WO2024212855A1 - Totally-enclosed fully-integrated nucleic acid testing micro-fluidic chip - Google Patents

Abstract

Disclosed is a totally-enclosed fully-integrated nucleic acid testing micro-fluidic chip, relating to the field of biomedical testing and the field of micro-fluidic chips, and comprising a sample loading layer, a sample injection layer, a gas channel layer, a reaction layer, and a sealing layer which are sealedly attached from top to bottom in sequence. A control assembly, a reagent assembly, a sample injection assembly and a valve assembly are integrated on the top surface of the sample loading layer; a waste liquid pool, an extraction pool and a testing area which are communicated in sequence are formed in the surface of the reaction layer; a central channel corresponding to the extraction pool and valve control channels used for the valve assembly to pass through are formed in the sample loading layer, the sample injection layer, and the gas channel layer; liquid cavities communicated with the central channel are formed in the surface of the sample injection layer, piercing bodies corresponding to the reagent assembly and the sample injection assembly are provided in the liquid cavities, and gas channels communicated with the central channel, the waste liquid pool and the testing area are formed in the sample injection layer and the gas channel layer. The provided nucleic acid testing chip is simple to operate and easily portable, multi-index testing meets the requirements of different application scenarios, and the present invention is particularly suitable for the field of in-vitro diagnosis POCT.

Description

A fully enclosed and fully integrated nucleic acid detection microfluidic chip Technical Field

The present invention relates to the fields of biomedical detection and microfluidic chips, and more specifically to a fully enclosed and fully integrated nucleic acid detection microfluidic chip.

Background Art

Nucleic acid testing is one of the important methods of in vitro diagnosis. It plays an important role in the diagnosis and prognosis of diseases. In addition, it is also widely used in other fields, such as public health, food safety, and forensic identification. The current disease diagnosis process based on nucleic acid analysis includes sample collection, transportation, and laboratory nucleic acid testing. The standard nucleic acid testing steps in the laboratory are divided into sample pretreatment, nucleic acid extraction and purification, nucleic acid amplification, and detection. Since the entire operation process involves the exposure of reagents and samples, strict requirements are placed on the operating environment. On the one hand, it needs to be completed in professional partitioned experiments, and on the other hand, professional operators are required to complete the operation. These restrictions hinder the popularization of nucleic acid testing in different scenarios and regions, such as secondary and lower community hospitals. The fully integrated nucleic acid testing system has the ability of "sample in-result out", which can effectively solve the problems of high laboratory construction and personnel investment, and further realize POCT (Point-of-Care Testing, also known as instant testing) applications.

Microfluidic chip technology uses microchannels, reaction chambers and other functional components to precisely control fluids, integrating basic operating units such as sample preparation, reaction, separation and detection for chemical, biological and medical analysis onto a chip a few centimeters in size, and automatically completing the entire analysis process. Compared with traditional analysis technologies, microfluidic chips and their supporting instruments have the advantages of miniaturization, low consumption and full automation, greatly reducing dependence on hardware equipment and professionals, and are particularly suitable for scenarios where a variety of medical facilities are not perfect and where rapid on-site testing is required.

From the perspective of technical advantages, the nucleic acid detection system based on microfluidic chips can effectively solve the pain points of traditional methods. However, there are still many challenges in realizing a fully integrated nucleic acid detection process on a microfluidic chip. The microfluidic chips currently proposed are more about realizing partial functions and do not fully meet the actual needs. It is difficult to replace traditional methods to meet the detection needs.

The requirements for a fully integrated nucleic acid detection microfluidic chip that can achieve "fast, convenient, accurate and cheap" include: 1. It includes all the processes and reagents of sample lysis-nucleic acid extraction and purification-amplification detection. The operator only needs to add the sample, and the sample goes in and the result comes out. The whole process is automated; 2. It is fully sealed. Sample leakage or aerosol contamination will cause infection of the operator and abnormal test results. The most effective way to avoid this is to isolate the entire chip environment from the external environment to truly achieve zero pores; 3. It is low-cost. Disposable microfluidic chips are used as consumables. Low material cost is a prerequisite for its popularization.

Therefore, proposing a highly practical, fully enclosed and fully integrated nucleic acid detection microfluidic chip is of great significance to disease diagnosis and monitoring, animal epidemics, food safety and other fields.

Summary of the invention

In view of this, the present invention provides a fully enclosed and fully integrated nucleic acid detection microfluidic chip, aiming to solve the above-mentioned technical problems.

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

A fully enclosed and fully integrated nucleic acid detection microfluidic chip, comprising a sample adding layer, a sample injection layer, an airway layer, a reaction layer and a sealing layer which are sealed and bonded in sequence from top to bottom;

The top surface of the sample adding layer is integrated with a control component, a reagent component, a sample injection component and a valve component;

The surface of the reaction layer is provided with a waste liquid pool, an extraction pool and a detection area which are connected in sequence;

The sample loading layer, the sample injection layer and the airway layer are provided with a central channel corresponding to the extraction pool, and a valve control channel for the valve assembly to pass through; the central channel controls the on-off of the gas path and the fluid channel by the up-and-down movement of the control assembly, and the valve assembly controls the on-and-off of the fluid channel between the waste liquid pool and the extraction pool, and between the extraction pool and the detection area by the up-and-down movement;

A liquid cavity connected to the central channel is provided on the surface of the injection layer, and a puncture body corresponding to the reagent component and the injection component is provided in the liquid cavity. A gas channel connected to the central channel, the waste liquid pool and the detection area is provided on the injection layer and the airway layer.

Through the above technical scheme, the present invention provides a fully enclosed and fully integrated nucleic acid detection microfluidic chip, on which reagents required for the entire nucleic acid detection process are pre-embedded and infused, and a gas internal circulation structure is provided. In a fully sealed state, in conjunction with dedicated instruments, it can automatically complete sample lysis, nucleic acid extraction and purification, amplification and real-time detection, truly realizing "sample in - result out"; the nucleic acid detection chip is easy to operate and carry, and its multi-index detection meets the needs of different application scenarios, making it particularly suitable for the field of in vitro diagnostic POCT.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the control component includes a threaded control rod, a slider and a piston; the slider is fixed on the sample loading layer, the control rod is threadedly connected to the slider, and the piston is fixed at the bottom end of the control rod.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the reagent components are all cylinders that can pierce the bottom membrane.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the injection assembly includes a barrel body, a screwable barrel cover threadedly connected to the top of the barrel body, and a sealing film fixed to the bottom opening of the barrel body.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the valve-controlled channel includes a through hole opened on the sample loading layer, a threaded through hole on the sample injection layer, and a valve channel opened on the airway layer.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the outer side wall of the valve assembly has threads.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the detection area includes a reconstitution pool connected to the extraction pool, the reconstitution pool is connected to the amplification pool through a sample inflow channel, the amplification pool is connected to the pores through an airway, and the pores are covered with a waterproof and breathable membrane.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the bottom surface of the extraction pool is sloped, and the height of the side away from the channel is higher than the side close to the channel, so as to play a guiding role.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, a gas chamber corresponding to the pore is opened on the airway layer, the top of the gas chamber is connected to the central channel, and a sealing cover is fixed to the inner wall of the bottom of the gas chamber through connecting ribs, the sealing cover corresponds to the pore, and the gas chamber is correspondingly provided with a downward pressure valve stem.

Preferably, in the above-mentioned fully enclosed and fully integrated nucleic acid detection microfluidic chip, the sample introduction layer and the airway layer are provided with a gas channel connecting the central channel and the waste liquid pool.

It can be seen from the above technical solutions that compared with the prior art, the present invention provides a fully sealed The closed fully integrated nucleic acid detection microfluidic chip has the following beneficial effects:

1. Fully sealed design: During the nucleic acid detection process, aerosol contamination caused by leakage of samples or amplification products will directly affect the accuracy of subsequent detection and even cause biological contamination to operators. The existing molecular POCT semi-open system has not truly achieved zero pollution. For this reason, the present invention uses a clever internal circulation design to achieve the completion of the entire nucleic acid analysis process under sealed conditions, eliminating aerosol contamination problems. In order to balance the pressure changes caused by the liquid flowing into the extruded gas, the upper end of the waste liquid pool is connected to the gas chamber at the upper end of the control component through the gas flow channel, and the other side of the chamber is also connected to the gas chamber on the amplification pool pore. The piston of the control component moves up and down to balance the pressure of the gas chamber at its upper end.

2. Integrated design of the entire process of nucleic acid analysis: From sample lysis, extraction and purification to qPCR, in vitro diagnosis is considered the gold standard for the diagnosis of infectious diseases. Traditional operations have extremely high requirements for the experimental environment and professional personnel. Molecular POCT technology based on microfluidics has outstanding advantages in shortening analysis time and saving manpower expenditure. However, due to the complexity of fluid manipulation and high technical barriers, this type of technology currently only realizes some standard nucleic acid analysis functions, greatly reducing the diagnostic value. The present invention adopts a multi-layer structure design and adopts a simple piston assembly, which can be used as both a liquid-driven control assembly and a fluid control valve assembly to realize the quantitative transfer of reagents and samples in the entire process of nucleic acid analysis.

3. Design of sealing cover of amplification pool: Quantification of reaction volume is a prerequisite for accurate analysis of nucleic acid. In the present invention, a hydrophobic breathable membrane is provided on the gas discharge hole inside the amplification pool. When the sample fills the amplification pool, the injection is stopped to achieve quantification. The nucleic acid increases exponentially during the amplification process, which is a high-risk stage for aerosol contamination. For double sealing, the sealing cover is integrated with the airway layer in advance through a clever soft connection design. The connecting rib breaks after pressing, and the sealing cover fits tightly against the hydrophobic membrane on the air hole under the action of the lower pressure rod, independently sealing the amplification area and preventing the reduction of the reaction volume under high temperature conditions.

4. Independent reagent cartridge design: The fully integrated POCT nucleic acid detection process involves multi-step liquid reagents such as cleaning solution, eluent, etc. The microfluidic chip chamber is directly filled with liquid and sealed for storage. It is difficult to avoid leakage during transportation, or a normally closed valve design is required to seal the liquid, which undoubtedly brings about problems of complex structure and difficult processing. The present invention uses an independent reagent cartridge to pre-package liquid reagents, which is connected to the chip body through a snap buckle. There is no need to use a valve structure to achieve stable storage of liquid reagents. When activated, only the reagent cartridge needs to be pressed, and the bottom membrane is pierced by the puncture needle on the chip to release the reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG1 is a schematic diagram of the structure of a microfluidic chip provided by the present invention;

FIG2 is an exploded view of the structure of the microfluidic chip provided by the present invention;

FIG3 is a schematic diagram of the structure of the sample loading layer in the microfluidic chip provided by the present invention;

FIG4 is a schematic diagram of the structure of the sample injection layer in the microfluidic chip provided by the present invention;

FIG5 is a schematic diagram of the airway layer structure in the microfluidic chip provided by the present invention;

FIG6 is a schematic diagram of the reaction layer structure in the microfluidic chip provided by the present invention;

FIG. 7 is a schematic diagram of the structure of the lysis liquid cartridge in the microfluidic chip provided by the present invention.

in:
1- sample layer; 2- sample injection layer; 3- airway layer; 4- reaction layer; 5- sealing layer; 6- reagent assembly; 7-
Injection assembly; 8-control assembly; 9-valve assembly; 10-press valve stem;
21-puncture body; 22-fluid channel; 23-threaded through hole; 24-first gas channel; 25-second gas channel; 26-first gas through hole;
31 - sealing cover; 32 - connecting rib; 33 - gas chamber; 34 - second gas through hole; 35 - valve channel;
36- third gas channel;
41-extraction pool; 42-waste liquid pool; 43-valve position; 44-microchannel; 45-redissolution pool; 46-sample inflow channel; 47-air hole; 48-airway; 49-amplification pool;
61-cleaning liquid cylinder 1; 62-cleaning liquid cylinder 2; 63-elution liquid cylinder;
71-lysate sample cartridge; 711-sealing film; 712-cartridge body; 713-twistable cartridge cover;
81-control rod; 82-slider; 83-piston;
91 - first valve assembly; 92 - second valve assembly.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention will be described below in conjunction with the accompanying drawings in the embodiments of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention.

Referring to Figures 1 to 6, an embodiment of the present invention discloses a fully enclosed and fully integrated nucleic acid detection microfluidic chip, comprising a sample loading layer 1, a sample injection layer 2, an airway layer 3, a reaction layer 4 and a sealing layer 5 which are sealed and bonded in sequence from top to bottom;

The top surface of the sample adding layer 1 is integrated with a control component 8, a reagent component 6, a sample injection component 7 and a valve component 9;

The surface of the reaction layer 4 is provided with a waste liquid pool 42, an extraction pool 41 and a detection area which are connected in sequence;

The sample loading layer 1, the sample injection layer 2 and the airway layer 3 are provided with a central channel corresponding to the extraction pool 41, and a valve-controlled channel for the valve assembly 9 to pass through; the central channel drives the reagent and sample to flow in and out of the amplification pool by the up and down movement of the control assembly 8, and the valve assembly 9 controls the opening and closing of the fluid channel between the waste liquid pool 42 and the extraction pool 41, and between the extraction pool 41 and the detection area by the up and down movement;

A liquid cavity connected to the central channel is provided on the surface of the sample injection layer 2, and a puncture body 21 corresponding to the reagent component 6 and the sample injection component 7 is provided in the liquid cavity. A gas channel connecting the central channel, the waste liquid pool 42 and the detection area is provided on the sample injection layer 2 and the airway layer 3.

Specifically, a control assembly 8 is fixed at the center slot of the sample loading layer 1, and the control assembly 8 is composed of a threaded control rod 81, a slider 82, and a piston 83. The slider 82 is fixed at the center slot of the sample loading layer 1, and the control rod 81 rotates in situ manually or by an instrument, and the piston 83 moves upward or downward under the transmission action of the slider 82.

Furthermore, a reagent assembly 6 and a sample injection assembly 7 are provided around one side of the control assembly 8. The reagent assembly 7 is composed of reagent cylinders filled with different reagents, including a cleaning liquid cylinder 1 61, a cleaning liquid cylinder 2 62 and an eluent cylinder 63, and the bottom is sealed with a pierceable bottom membrane.

7 , the sample injection assembly 7 is composed of a sealing film 711 , a barrel 712 and a screwable barrel cover 713 , and the interior is filled with lysis solution.

Furthermore, a valve assembly 9 and a pressing rod 10 are provided around the other side of the control assembly 8 , and the valve assembly 9 includes a first valve assembly 91 and a second valve assembly 92 .

The center of the sampling layer 2 is a circular through hole, which serves as a passage for the piston 83 of the control component 8 to move up and down. The central through hole is connected to the third gas channel 36 and the first gas through hole 26 through the upper first gas channel 24 and the second gas channel 25 respectively.

Furthermore, a plurality of piercing bodies 21 are arranged around the central through hole of the sample adding layer 2 and at the upper and lower corresponding positions of the reagent assembly 6 and the sample injection assembly 7. Under the action of pressure, the piercing bodies 21 pierce the bottom film of the reagent cartridge to release the internal reagent. A fluid channel 22 is arranged at the bottom of the sample adding layer 2 to connect the liquid cavity and the central through hole respectively. A threaded through hole 23 is arranged at the other side of the central through hole and at the upper and lower corresponding positions of the valve assembly 9, which matches the thread on the valve assembly 9.

The airway layer 3 is composed of multiple groups of through holes, including a central circular through hole, which runs through the upper and lower parts of the piston 83 of the control component 8 and serves as a channel for the up and down movement. A second gas through hole 34, a valve channel 35 and a gas chamber 33 are arranged around the central through hole, and the upper layer of the gas chamber 33 is connected to the third gas channel 36.

Furthermore, a sealing cover 31 is fixed to the inner wall of the gas chamber 33 through a connecting rib 32. Under the action of pressure, the lower pressure rod 10 is pressed down along the through hole to the sealing cover 31. During the continued downward pressing process, the connecting rib 32 breaks and releases the sealing cover 31. Under the continuous downward pressing action of the lower pressure rod 10, the sealing cover 31 is pushed to the position to be sealed.

The reaction layer 4 includes an extraction pool 41 and a waste liquid pool 42 , which are connected by a microchannel 44 . A valve position 43 is provided in the middle of the microchannel 44 . When the first valve assembly 91 reaches the valve position 43 , the microchannel 44 here is sealed to block the extraction pool 41 and the waste liquid pool 42 .

Furthermore, the other side of the extraction pool 41 is connected to the re-dissolution pool 45 through a microchannel 44. A valve position 43 of the second component 92 is provided on the microchannel 44. The bottom surface of the extraction pool 41 close to the valve position 43 is higher than the other side, which plays a role in guiding the liquid.

Furthermore, under the downward pressure of the control component 8 , the sample in the extraction pool 41 is evenly distributed to multiple amplification pools 49 through the sample inflow channel 48 , and the gas in the amplification pools 49 and the channel flows into the gas chamber 33 from the pores 47 .

Furthermore, a waterproof and breathable membrane is provided on the air hole 47, which is breathable but not water-permeable.

Specifically, during the sample nucleic acid detection process, the specific workflow of the chip of the present invention is as follows:

First, open the cylinder cover 713 of the injection assembly 7, add a quantitative amount of liquid sample and seal it, put the chip into the matching instrument, set the parameters and start the operation with one button, and the following actions will be automatically completed on the instrument:

S1. Sample lysis: Press the injection component 7, and the corresponding puncture body 21 on the sample loading layer 2 pierces the sealing film 711 of the lysis sample tube 71 containing the sample, releasing the lysis solution to flow into the extraction pool 41 through the microchannel. The control component 8 can be pulled upward appropriately to achieve the complete transfer of the lysis solution. In order to fully lyse the sample, an ultrasonic module can also be set at the bottom of the extraction pool 41 to improve the lysis efficiency. The lysed nucleic acid is captured by the magnetic beads pre-embedded in the extraction pool 41. After sufficient capture, the electromagnet on the side of the amplification pool 41 is opened to adsorb the magnetic beads, open the first valve component 91, and push the control component 8 to send the lysis solution into the waste liquid pool 42 through the microchannel 44. Under the action of pressure, Part of the gas in the waste liquid pool 42 is transferred from the upper second gas through hole 34 to the upper chamber of the piston 83 of the control component 8.

S2, nucleic acid extraction and purification: Press the top of the cleaning liquid cylinder 61, the bottom membrane is pierced by the corresponding puncture body 21 on the sample layer 2, the internal cleaning liquid is released and flows into the extraction pool 41, the control component 8 can be pulled upward appropriately to realize the complete transfer of the cleaning liquid, or the cleaning liquid can be released in multiple times through the stroke of the control component 8, and ultrasonic energy is applied to the bottom of the extraction pool 41, so that the impurities except nucleic acids in the extraction pool 41 are cleaned, the electromagnet on the side of the amplification pool 41 is turned on, the magnetic beads are adsorbed, the first valve component 91 is opened, and the control component 8 is pushed to send the cleaning liquid into the waste liquid pool 42 through the microchannel 44. Under the action of pressure, part of the gas in the waste liquid pool 42 is transferred from the second gas through hole 34 above to the upper chamber of the piston 83 of the control component 8. The cleaning liquid in the cleaning liquid cylinder 62 is released in the same way to complete the cleaning.

S3, elution injection: press the top of the eluent cartridge 63, the corresponding puncture body 21 on the sample loading layer 2 pierces the bottom membrane of the eluent cartridge 63 containing the sample, and the eluent is released to flow into the extraction pool 41 through the microchannel. The control component 8 can be pulled upward appropriately to achieve the complete transfer of the lysate, apply ultrasonic energy to the bottom of the extraction pool 41, and then control the temperature of the extraction pool at about 50°C to improve the elution efficiency. The electromagnet on the side of the amplification pool 41 is turned on to adsorb the magnetic beads, open the second valve assembly 92, and push the control assembly 8 to transfer the eluent through the sample inflow channel 46 to the eight parallel amplification pools 49. The gas in the amplification pool 49 and the channel is transferred to the gas chamber 33 above through the pores 47, and the internal circulation is realized through the third gas channel 36 and the first gas channel 24.

S4, PCR amplification and real-time detection: After the sample fills the amplification pool 49, the lower pressure rod 10 is pushed downward, and the sealing cover 31 is released from the inside of the gas chamber 33 under the action of pressure to reach above the air hole 47. Under the squeezing action of the lower pressure rod 10, the sealing cover presses the waterproof and breathable membrane on the air hole 47, seals the amplification system, starts the cycle temperature control, and starts the amplification and real-time fluorescence detection.

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.

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 is not limited to the embodiments shown herein, but It is to be consistent with the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

A fully enclosed and fully integrated nucleic acid detection microfluidic chip, characterized in that it comprises a sample loading layer (1), a sample injection layer (2), an airway layer (3), a reaction layer (4) and a sealing layer (5) which are sealed and bonded in sequence from top to bottom; The top surface of the sample adding layer (1) is integrated with a control component (8), a reagent component (6), a sample injection component (7) and a valve component (9); The surface of the reaction layer (4) is provided with a waste liquid pool (42), an extraction pool (41) and a detection area which are connected in sequence; The sample loading layer (1), the sample injection layer (2) and the airway layer (3) are provided with a central channel corresponding to the extraction pool (41), and a valve-controlled channel for the valve assembly (9) to pass through; the central channel controls the on-off of the gas path and the fluid channel by the up-and-down movement of the control assembly (8), and the valve assembly (9) controls the on-and-off of the fluid channel between the waste liquid pool (42) and the extraction pool (41), and between the extraction pool (41) and the detection area by the up-and-down movement; The surface of the sample injection layer (2) is provided with a liquid cavity connected to the central channel, and the liquid cavity has a puncture body (21) corresponding to the reagent component (6) and the sample injection component (7). The sample injection layer (2) and the airway layer (3) are provided with a gas channel connected to the central channel, the waste liquid pool (42) and the detection area. According to a fully enclosed and fully integrated nucleic acid detection microfluidic chip according to claim 1, it is characterized in that the control component (8) comprises a threaded control rod (81), a slider (82) and a piston (83); the slider (82) is fixed on the sample loading layer (1), the control rod (81) is threadedly connected to the slider (82), and the piston (83) is fixed at the bottom end of the control rod (81). A fully enclosed and fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that the reagent components (6) are all cylinders that can pierce the bottom membrane. A fully enclosed and fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that the injection assembly (7) includes a barrel (712), a screwable barrel cover (713) threadedly connected to the top of the barrel (712), and a sealing film (711) fixed to the bottom opening of the barrel (712). A fully enclosed and fully integrated nucleic acid detection microfluidic chip according to claim 1, characterized in that the valve-controlled channel includes a through hole opened on the sample loading layer (1), a threaded through hole (23) of the sample injection layer (2), and a valve channel (35) opened on the airway layer (3). According to claim 5, a fully enclosed and fully integrated nucleic acid detection microfluidic chip, The characteristic is that the outer side wall of the valve assembly (9) has threads. A fully enclosed and fully integrated nucleic acid detection microfluidic chip according to any one of claims 1-6, characterized in that the detection area includes a re-dissolution pool (45) connected to the extraction pool (41), the re-dissolution pool (45) is connected to the amplification pool (49) through a sample inflow channel (46), the amplification pool (49) is connected to the pore (47) through an airway (48), and the pore (47) is covered with a waterproof and breathable membrane. According to claim 7, a fully enclosed and fully integrated nucleic acid detection microfluidic chip is characterized in that the bottom surface of the extraction pool (41) is sloped, with the side away from the channel being higher than the side close to the channel. According to claim 7, a fully enclosed and fully integrated nucleic acid detection microfluidic chip is characterized in that a gas chamber (33) corresponding to the air hole (47) is opened on the airway layer (3), the top of the gas chamber (33) is connected to the central channel, and a sealing cover (31) is fixed to the inner wall of the bottom of the gas chamber (33) through a connecting rib (32), the sealing cover (31) corresponds to the air hole (47), and the gas chamber (33) is correspondingly provided with a downward pressure valve stem (10). A fully enclosed and fully integrated nucleic acid detection microfluidic chip according to claim 7, characterized in that the sample introduction layer (2) and the airway layer (3) have a gas channel connecting the central channel and the waste liquid pool (42).