second adhesive layer, and the control channel passes through the top of a plurality of inflow channels in sequence; a sample loading pool is provided on the sample loading layer, and an exhaust hole corresponding to the gas filter pool is provided on the sample loading layer; an air inlet channel corresponding to the air inlet through hole is provided on the sample loading layer, and an air bag is fixed in the air inlet channel; a flow stop portion corresponding to a plurality of gas filter pools is provided on the top surface of the sample loading layer; and a raised pressure plug is provided on the inner side of the pressure cover.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical detection and microfluidic chips, and more specifically to a fully enclosed microfluidic chip for nucleic acid amplification detection. Background Art

[0002] As one of the main methods of in vitro diagnosis, nucleic acid testing is the gold standard for screening, prevention and control of infectious diseases. Conventional PCR nucleic acid testing experiments require each step to be completed in a partitioned laboratory, and require professional operators and expensive analytical instruments, which cannot be met by primary medical institutions.

[0003] POCT (Point-of-Care Testing) refers to rapid testing and diagnosis performed at the patient's side. Its technical advantages are mainly reflected in: simple operation, no need for professionals; reduced sample transmission, saving testing time; portable instruments and reagents. Nucleic acid testing products based on POCT technology can be said to have the characteristics of high sensitivity and high specificity of molecular diagnosis and the advantages of POCT being small, portable and fast, which is more conducive to the deployment of primary medical institutions, airports, communities and other places.

[0004] Microfluidic chip technology integrates the basic operating units of biological, chemical and medical analysis processes such as sample preparation, reaction, separation and detection into a chip of several centimeters in size, and automatically completes the entire analysis process. It has the advantages of small size, low sample consumption and fast analysis speed. It has broad application prospects in the functional integration of POCT products, especially nucleic acid analysis POCT products with complex processes and high system sealing requirements.

[0005] Through microfluidic manipulation technology, trace liquid samples can be distributed to different reaction pools on the microfluidic chip to achieve simultaneous detection of multiple pathogens. The heating process in the nucleic acid amplification process will accelerate the evaporation of trace sample reagents and the cross-liquidity between different reaction pools, causing interference with the result detection. Therefore, the separation of different reaction pools during the amplification process plays an important role. Furthermore, the application occasions of microfluidic chips used for POCT nucleic acid detection are non-professional environments such as communities and families, which do not have the ability to prevent aerosol pollution. Therefore, the sealing of such microfluidic chips is also an important performance evaluation indicator.

[0006] Microvalve is one of the most important functional units in microfluidic chips for realizing microfluidic manipulation. Its basic function is to realize the conduction and isolation of microfluidic channels. Designing different valve structures in microchannels has become the main technical approach for the current development of nucleic acid detection microfluidic chips to achieve amplification pool isolation, including torque microvalves and phase change microvalves, but both require peripheral instruments to provide actuation force to complete the opening and closing of valves one by one, resulting in an increase in the number of manipulation steps with the number of reaction pools, high requirements for instrument automation, complex chip processing technology, and high cost.

[0007] The pneumatic valve proposed by Quake's research group in 2000 has the advantages of simple structure, fast response speed, and easy large-scale integration. It is very suitable for realizing the one-time partition of multiple amplification pools for nucleic acid detection. However, the reason why this technology has not yet been applied in the field of POCT molecular detection is that the air pressure source of the pneumatic valve is difficult to integrate into the microfluidic chip. After the detection is completed, the chip needs to be separated from the external air pressure source. The anti-aerosol contamination function of the pneumatic valve will also fail. In addition, it is difficult to avoid the problem of biological contamination caused by gas overflow by relying only on a single-layer pneumatic valve during the amplification process.

[0008] Therefore, how to provide a fully enclosed microfluidic chip integrated with an equipment-free pneumatic valve that is of great value to the development of the POCT nucleic acid detection field is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0009] In view of this, the present invention provides a fully enclosed microfluidic chip for nucleic acid amplification detection, aiming to solve the above technical problems.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] A fully enclosed microfluidic chip for nucleic acid amplification detection, comprising a reaction layer, a first adhesive layer, an elastic film, a second adhesive layer and a sample loading layer which are sequentially attached from bottom to top, wherein a pressure cover is snap-fitted on the top of the sample loading layer;

[0012] The middle part of the top surface of the reaction layer is provided with an injection port, a plurality of reaction pools arranged around the injection port, and a number of gas filtering pools arranged around the plurality of reaction pools, which are the same as the number of the reaction pools; the injection port is connected to the reaction pools through inflow channels, respectively, and the reaction pools are connected to the gas filtering pools through outflow channels, respectively;

[0013] The first adhesive layer is provided with conformal notches corresponding to the injection port, the inflow channel, the reaction pool, the outflow channel and the gas filter pool;

[0014] The elastic film is provided with a first through hole corresponding to the injection port and a second through hole corresponding to the gas filter pool;

[0015] The second adhesive layer is provided with a third through hole corresponding to the injection port and a fourth through hole corresponding to the gas filter pool; the second adhesive layer is also provided with an air intake through hole and a control channel connected to the air intake through hole, and the control channel passes through the top of the plurality of inflow channels in sequence;

[0016] The sample loading layer is provided with a sample loading pool which is connected vertically and corresponds to the sample inlet, and the sample loading layer is provided with exhaust holes which are connected vertically and correspond to the plurality of gas filtering pools; the sample loading layer is provided with an air inlet channel which corresponds to the air inlet through hole, and an air bag is fixed in the air inlet channel; the top surface of the sample loading layer is provided with a flow stopper which corresponds to the plurality of gas filtering pools;

[0017] The inner side of the gland is provided with a raised pressure plug for pushing the airbag.

[0018] Through the above technical scheme, the present invention provides a fully enclosed microfluidic chip for nucleic acid amplification detection, which is internally integrated with a pneumatic valve and an airbag structure for controlling the pneumatic valve, and can achieve the isolation of the reaction pool and the full closure of the chip without the need for peripheral driving instruments and equipment, thereby effectively avoiding evaporation, cross-liquid and aerosol pollution problems during nucleic acid detection. The chip is small in size, simple in sample addition, and has good sealing performance, and is very suitable for POCT nucleic acid detection, such as PCR amplification, LAMP, RPA, CRISPR isothermal amplification, etc. In addition, the chip has a regular appearance, a simple structure, and no requirements for equipment in the fluid drive and sealing process, so it is easy to match with different systems, and can also meet the requirements of batch manufacturing and low cost.

[0019] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the side wall of the pressure cover is connected to the side wall of the sample loading layer via a flexible hinge, so as to achieve an integrated connection between the pressure cover and the sample loading layer.

[0020] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the inner side wall of the gland has a plurality of buckles, and the side wall of the sample loading layer has a plurality of slots that match the buckles. The slots on the gland completely match the buckles on the sample loading layer, and the gland assists in squeezing and sealing the sample loading layer, so that the sample added to the chip is completely isolated from the external environment.

[0021] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the middle part of the inner side of the gland has a raised embedded ring, a sealing gasket is embedded in the embedded ring, and the sealing gasket is pressed tightly at the top opening of the sample pool. The sealing gasket is tightly attached to the top of the sample pool to seal the sample pool.

[0022] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the top surface of the sample loading layer has a sealing ring pressed between the stopper and the gland; the sealing ring is provided with an escape hole for escaping the pressure plug. The sealing ring is in close contact with the stopper to seal the exhaust hole.

[0023] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the number of the reaction pools is 4, and they are evenly distributed on the reaction layer in the circumferential direction. In practical applications, the number of reaction pools can be increased accordingly according to demand to achieve joint detection of multiple pathogens.

[0024] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the reaction layer, the first adhesive layer, the elastic film, the second adhesive layer, the sample loading layer and the gland are all circular, so that the overall structure is more regular and more convenient to use.

[0025] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the flow stop portion is an annular hydrophobic breathable membrane; or the flow stop portion is an annular water-absorbing membrane, and both the hydrophobic breathable membrane and the water-absorbing membrane have anti-interference through holes for avoiding the pressure plug; or the flow stop portion is a waste liquid pool formed on the top surface of the sample loading layer and connected to the plurality of exhaust holes, and the waste liquid pool avoids the air inlet channel.

[0026] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the elastic film material may be polydimethylsiloxane, silicone rubber, memory alloy or polytetrafluoroethylene.

[0027] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the control channel and the inflow channel are arranged to intersect, and the elastic film side wall of the control channel is a common wall where the control channel and the inflow channel intersect.

[0028] Preferably, in the above-mentioned fully enclosed microfluidic chip for nucleic acid amplification detection, the material of the airbag can be polydimethylsiloxane, silicone rubber, memory alloy or polytetrafluoroethylene, and its volume can be reduced or increased under the action of pressure.

[0029] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a fully enclosed microfluidic chip for nucleic acid amplification detection, which has the following beneficial effects:

[0030] 1. The pneumatic microvalve and the air pressure source are integrated in the chip. The side wall above the reaction layer is composed of an elastic film. After the sample is injected, the elastic film above the inflow channel is deformed and blocked by changing the air pressure, so that multiple reaction pools with different detection indicators can be separated at the same time. Furthermore, the airbag structure is integrated in the chip, and the closing action can realize the squeezing of the airbag to provide an air pressure source for the air pressure valve without any external air source equipment. The simple and ingenious design breaks through the application limitations of pneumatic microvalves in POCT nucleic acid detection microfluidic chips, which is a milestone in this technical field.

[0031] 2. The sealing component is set up to fix the sealing gasket and the sealing ring inside the pressure cover. The positions of the sealing gasket and the sealing ring correspond to the sample injection port and the exhaust hole respectively. It only needs to be simply closed. With the assistance of the buckle on the outer periphery of the pressure cover, the pressure cover keeps squeezing the soft sealing ring to achieve complete sealing of the internal channel of the chip, keep the amplification product isolated from the external environment during the nucleic acid amplification process, and eliminate the aerosol contamination problem in an extremely simple way.

[0032] 3. It adopts a multi-channel microfluidic system. The sample flows from the central sample inlet into multiple channels distributed in a circle. You only need to align the sample tube with the sample pool and squeeze it to achieve the distribution of the sample in multiple reaction pools. No complicated peripheral equipment is required to complete the sample addition process. The operation is convenient and fast, and even personnel without relevant professional background can operate it. It is very suitable for POCT scenarios.

[0033] 4. A closed membrane or waste liquid pool is set above the pores. During the extrusion sampling process, the liquid sample flows into the channel and the reaction pool, and the gas in the channel and the reaction pool is discharged from the pores. The pore-sealing membrane has the function of being breathable but impermeable or saturated with water. After the sample fills the reaction pool, it can prevent the sample from continuing to flow out of the pores. The waste liquid pool can carry the liquid sample that overflows from the amplification chamber in advance. Whether it is a closed membrane or a waste liquid pool, accurate quantification of each amplification reaction pool is guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0035] Figure 1 The accompanying drawing is an exploded structural diagram of the microfluidic chip provided by the present invention;

[0036] Figure 2 The accompanying drawing is a schematic diagram of the cross-sectional structure of the microfluidic chip provided in Example 1 of the present invention;

[0037] Figure 3 The accompanying drawing is a schematic diagram of the cross-sectional structure of a microfluidic chip provided in Example 2 of the present invention;

[0038] Figure 4 The accompanying drawing is a schematic diagram of the cross-sectional structure of the microfluidic chip provided in Example 3 of the present invention.

[0039] in:

[0040] 1-reaction layer;

[0041] 11-inlet; 12-reaction cell; 13-gas filter cell; 14-inflow channel; 15-outflow channel;

[0042] 2- first adhesive layer;

[0043] 21-conforming notch;

[0044] 3- Elastic film;

[0045] 31-first through hole; 32-second through hole;

[0046] 4- second adhesive layer;

[0047] 41-third through hole; 42-fourth through hole; 43-air intake through hole; 44-control channel;

[0048] 5- add sample layer;

[0049] 51-sample adding pool; 52-exhaust hole; 53-air inlet channel; 54-air bag; 55-card slot;

[0050] 6- gland;

[0051] 61-pressure plug; 62-flexible hinge; 63-buckle; 64-embedded ring;

[0052] 7- flow stop;

[0053] 71-hydrophobic breathable membrane; 72-water absorbing membrane; 73-waste liquid pool; 74-anti-interference through hole;

[0054] 8-Sealing gasket;

[0055] 9- Sealing ring;

[0056] 91-Avoidance hole. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] See attached Figure 1 To Attachment Figure 4 The embodiment of the present invention discloses a fully enclosed microfluidic chip for nucleic acid amplification detection, comprising a reaction layer 1, a first adhesive layer 2, an elastic film 3, a second adhesive layer 4 and a sample loading layer 5 which are sequentially attached from bottom to top, and a pressure cover 6 is snap-fitted on the top of the sample loading layer 5;

[0059] The middle part of the top surface of the reaction layer 1 is provided with an injection port 11, a plurality of reaction pools 12 arranged around the injection port 11, and a number of gas filtering pools 13 arranged around the plurality of reaction pools 12, the number of which is the same as the number of the reaction pools 12; the injection port 11 is connected to the reaction pools 12 through an inflow channel 14, respectively, and the reaction pools 12 are connected to the gas filtering pools 13 through an outflow channel 15, respectively;

[0060] The first adhesive layer 2 is provided with a profiled notch 21 corresponding to the sample inlet 11, the inflow channel 14, the reaction pool 12, the outflow channel 15 and the gas filter pool 13;

[0061] The elastic film 3 is provided with a first through hole 31 corresponding to the sample inlet 11 and a second through hole 32 corresponding to the gas filter pool 13;

[0062] The second adhesive layer 4 is provided with a third through hole 41 corresponding to the sample inlet 11, and a fourth through hole 42 corresponding to the gas filter pool 13; the second adhesive layer 4 is also provided with an air inlet through hole 43, and a control channel 44 connected to the air inlet through hole 43, and the control channel 44 passes through the top of the plurality of inflow channels 14 in sequence;

[0063] The sample adding layer 5 is provided with a sample adding pool 51 which is connected from top to bottom and corresponds to the sample inlet 11, and the sample adding layer 5 is provided with an exhaust hole 52 which is connected from top to bottom and corresponds to the multiple air filtering pools 13; the sample adding layer 5 is provided with an air inlet channel 53 which corresponds to the air inlet through hole 43, and an air bag 54 is fixed in the air inlet channel 53; the top surface of the sample adding layer 5 is provided with a flow stopper 7 which corresponds to the multiple air filtering pools 13;

[0064] The inner side of the pressure cover 6 has a raised pressure plug 61 for pushing the air bag 54 .

[0065] In order to further optimize the above technical solution, the side wall of the pressure cover 6 is connected to the side wall of the sample loading layer 5 via a flexible hinge 62 .

[0066] In order to further optimize the above technical solution, the inner side wall of the pressure cover 6 has a plurality of buckles 63 , and the side wall of the sample loading layer 5 has a plurality of slots 55 that cooperate with the buckles 63 .

[0067] In order to further optimize the above technical solution, a raised embedding ring 64 is provided in the middle of the inner side of the pressure cover 6 , and a sealing gasket 8 is embedded in the embedding ring 64 . The sealing gasket 8 is pressed tightly against the top opening of the sample loading pool 51 .

[0068] In order to further optimize the above technical solution, the top surface of the sample loading layer 5 has a sealing ring 9 pressed between the flow stop portion 7 and the pressure cover 6; the sealing ring 9 is provided with an escape hole 91 for escaping the pressure plug 61.

[0069] In order to further optimize the above technical solution, the number of reaction pools 12 is 4 and they are evenly distributed on the reaction layer 1 in the circumferential direction.

[0070] In order to further optimize the above technical solution, the reaction layer 1, the first adhesive layer 2, the elastic film 3, the second adhesive layer 4, the sample adding layer 5 and the pressure cover 6 are all circular. Embodiment 1:

[0071] See attached Figure 2 When adding samples, the sample tube is aligned with the sample pool 51, and the liquid sample of the sample tube enters the reaction pool 12 by squeezing the sample tube, and the gas in the channel and the reaction pool is discharged through the exhaust hole 52. The hydrophobic breathable membrane 71 on the exhaust hole 52 is breathable but not water-permeable. When the sample fills the reaction pool 12 and reaches the exhaust hole 52, the hydrophobic breathable membrane 71 prevents the sample from continuing to flow in. When all channel samples reach this state, the sample addition action is completed. Before the amplification reaction is started, the pressure cover 6 is closed. When the pressure plug 61 on the pressure cover 6 contacts the air bag 54 and squeezes the air bag 54, the pressure in the control channel 44 increases, and the elastic film 3 above the corresponding inflow channel 14 is deformed, and the concave concave blocks the inflow channel 14, completing the partition between different amplification chambers. At the same time, the buckle 63 on the pressure cover 6 is snapped into the card slot 55 on the sample loading layer 5, completing the full sealing of the entire chip, and the amplification system in the chip is completely isolated from the external environment. Embodiment 2:

[0072] See attached Figure 3 This embodiment optimizes the structure of the flow stop 7. When the sample is squeezed, the liquid sample fills the reaction pool 12 and reaches the exhaust hole 52. When the sample is added, the water absorption membrane 72 on the exhaust hole 52 absorbs the liquid sample. When the water absorption membrane 72 is saturated with liquid, the sample can be stopped from continuing to flow in. When all channel samples reach this state, the sample addition action is completed. Before the amplification reaction is started, the pressure cover 6 is closed, and the pneumatic valve is started to block the inflow channel 14 to achieve the isolation of different reaction pools 12, and at the same time, the whole chip is fully sealed. Embodiment 3:

[0073] See attached Figure 4The present embodiment optimizes the structure of the flow stopper 7. A circular waste liquid pool 73 is provided above the sample loading layer 5. The waste liquid pool 73 is connected to all the exhaust holes 52. When the sample is squeezed, the liquid sample fills the reaction pool 12 and reaches the exhaust hole 52, and then flows into the waste liquid pool 73. The sample loading process is completed after the liquid samples in all the reaction pools 12 are filled and flow into the waste liquid pool 73. Before the amplification reaction is started, the pressure cover 6 is closed, and the pneumatic valve is started to block the inflow channel 14 to achieve the isolation of different reaction pools 12, and at the same time, the entire chip is fully sealed.

[0074] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0075] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fully enclosed microfluidic chip for nucleic acid amplification detection, It is characterized in that It comprises a reaction layer (1), a first adhesive layer (2), an elastic film (3), a second adhesive layer (4) and a sample loading layer (5) which are sequentially attached from bottom to top, wherein the top of the sample loading layer (5) is snap-fitted with a pressure cover (6); The reaction layer (1) is provided with a sample inlet (11) in the middle of the top surface, and a plurality of reaction pools (12) arranged around the sample inlet (11), and a number of gas filter pools (13) arranged around the plurality of reaction pools (12) being the same as the number of the reaction pools (12); the sample inlet (11) and the reaction pools (12) are respectively connected via inflow channels (14), and the reaction pools (12) and the gas filter pools (13) are respectively connected via outflow channels (15); The first adhesive layer (2) is provided with conformal notches (21) corresponding to the sample inlet (11), the inflow channel (14), the reaction pool (12), the outflow channel (15) and the gas filter pool (13); The elastic film (3) is provided with a first through hole (31) corresponding to the sample inlet (11), and a second through hole (32) corresponding to the gas filter pool (13); The second adhesive layer (4) is provided with a third through hole (41) corresponding to the sample inlet (11), and a fourth through hole (42) corresponding to the gas filter pool (13); the second adhesive layer (4) is also provided with an air intake through hole (43) and a control channel (44) connected to the air intake through hole (43), and the control channel (44) passes through the top of the plurality of inflow channels (14) in sequence; The sample loading layer (5) is provided with a sample loading pool (51) which is connected vertically and corresponds to the sample inlet (11); the sample loading layer (5) is provided with an exhaust hole (52) which is connected vertically and corresponds to the plurality of gas filtering pools (13); the sample loading layer (5) is provided with an air intake channel (53) which corresponds to the air intake through hole (43); an air bag (54) is fixed in the air intake channel (53); and the top surface of the sample loading layer (5) has a flow stopper (7) which corresponds to the plurality of gas filtering pools (13); The inner side of the gland (6) has a protruding pressure plug (61) for pushing the airbag (54); The flow-stopping portion (7) is an annular hydrophobic and breathable membrane (71), and the hydrophobic and breathable membrane (71) is provided with an anti-interference through hole (74) for avoiding the pressure plug (61); Alternatively, the flow-stopping portion (7) is an annular water-absorbing membrane (72), and the water-absorbing membrane (72) has an anti-interference through hole (74) for avoiding the pressure plug (61); Alternatively, the flow stop portion (7) is a waste liquid pool (73) formed on the top surface of the sample loading layer (5) and connected to the plurality of exhaust holes (52), and the waste liquid pool (73) avoids the air inlet channel (53); The side wall of the pressure cover (6) is connected to the side wall of the sample loading layer (5) via a flexible hinge (62); The middle part of the inner side of the pressure cover (6) has a raised embedded ring (64), a sealing gasket (8) is embedded in the embedded ring (64), and the sealing gasket (8) is pressed tightly against the top opening of the sample loading pool (51).

2. A fully enclosed microfluidic chip for nucleic acid amplification detection according to claim 1, It is characterized in that The inner side wall of the pressure cover (6) has a plurality of buckles (63), and the side wall of the sample loading layer (5) has a plurality of slots (55) that cooperate with the buckles (63).

3. A fully enclosed microfluidic chip for nucleic acid amplification detection according to claim 1, It is characterized in that The top surface of the sample loading layer (5) has a sealing ring (9) pressed between the flow stop portion (7) and the pressure cover (6); the sealing ring (9) is provided with an escape hole (91) for evading the pressure plug (61).

4. A fully enclosed microfluidic chip for nucleic acid amplification detection according to claim 1, It is characterized in that The number of the reaction pools (12) is 4, and they are evenly distributed in the circumferential direction on the reaction layer (1).

5. A fully enclosed microfluidic chip for nucleic acid amplification detection according to claim 1, It is characterized in that The reaction layer (1), the first adhesive layer (2), the elastic film (3), the second adhesive layer (4), the sample addition layer (5) and the pressure cover (6) are all circular.

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