Closed nucleic acid extraction, amplification and detection fully-integrated device

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a closed nucleic acid extraction, amplification and detection fully-integrated device. Comprising an injector type nucleic acid extraction, enrichment and amplification assembly, a detection card box, a three-way valve and a sealing cover, wherein the nucleic acid extraction, enrichment and amplification assembly comprises a chitosan modified paper sheet used for nucleic acid enrichment and an injector, the chitosan modified paper sheet is fixed in a needle cylinder of the injector through a rubber plug, and a piston of the injector is isolated from the chitosan modified paper sheet through isolation liquid; the detection card box is internally provided with at least one test strip, a box body is provided with a vent hole, the vent hole is covered with a film, and the film is breathable and waterproof; when an amplification product is loaded, one end of the three-way valve is connected with the detection card box, the other end of the three-way valve is connected with the nucleic acid extraction, enrichment and amplification assembly, and the third end of the three-way valve serves as a liquid inlet; during detection, the sealing cover is connected with the third end of the three-way valve.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular relates to a closed nucleic acid extraction, amplification and detection fully integrated device. Background Art

[0002] Infectious diseases caused by pathogenic microorganisms have an acute onset and rapid progression. It is crucial to identify pathogens immediately and quickly and take appropriate treatment measures for the diagnosis and prevention of infectious diseases. In addition, the superposition of multiple viruses can lead to more serious mixed infections, which can cause more serious symptoms than a single viral infection, posing greater challenges to clinical treatment and placing higher demands on rapid and accurate multiple virus detection technologies. With the development of detection technology, the concept of "point-of-care testing" has been proposed. Compared with existing detection methods, nucleic acid-based molecular point-of-care testing (POCT) technology has the characteristics of high sensitivity, fast analysis speed, small equipment size, and low detection cost, and plays an important role in responding to new and emerging infectious diseases.

[0003] Nucleic acid amplification in nucleic acid detection technology mainly includes PCR technology and isothermal amplification technology, among which isothermal amplification technology includes LAMP, RPA and RCA. Isothermal amplification technology does not require complex thermal cycling equipment, but only requires simple constant temperature water bath equipment, which makes the operation easier and has lower requirements on environmental conditions, and is suitable for environments such as operating rooms and on-site. For example, RPA can quickly amplify nucleic acids at a constant temperature of about 37°C, and a large amount of nucleic acid amplification can be achieved within 15-60 minutes.

[0004] The detection of amplification products in nucleic acid detection technology is crucial and is a key step in the field of molecular diagnosis. The detection of amplification products mainly includes gel electrophoresis technology, real-time fluorescence detection technology, lateral flow test strip technology, etc. Among them, lateral flow test strips with advantages such as light weight, portability, storage of dry reagents, and fluid flow controlled by the capillary force of the substrate without the need for other external pumps have shown great potential in the field of instant diagnosis. They can significantly reduce the demand for external instruments and provide a user-friendly result reading method. As a classic POCT diagnostic method, test strips have been used for the detection of influenza, molecular biomarkers of complex diseases, and other infectious diseases, providing a cost-effective and easy-to-use method for disease diagnosis. Recombinase polymerase amplification combined with lateral flow test strips (RPA-LFD) is an innovative detection method suitable for practical applications. RPA-LFD has the advantages of being intuitive and convenient, and has received widespread attention. This method not only avoids the risk of cross-contamination, but also does not require complex and bulky laboratory equipment and cumbersome processing steps.

[0005] However, when chromatographic test strips are used for molecular diagnosis, they usually require complex nucleic acid extraction, enrichment and amplification steps before sample loading and testing, and have limitations in complex fluid processing. In order to combine nucleic acid extraction and amplification steps with test strip detection, it is necessary to design more complex fluid control components and add some special pumps, valves and reactor structures to the microfluidic control components, but they usually require power supply or use batteries, and require complex processes to be integrated into the microfluidic chip, making the entire system bulky and complex, limiting its application as a POCT method. Disposable syringes can be used as vacuum sources or pressure sources, have the advantages of low cost and simple manufacturing process, and show great potential in the pre-treatment of samples.

[0006] (1) Most nucleic acid-based isothermal amplification technologies require opening the tube to transfer the amplification product after the amplification reaction is completed, and the tube also needs to be opened again when testing with a test strip, which increases the risk of product contamination and operational difficulty;

[0007] (2) From the perspective of system complexity, the design of an integrated detection system based on nucleic acid amplification and lateral flow test strips is relatively complex, usually requiring the design of more complex fluid control components, the addition of some special pumps, valves and reactor structures, and the need for complex process integration, making the entire system bulky and complex, and increasing the corresponding manufacturing and testing costs;

[0008] (3) Most of the current lateral flow test strips are for the detection of a single target, which is difficult to meet the urgent needs of multiple detection of complex safety issues. Achieving the detection of multiple targets or multiple targets while maintaining high sensitivity and specificity is still a technical problem that needs to be solved urgently. Summary of the invention

[0009] In view of this, the present invention provides a closed fully integrated device for nucleic acid extraction, amplification and detection, which can effectively enrich nucleic acids and well combine RPA-based amplification with test strip-based detection reactions, and has low cost, light weight, easy portability and simple operation.

[0010] The technical solution for implementing the present invention is as follows:

[0011] In a first aspect, the present invention provides a closed nucleic acid extraction, amplification and detection fully integrated device, including a syringe-type nucleic acid extraction, enrichment and amplification component, a detection cartridge, a three-way valve and a sealing cover; wherein,

[0012] The nucleic acid extraction, enrichment and amplification component comprises a chitosan modified paper sheet for nucleic acid enrichment and a syringe, wherein the chitosan modified paper sheet is fixed in the syringe barrel through a rubber stopper, and an isolation liquid is used to isolate the piston of the syringe and the chitosan modified paper sheet;

[0013] At least one test strip is arranged in the detection card box, and a vent hole is arranged on the box body, and the vent hole is covered with a film, and the film is air-permeable and water-impermeable;

[0014] When the amplification product is loaded, one end of the three-way valve is connected to the detection card box, and the other end is connected to the nucleic acid extraction, enrichment and amplification component, and the third end of the three-way valve is used as the liquid inlet; when testing, the sealing cover is connected to the third end of the three-way valve.

[0015] Furthermore, in the present invention, mineral oil is used to isolate between the chitosan modified paper sheet and the piston of the syringe.

[0016] Furthermore, the present invention clamps the chitosan modified paper sheet on the rubber plug, and then plugs the rubber plug into the syringe barrel of the syringe.

[0017] Furthermore, the detection card box of the present invention is a chip-type structure, including an upper chip, a lower chip and at least one built-in test strip; the lower chip is provided with strip grooves corresponding to the number of test strips for placing test strips, and the lower chip is also provided with a breathable chamber, the ventilation hole enables the breathable chamber to communicate with the outside, and the film is fixed in the breathable chamber.

[0018] Furthermore, the test strip of the present invention comprises a sample pad, an NC membrane and an absorbent pad, wherein the sample pad is 2.0 cm long, 1.6 cm wide and 0.3 mm high, the NC membrane with C lines and T lines is 6.0 cm long, each 0.4 cm wide, and is used to display the test results, the interval between the three parallel strips is 0.1 cm, and the absorbent pad is 2.0 cm long and 0.4 cm wide.

[0019] Furthermore, the upper chip of the present invention is provided with an amplification product loading hole, and a Luer connector is provided at a position corresponding to the amplification product loading hole on the outer surface of the detection cartridge for connecting with a three-way valve via a hose.

[0020] Furthermore, the nucleic acid extraction, enrichment and amplification components of the present invention are fixed on the detection card box.

[0021] In the second aspect, a detection method based on a closed nucleic acid extraction, amplification and detection fully integrated device, the specific process is:

[0022] Step 1: Using the nucleic acid extraction, enrichment and amplification component to absorb the lysis product containing the sample to be tested, the nucleic acid released by lysis is adsorbed on the chitosan modified paper, and then the syringe piston is pushed to discharge impurities in the lysis product;

[0023] Step 2: Using the nucleic acid extraction, enrichment and amplification component to absorb the RPA amplification mixed solution, and placing it in a constant temperature device to perform an RPA amplification reaction;

[0024] Step 3, connecting the detection cartridge, the three-way valve and the nucleic acid extraction, enrichment and amplification component, using the nucleic acid extraction, enrichment and amplification component to absorb the loading buffer and the recognition probe mixed solution from the liquid inlet of the three-way valve, and then loading the amplification product onto the sample pad of the test strip in the detection cartridge;

[0025] Step 4: Connect a sealing cover to the liquid inlet of the three-way valve connected to the detection cartridge to form a closed reaction environment, thereby completing the detection of the nucleic acid amplification product.

[0026] Furthermore, the RPA amplification mixed solution of the present invention includes an RPA amplification system and an elution solution Tris-HCl, and the RPA amplification system includes primers, buffer, ddH2O, recombinase, single-strand binding protein, strand displacement DNA polymerase and magnesium acetate.

[0027] Furthermore, the RPA amplification reaction of the present invention is: performing the RPA amplification reaction in a constant temperature device at 37° C., and the reaction time is 30 minutes.

[0028] Beneficial effects:

[0029] First, this device can effectively enrich nucleic acids, so that the RPA-based amplification and the test strip-based detection reaction are well combined, and it can achieve accurate diagnosis with fully integrated, micro-nano reaction system, high sensitivity and high specificity, and multi-target parallel detection. It has high clinical application value and practical potential in on-site detection of various bacteria or viruses.

[0030] Second, this device has strong biosafety. Through closed operation, it can effectively control the spread of infectious samples and protect operators and laboratory environment, which is especially important when dealing with highly pathogenic pathogens.

[0031] Third, the device reduces the possibility of exposure to aerosol pollutants and avoids multiple manual operation steps, such as extraction of nucleic acids, transfer of amplification products, mixing of amplification products with loading buffer, and complex loading and detection operations; it is easy to operate and does not require professional operation, and non-professional background personnel can perform the test.

[0032] Fourth, the device has the advantages of low cost, light weight, easy to carry, simple operation, fast and green environmental protection; it avoids professional laboratories and complex and large instruments and equipment, and the entire system is very suitable for on-site detection.

[0033] Fifth, the detection method based on this device shortens the detection time and improves the detection efficiency; the detection and production costs are very low and can be mass-produced; at the same time, the multiple detection method can reduce the sample demand, which is especially valuable for trace samples. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 1 Schematic diagram of a fully integrated device for closed nucleic acid extraction, amplification and detection (front)

[0036] Figure 2 Schematic diagram of a fully integrated device for closed nucleic acid extraction, amplification and detection (back)

[0037] Figure 3 Schematic diagram of the detection card box structure containing the test strip

[0038] Figure 4 Schematic diagram of the lower layer and ventilation holes of the detection card box

[0039] Figure 5 Schematic diagram of the connection between the test cartridge and the syringe

[0040] Among them, 1- a syringe-type nucleic acid extraction, enrichment and amplification component; 2- a detection card box containing a test strip; 3- a three-way valve; 4- a connecting hose; 5- a Luer connector; 6- a slot for fixing the syringe; 7- a screw for fixing the slot; 8- a liquid inlet; 9- a sample pad; 10- an NC membrane; 11- a water-absorbent pad; 12- amplification product loading hole; 13- a lower chip; 14- an upper chip; 15- a breathable chamber; 16- a vent; 17- a rubber plug with a chitosan-modified paper sheet; 18- a piston; 19- a syringe. DETAILED DESCRIPTION

[0041] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0042] It should be noted that the following embodiments and features in the embodiments may be combined with each other in the absence of conflict; and, based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without making any creative work are within the scope of protection of the present disclosure.

[0043] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein may be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on the present disclosure, it should be understood by those skilled in the art that an aspect described herein may be implemented independently of any other aspect, and two or more of these aspects may be combined in various ways. For example, any number of aspects described herein may be used to implement the device and / or practice the method. In addition, other structures and / or functionalities other than one or more of the aspects described herein may be used to implement this device and / or practice this method.

[0044] To prevent contamination caused by opening the cover, Figure 1-5 As shown, the embodiment of the present application is a closed nucleic acid extraction, amplification and detection fully integrated device, including a syringe-type nucleic acid extraction, enrichment and amplification component 1, a detection cartridge 2, a three-way valve 3 and a sealing cover; wherein,

[0045] The nucleic acid extraction, enrichment and amplification assembly 1 comprises a chitosan modified paper sheet for nucleic acid enrichment and a syringe 19, wherein the chitosan modified paper sheet is fixed in the syringe barrel of the syringe 19 by a rubber stopper 17, and an isolation liquid is used to isolate the piston 18 of the syringe 19 and the chitosan modified paper sheet;

[0046] At least one test strip is arranged in the detection card box 2, and a vent hole is arranged on the box body 2, and the vent hole is covered with a film, and the film is air-permeable and water-impermeable;

[0047] When the amplification product is loaded, one end of the three-way valve 3 is connected to the detection card box 2, and the other end is connected to the nucleic acid extraction, enrichment and amplification component 1, and the third end of the three-way valve 3 serves as the liquid inlet; when testing, the sealing cover is connected to the third end (liquid inlet) of the three-way valve.

[0048] The nucleic acid extraction, enrichment and amplification components of this embodiment are connected to the detection cartridge through a three-way valve to ensure that the closed state is maintained when the amplified product is loaded, and the user is allowed to control the volume of the distributed solution, inject the solution in the syringe into the sample pad of the test strip in the detection cartridge, and combine these two main modules into a new fully integrated nucleic acid analysis system. The opening of the detection cartridge adopts a Luer connector 5, which can be connected to a corresponding hose, and the other end of the hose is connected to the three-way valve. This connection simplifies the control of fluid flow and has strong adaptability. It can be used in conjunction with a variety of microfluidic chips and experimental devices, increasing the compatibility of the system; the three-way valve can realize the switching, introduction or stopping of different pipelines, increasing the flexibility and versatility of the system.

[0049] In another embodiment of the present application, mineral oil (such as paraffin oil) is used to isolate the chitosan modified paper sheet and the piston 18 of the syringe 19; the use of mineral oil (such as paraffin oil) can not only play a role in sealing and resisting pollution and interference, but also promote the complete discharge of liquid to avoid residue in the syringe, which can minimize reagent consumption and reduce experimental costs.

[0050] In another embodiment of the present application, the chitosan modified paper sheet is clamped on the rubber plug 17, and then the rubber plug is tightly inserted into the barrel of the syringe 19, so as to achieve relative fixation of the chitosan modified paper sheet in the syringe.

[0051] In another embodiment of the present application, the chip-type detection card box 2 includes an upper chip 14, a lower chip 13 and at least one built-in test strip; the lower chip 13 is provided with strip grooves corresponding to the number of test strips for placing the test strips, and the lower chip 13 is also provided with a breathable chamber 15, the vent hole 16 makes the breathable chamber communicate with the outside, and the film is fixed in the breathable chamber.

[0052] In specific implementation, three parallel strips can be set in the test card box, and the three strips are respectively located in the three strip grooves of the lower chip chamber, and the upper and lower chips are fixed by 3M adhesive. The lower chip is etched with a test strip placement area and a circular air-permeable chamber, and the circular chamber can fix the air-permeable and water-impermeable membrane to ensure the injection of liquid and prevent aerosol contamination.

[0053] In another embodiment of the present application, the upper chip is engraved with a fixed loading hole 12, and a Luer connector is used at the opening of the detection cartridge, and the connector can be connected to a corresponding hose for injecting the amplification product into the sample pad.

[0054] In another embodiment of the present application, the test strip includes a sample pad, an NC membrane and an absorbent pad, wherein the sample pad is 2.0 cm long, 1.6 cm wide and 0.3 mm high, the NC membrane with C lines and T lines is 6.0 cm long, each 0.4 cm wide, and is used to display the test results, the interval between the three parallel strips is 0.1 cm, and the absorbent pad is 2.0 cm long and 0.4 cm wide.

[0055] In this embodiment, when the RPA amplification product is injected into (loaded with) the test cartridge through the nucleic acid extraction, enrichment and amplification components, the amplification product detection based on the test strip combines the recombinase polymerase amplification and the multiple nucleic acid lateral flow test strip, and can detect multiple pathogenic microorganisms at the same time. The RPA primers labeled with nucleic acids at both ends are used to generate double-tailed RPA amplicons, which are first recognized by the colloidal gold-labeled probes, and then captured by the nucleic acid probes pre-labeled on the three test lines of the test strip, forming three test lines visible to the naked eye. At the same time, the recognition probe can also be captured by the control line on the test strip as a control.

[0056] The present application embodiment is based on a detection method of a closed nucleic acid extraction, amplification and detection fully integrated device, and the specific process is as follows:

[0057] Step 1: Using the nucleic acid extraction, enrichment and amplification component to absorb the lysis product containing the sample to be tested, the nucleic acid released by the lysis is adsorbed on the chitosan modified paper, and then the syringe piston is pushed to discharge the impurities of the lysis product;

[0058] Step 2: Using the nucleic acid extraction, enrichment and amplification component to absorb the RPA amplification mixed solution, and placing it in a constant temperature device to perform an RPA amplification reaction;

[0059] Step 3, connecting the detection cartridge, the three-way valve and the nucleic acid extraction, enrichment and amplification component, using the nucleic acid extraction, enrichment and amplification component to absorb the loading buffer and the recognition probe mixed solution from the liquid inlet of the three-way valve, and then loading the amplification product onto the sample pad of the test strip in the detection cartridge;

[0060] Step 4: Connect a sealing cover to the liquid inlet of the three-way valve connected to the detection cartridge to form a closed reaction environment, thereby completing the detection of the nucleic acid amplification product.

[0061] In actual use, the following steps are mainly included: after the sample to be tested is evenly mixed with other reagents such as microbial lysis solution in a centrifuge tube, it is allowed to stand at room temperature for 10 minutes, 200 μL of lysis product is drawn with a syringe, and the solution is discharged after pushing and pulling the piston 3 times. At this time, the nucleic acid released by lysis is adsorbed on the chitosan-modified cellulose paper, and then 90 μL RPA amplification mixed solution (RPA amplification system and eluent Tris-HCl, RPA amplification system is primers, buffer, ddH2O, recombinase, single-strand binding protein, strand displacement DNA polymerase and magnesium acetate) is drawn, and the RPA amplification reaction can be carried out using a simple 37°C constant temperature device. After 30 minutes of reaction, 210 μL of mixed solution (colloidal gold-labeled recognition probe and loading buffer) is drawn through the liquid inlet, and then the liquid in the syringe is pushed onto the sample pad of the test strip using a piston, and then the liquid inlet of the three-way valve is connected with a sealing cap to seal the liquid inlet to construct a closed reaction system. After 5 minutes of loading, the test results can be directly observed. The test cassette can perform nine different nucleic acid tests at the same time and conveniently display the results in a transparent test cassette. It is simple to operate, has clear results, fast detection speed, high detection throughput, and is easy to observe visually. The closed design avoids interference and contamination.

[0062] At present, the visualization of the results of amplification products is based on colloidal gold test strips. By coupling colloidal gold to the recognition probe and using colloidal gold as a tracer marker for detection, fluorescent microspheres can be coupled to the probe to form a fluorescent probe or other fluorescent markers (such as quantum dots and upconversion nanoparticles) can be coupled to form a fluorescent probe to prepare a fluorescent test strip to adapt to a variety of detection conditions and be more widely used in clinical detection. The test strip uses a nucleic acid complementary pairing recognition method to capture specific targets, which improves the specificity of the test and reduces the cost of the test.

[0063] This embodiment has the following effects:

[0064] The entire process is carried out in a fully sealed system, which simplifies the operation process, improves the reaction speed, and reduces the risk of contamination. The system forms a closed internally interconnected space in the visual mixing container, which greatly reduces aerosol contamination. The nucleic acid amplification reaction can occur at room temperature, and an instrument-free molecular detection is adopted, which avoids dependence on PCR laboratories and complex instruments, and the results can be read by the naked eye. The system is simple, lightweight, and easy to carry, which can realize portable detection of microorganisms and has strong usability. The goal of multiple detection is achieved. Compared with single-target detection, multi-channel detection can reduce detection time and cost, improve detection efficiency, and is very suitable for on-site detection.

[0065] In summary, the above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A closed nucleic acid extraction, amplification and detection fully integrated device, characterized in that: It includes a syringe-type nucleic acid extraction, enrichment and amplification component, a detection card box, a three-way valve and a sealing cover; wherein, The nucleic acid extraction, enrichment and amplification component comprises a chitosan modified paper sheet for nucleic acid enrichment and a syringe, wherein the chitosan modified paper sheet is fixed in the syringe barrel through a rubber stopper, and an isolation liquid is used to isolate the piston of the syringe and the chitosan modified paper sheet; At least one test strip is arranged in the detection card box, and a vent hole is arranged on the box body, and the vent hole is covered with a film, and the film is air-permeable and water-impermeable; When the amplification product is loaded, one end of the three-way valve is connected to the detection card box, and the other end is connected to the nucleic acid extraction, enrichment and amplification component, and the third end of the three-way valve is used as the liquid inlet; when testing, the sealing cover is connected to the third end of the three-way valve.

2. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 1, characterized in that: Mineral oil was used to insulate between the chitosan-modified paper sheet and the plunger of the syringe.

3. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 1, characterized in that: The chitosan modified paper sheet is clamped on the rubber plug, and then the rubber plug is tightly inserted into the syringe barrel of the syringe.

4. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 1, characterized in that: The detection card box is a chip-type structure, including an upper chip, a lower chip and at least one built-in test strip; the lower chip is provided with strip grooves corresponding to the number of test strips for placing test strips, and the lower chip is also provided with a breathable chamber, the ventilation hole enables the breathable chamber to communicate with the outside, and the film is fixed in the breathable chamber.

5. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 4, characterized in that: The test strip includes a sample pad, an NC membrane and a water absorbent pad, wherein the sample pad is 2.0 cm long, 1.6 cm wide and 0.3 mm high, the NC membrane with C lines and T lines is 6.0 cm long, each 0.4 cm wide, and is used to display the test results, the interval between the three parallel strips is 0.1 cm, and the water absorbent pad is 2.0 cm long and 0.4 cm wide.

6. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 1, characterized in that: The upper chip is provided with an amplification product loading hole, and the outer surface of the detection cartridge is provided with a Luer connector at a position corresponding to the amplification product loading hole, which is used to be connected to the three-way valve through a hose.

7. The closed nucleic acid extraction, amplification and detection fully integrated device according to claim 1, characterized in that: The nucleic acid extraction, enrichment and amplification components are fixed on the detection card box.

8. A detection method based on a closed nucleic acid extraction, amplification and detection fully integrated device, characterized in that: The specific process is: Step 1: Using the nucleic acid extraction, enrichment and amplification component to absorb the lysis product containing the sample to be tested, the nucleic acid released by lysis is adsorbed on the chitosan modified paper, and then the syringe piston is pushed to discharge impurities in the lysis product; Step 2: Using the nucleic acid extraction, enrichment and amplification component to absorb the RPA amplification mixed solution, and placing it in a constant temperature device to perform an RPA amplification reaction; Step 3, connecting the detection cartridge, the three-way valve and the nucleic acid extraction, enrichment and amplification component, using the nucleic acid extraction, enrichment and amplification component to absorb the loading buffer and the recognition probe mixed solution from the liquid inlet of the three-way valve, and then loading the amplification product onto the sample pad of the test strip in the detection cartridge; Step 4: Connect a sealing cover to the liquid inlet of the three-way valve connected to the detection cartridge to form a closed reaction environment, thereby completing the detection of the nucleic acid amplification product.

9. The detection method based on the closed nucleic acid extraction, amplification and detection fully integrated device according to claim 8, characterized in that: The RPA amplification mixed solution comprises an RPA amplification system and an elution solution Tris-HCl, and the RPA amplification system comprises primers, a buffer, ddH2O, a recombinase, a single-strand binding protein, a strand displacement DNA polymerase and magnesium acetate.

10. The detection method based on the closed nucleic acid extraction, amplification and detection fully integrated device according to claim 8, characterized in that: The RPA amplification reaction is as follows: the RPA amplification reaction is performed in a 37° C. constant temperature device, and the reaction time is 30 minutes.

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