A closed nucleic acid amplification detection device and method

Abstract

The application provides a closed nucleic acid amplification detection device and method, which comprises an amplification unit and a detection unit. The amplification unit has a reaction cavity, and the detection unit is arranged in a relative rotating manner on the amplification unit and is in sealed cooperation with the amplification unit. A sample adding port and a first channel are arranged on the amplification unit, and the end of the first channel forms a liquid outlet interface. A second channel, a detection area and an exhaust structure are arranged on the detection unit, and the end of the second channel forms a liquid inlet interface. The detection unit can be switched between a first working position, a second working position and a third working position in rotation, so that the liquid outlet interface and the liquid inlet interface are in a misaligned sealing state, an aligned communication state and a misaligned sealing state again. The reaction cavity is isolated from the detection area during amplification. After the amplification is completed, the two are communicated, and the amplification product enters the detection area under the driving of thermal expansion pressure and capillary force. After liquid filling is completed, the detection area forms a closed detection cavity in the misaligned sealing state again. The application realizes closed integrated operation of amplification and detection, does not need external pumps and valves, can reduce the pollution risk and improve the detection reliability.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostics and nucleic acid detection technology, specifically relating to an integrated closed consumable for nucleic acid amplification and detection and its usage method, and particularly to a device and method for achieving a closed operation of the entire process of amplification, transfer and detection through coupling of a vertical thermal convection PCR reaction column and a rotatable detection disk. Background Technology

[0002] Polymerase chain reaction (PCR) is the most widely used in vitro nucleic acid amplification technique in nucleic acid detection. Conventional PCR testing procedures typically include nucleic acid extraction, PCR amplification, amplification product transfer, and fluorescence detection. Each step needs to be performed separately in different equipment or areas, making the process complex, time-consuming, and demanding on skilled operators, thus failing to meet the needs of rapid on-site testing.

[0003] To address these issues, microfluidic nucleic acid detection technologies in recent years have integrated amplification, liquid transfer, and detection structures onto a single chip or disk. For example, some disk-type detection chips use centrifugal force generated by rotation to distribute the amplification solution to each detection unit; some integrated chips use membrane valves, wax valves, pneumatic valves, or external pumps to control the transfer of liquid between the amplification chamber and the detection area. Furthermore, thermal convection PCR technology utilizes the density difference created by the temperature difference between the upper and lower parts of the reaction solution to drive the liquid to circulate within the reaction chamber, completing PCR amplification without repeated heating and cooling, thus simplifying temperature control to some extent.

[0004] However, the aforementioned existing technologies still have their own shortcomings. In the amplification product transfer stage, traditional PCR tubes or plates require opening and pipetting after amplification. Given the high concentration of amplified products, opening the cap easily creates aerosol contamination, leading to false positive results. Regarding the isolation and connectivity control between the amplification chamber and the detection zone, if they are always connected, the probes or reagents pre-placed in the detection zone may prematurely contact the PCR reaction system, affecting amplification efficiency. If they are completely isolated, additional mechanisms such as puncture, squeezing, or valve control are required for liquid transfer, increasing the complexity of consumables and instruments.

[0005] Regarding fluid-driven methods, solutions relying on pumps, valves, or complex microchannels are difficult to manufacture and costly, while solutions relying solely on capillary or centrifugal forces are often unreliable in terms of timing control during transfer and sealing after transfer. In terms of filling the detection zone with liquid, insufficient venting can easily leave air bubbles, affecting the uniformity of probe reaction and the accuracy of fluorescence detection.

[0006] In terms of reaction chamber structure, the reaction chamber height of sheet-like microfluidic chips is relatively small, making it difficult to form a significant vertical temperature gradient and stable thermal convection circulation. Therefore, repeated heating and cooling programs using an external temperature control module are usually still required. Furthermore, if the detection zone is not effectively isolated from the amplification chamber and the external environment after the amplification solution enters, there is a risk of liquid backflow, evaporation, or leakage, making it impossible to maintain a stable closed reaction environment during the probe reaction and fluorescence reading stages.

[0007] Therefore, the existing technology lacks an integrated nucleic acid detection device and method that can reliably isolate the reaction chamber from the detection area during the amplification stage, achieve closed liquid transfer without external pumps or valves after amplification, and allow the detection area to form an independent closed chamber again after transfer, while achieving efficient thermal convection PCR amplification through a vertical reaction chamber. Summary of the Invention

[0008] The purpose of this invention is to provide a closed nucleic acid amplification detection device and method to solve the problem mentioned in the background art of the lack of an integrated nucleic acid detection solution for efficient thermal convection PCR amplification.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A closed nucleic acid amplification and detection device, comprising:

[0011] The amplification unit has a reaction chamber for accommodating nucleic acid samples and PCR reaction systems;

[0012] A detection unit is rotatably disposed relative to the amplification unit and is sealed to the amplification unit;

[0013] The amplification unit is provided with a sample inlet and a first channel that are connected to the reaction chamber, and the end of the first channel forms a liquid outlet.

[0014] The detection unit is provided with a second channel and at least one detection area for performing the detection reaction. The end of the second channel forms a liquid inlet. The detection unit is also provided with an exhaust structure that communicates with the detection area.

[0015] The detection unit can rotate and switch between a first working position, a second working position, and a third working position relative to the amplification unit;

[0016] In the first working position, the liquid outlet and the liquid inlet are staggered and sealed to isolate the reaction chamber from the detection area;

[0017] In the second working position, the liquid outlet is aligned and connected with the liquid inlet, so that the amplification product in the reaction chamber enters the detection area through the first channel, the liquid outlet, the liquid inlet, and the second channel;

[0018] In the third working position, the liquid outlet and the liquid inlet are again staggered and sealed, so that the detection area forms a closed detection chamber.

[0019] As a further preferred technical solution, the amplification unit is a vertical PCR reaction column, the reaction chamber is a vertically extending columnar inner cavity, and the detection unit is a detection disk disposed below the vertical PCR reaction column.

[0020] As a further preferred technical solution, the vertical PCR reaction column is provided with at least one internal partition. The internal partition extends along the height direction of the vertical PCR reaction column, and the upper end of the internal partition forms a top communication gap with the top wall of the columnar inner cavity, and the lower end of the internal partition forms a bottom communication gap with the bottom wall of the columnar inner cavity, so as to guide the reaction solution to form a thermal convection circulation around the internal partition under the vertical temperature gradient.

[0021] As a further preferred technical solution, the detection disk is provided with a flow channel, the flow channel connects the second channel and the detection area, and at least a portion of the inner wall of the flow channel is provided with a hydrophilic flow guiding layer, which is used to assist the amplification product to flow from the second channel to the detection area through capillary action.

[0022] As a further preferred technical solution, the exhaust structure includes a waste liquid area and an exhaust channel. The waste liquid area is connected to the detection area through the exhaust channel. The waste liquid area is used to receive the discharged air and excess liquid during the filling process of the detection area. The waste liquid area is provided with a liquid-absorbing material and / or a hydrophobic and breathable membrane.

[0023] As a further preferred technical solution, the detection disk is provided with multiple detection areas, and the multiple detection areas are respectively connected to the second channel through branch guide channels. Each detection area is used to pre-set the same or different detection probes to realize parallel detection of single targets or multiple targets.

[0024] As a further preferred technical solution, a sealing contact structure is provided between the lower end of the vertical PCR reaction column and the upper surface of the detection disk. The sealing contact structure is any one of a planar contact sealing surface, an annular boss, an elastic sealing ring, or a compression sealing surface, which is used to maintain the seal between the vertical PCR reaction column and the detection disk during the rotation of the detection disk.

[0025] As a further preferred technical solution, the detection disk is provided with a rotation positioning hole for cooperating with an external rotation drive module to drive the detection disk to rotate.

[0026] A method for nucleic acid detection using the aforementioned closed nucleic acid amplification and detection device includes the following steps: Step S1: Place the detection unit in the first working position, so that the liquid outlet and the liquid inlet are in a misaligned and sealed state; Step S2: Add nucleic acid sample and PCR reaction system into the reaction chamber through the sample addition port, and exhaust air from the reaction chamber through the exhaust port provided on the amplification unit; Step S3: Seal the sample inlet and the vent hole to form a closed amplification chamber in the reaction chamber; Step S4: Apply a temperature gradient to the reaction chamber to create thermal convection circulation of the reaction solution within the reaction chamber for PCR amplification; Step S5: After amplification is complete, rotate the detection unit to the second working position so that the liquid outlet interface is aligned and connected with the liquid inlet interface; Step S6: Using the driving force generated in the reaction chamber, the amplification product is driven to enter the detection area through the first channel, the liquid outlet, the liquid inlet, and the second channel; Step S7: Rotate the detection unit to the third working position, so that the liquid outlet and the liquid inlet are misaligned and sealed again, thus sealing the detection area; Step S8: Apply detection reaction conditions to the detection region to cause the amplification product to react specifically with the pre-placed detection reagent and generate a detectable signal; Step S9: Read the signal in the detection area through the signal reading module and output the detection result.

[0027] As a further preferred technical solution, a first temperature is applied to the upper part of the amplification unit, and a second temperature higher than the first temperature is applied to its lower part to form the temperature gradient; the first temperature is 55°C to 65°C, and the second temperature is 90°C to 98°C; in step S8, the detection reaction condition is a detection reaction temperature of 50°C to 65°C; in step S6, the driving force includes the thermal expansion pressure in the reaction chamber and / or the capillary force in the detection unit; air and / or excess amplification products in the detection area enter the waste liquid area through the exhaust channel to reduce the residual bubbles in the detection area; in step S9, the signal reading module is a fluorescence detection module, which detects the fluorescence intensity, fluorescence color, or fluorescence change rate in the detection area, and outputs positive, negative, or quantitative results according to preset interpretation rules.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] First, by rotating the detection disk relative to the amplification unit between the first working position, the second working position, and the third working position, the first channel and the second channel can switch between misaligned sealing, aligned connection, and misaligned sealing again. This allows the amplification and detection processes to be completed within the same sealed consumable, avoiding aerosol contamination caused by opening the cap during the transfer of amplification products and reducing the risk of false positives.

[0030] Secondly, by keeping the first and second channels in a staggered and sealed state during the amplification stage, the reaction chamber and the detection area are isolated from each other, preventing the reagents in the detection area from coming into premature contact with the PCR reaction system and ensuring that the PCR amplification reaction is not disturbed.

[0031] Third, by re-sealling the first and second channels after the detection area is filled with liquid, a closed detection chamber is formed in the detection area, which avoids backflow, evaporation or leakage of the amplification solution and ensures the sealing reliability of the detection area during the probe reaction and fluorescence reading stages.

[0032] Fourth, by setting up a waste liquid area and an exhaust channel connected to the detection area inside the detection disk, air and excess liquid are discharged when the amplification solution enters the detection area, reducing the residual air bubbles in the detection area and improving the accuracy of fluorescence detection.

[0033] Fifth, the internal partition inside the vertical PCR column guides the reaction solution to form a thermal convection circulation under the action of the temperature difference between the top and bottom, realizing the transfer of amplification solution without the need for external pumps and valves, reducing the difficulty of consumable processing and the complexity of instrument control. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the overall structure of a closed nucleic acid amplification and detection device according to the present invention;

[0035] Figure 2 This is a schematic diagram of the vertical PCR reaction column structure of the present invention;

[0036] Figure 3 This is a schematic diagram of the detection disk structure of the present invention.

[0037] In the diagram: 1. Vertical PCR reaction column; 2. Sample loading port; 3. Exhaust port; 4. Internal partition;

[0038] 5. Top connecting gap; 6. Bottom connecting gap; 7. First channel; 8. Liquid outlet; 9. Detection disc; 10. Liquid inlet; 11. Second channel; 12. Detection area; 13. Waste liquid area; 14. Rotary positioning hole; 15. Annular sealing ring. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Please see Figures 1 to 3 The present invention provides a closed nucleic acid amplification and detection device, including a vertical PCR reaction column 1 and a detection disk 9.

[0041] The vertical PCR reaction column 1 is a vertically positioned cylindrical cavity with a vertically extending columnar inner cavity, made of polypropylene material, with a volume of 50 μL. The upper end of the vertical PCR reaction column 1 has a sample loading port 2 and an vent 3. Sample loading port 2 is used to add nucleic acid samples and the PCR reaction system, while vent 3 is used to expel air from the column. After sample loading, sample loading port 2 and vent 3 are sealed with a pressure-sensitive sealing membrane to form a closed amplification chamber.

[0042] An internal partition 4 is installed inside the vertical PCR reaction column 1, extending along the height of the column. The upper end of the internal partition 4 forms a top communication gap 5 with the top wall of the column's inner cavity, and the lower end of the internal partition 4 forms a bottom communication gap 6 with the bottom wall of the column's inner cavity. The internal partition 4 divides the internal space of the column into two flow regions, allowing the reaction solution to circulate through thermal convection around the internal partition 4 under a vertical temperature gradient.

[0043] A first channel 7 is provided on the lower side wall of the vertical PCR reaction column 1, and an outlet 8 is formed at the end of the first channel 7. The outlet 8 is located in the sealed contact area between the vertical PCR reaction column 1 and the detection disk 9.

[0044] The detection disk 9 is a disc-shaped structure made of transparent polycarbonate material. It is rotatably positioned below the vertical PCR reaction column 1 and is sealed to it. The detection disk 9 has a second channel 11, a flow channel, a detection zone 12, a waste liquid zone 13, and a rotation positioning hole 14. The end of the second channel 11 forms a liquid inlet 10. The flow channel connects the second channel 11 and the detection zone 12. At least a portion of the inner wall of the flow channel has a hydrophilic flow-guiding layer to facilitate the flow of amplification products from the second channel 11 to the detection zone 12 via capillary action. The detection zone 12 is used for the detection reaction, and a fluorescent probe layer is fixed at its bottom. The waste liquid zone 13 is connected to the detection zone 12 through an exhaust channel and is used to receive the discharged air and excess liquid during the filling process of the detection zone 12. The waste liquid zone 13 contains absorbent material. An annular sealing ring 15 (which can also be any one of a flat sealing surface, an annular boss, an elastic sealing ring, or a compressed sealing surface) is provided between the lower end of the vertical PCR reaction column 1 and the upper surface of the detection disk 9, so that the two maintain sealed contact during relative rotation.

[0045] The detection disk 9 can rotate and switch between the first working position, the second working position, and the third working position relative to the vertical PCR reaction column 1. The rotation positioning hole 14 is used to cooperate with an external rotation drive module to drive the detection disk 9 to rotate.

[0046] In the first working position, the liquid outlet 8 and the liquid inlet 10 are staggered and sealed, isolating the reaction chamber from the detection area 12. In the second working position, the liquid outlet 8 and the liquid inlet 10 are aligned and connected, and the amplification product in the reaction chamber enters the detection area 12 through the first channel 7, the liquid outlet 8, the liquid inlet 10, and the second channel 11. In the third working position, the liquid outlet 8 and the liquid inlet 10 are again staggered and sealed, forming a closed detection chamber in the detection area 12.

[0047] In use, first position the detection disk 9 in the first working position. Add the nucleic acid sample and PCR reaction system to the vertical PCR reaction column 1 through the sample loading port 2. Air inside the column is expelled through the vent 3. After sample loading, seal the sample loading port 2 and the vent 3 with a pressure-sensitive sealing membrane.

[0048] Place the consumables into the instrument. The instrument applies a first temperature to the upper part of the vertical PCR reaction column 1 via a first temperature control module, and a second temperature to the lower part of the vertical PCR reaction column 1 via a second temperature control module. The second temperature is higher than the first temperature. The first temperature is 55℃ to 65℃, preferably 60℃; the second temperature is 90℃ to 98℃, preferably 95℃. The density of the PCR reaction solution changes due to the temperature difference; the density is lower in the high-temperature region and higher in the low-temperature region, and the liquid forms natural convection under gravity. Guided by the internal partition 4, the reaction solution flows upwards on one side and downwards on the other, forming a stable circulation path. The reaction solution completes nucleic acid denaturation in the high-temperature region and primer annealing and extension in the low-temperature region. After multiple cycles, PCR amplification is completed.

[0049] After amplification, the instrument's rotating shaft is inserted into the rotating positioning hole 14 of the detection disk 9, causing the detection disk 9 to rotate to the second working position, aligning and connecting the outlet port 8 and the inlet port 10. After PCR amplification, the vertical PCR reaction column 1 is at a high temperature, and the reaction liquid and gas are under thermal expansion pressure. At the same time, the hydrophilic guide layer on the inner wall of the guide channel reduces the surface tension of the liquid, generating capillary suction. Under the combined action of thermal expansion pressure and capillary force, the amplification solution enters the detection disk 9 through the first channel 7, outlet port 8, inlet port 10, and second channel 11, and flows into the detection zone 12 through the guide channel. Air in the detection zone 12 enters the waste liquid zone 13 through the exhaust channel, and excess amplification solution also enters the waste liquid zone 13 and is absorbed by the absorbent material, thereby reducing the residual air bubbles in the detection zone 12.

[0050] After the detection area 12 is filled with amplification solution, the instrument continues to drive the detection disk 9 to rotate to the third working position, so that the liquid outlet 8 and the liquid inlet 10 are misaligned and sealed again, and the detection area 12 forms a closed detection chamber.

[0051] Subsequently, the instrument applies a detection reaction temperature to the detection area 12 via the third temperature control module, causing the amplification products to specifically react with the pre-placed fluorescent probe layer, generating a detectable fluorescent signal. The detection reaction temperature is 50℃ to 65℃, preferably 60℃. The specific reaction can be a nucleic acid hybridization reaction, strand substitution reaction, probe substitution reaction, or fluorescence release reaction, with a reaction time of 5 minutes to 15 minutes.

[0052] The fluorescence detection module reads the fluorescence signal in the detection area 12, detects the fluorescence intensity, fluorescence color or fluorescence change rate, and outputs positive, negative or quantitative results according to the preset interpretation rules.

[0053] When multiple targets need to be detected simultaneously, multiple detection zones 12 can be set on the detection disk 9. Each detection zone 12 is connected to the second channel 11 through a branched flow channel, and different detection probes are pre-placed in each detection zone 12. After amplification, the detection disk 9 rotates to the second working position, and the amplification solution is simultaneously distributed to each detection zone 12 through the branched flow channel. Each detection zone 12 performs independent probe reaction and fluorescence detection, realizing parallel detection of multiple targets.

[0054] As an alternative to the internal partition 4, a spiral flow-guiding partition can be used, allowing the reaction solution to circulate between the high-temperature and low-temperature regions along a spiral path, enhancing the mixing effect. Alternatively, two or more vertically arranged partitions can be used to form multiple circulation channels inside the vertical PCR reaction column 1, improving the stability of the reaction solution circulation.

[0055] As an alternative embodiment to the waste liquid zone 13, a hydrophobic and breathable membrane can be provided above the waste liquid zone 13 to allow air to escape while preventing liquid from passing through, thereby further improving the sealing safety. The liquid-absorbing material in the waste liquid zone 13 can be any one of cellulose paper, glass fiber, porous polymer, or hydrophilic sponge.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A closed nucleic acid amplification and detection device, characterized in that, include: The amplification unit has a reaction chamber for accommodating nucleic acid samples and PCR reaction systems; A detection unit is rotatably disposed relative to the amplification unit and is sealed to the amplification unit; The amplification unit is provided with a sample inlet and a first channel (7) that are connected to the reaction chamber, and the end of the first channel (7) forms a liquid outlet (8). The detection unit is provided with a second channel (11) and at least one detection area (12) for performing the detection reaction. The end of the second channel (11) forms a liquid inlet (10). The detection unit is also provided with an exhaust structure that communicates with the detection area (12). The detection unit can rotate and switch between a first working position, a second working position, and a third working position relative to the amplification unit; In the first working position, the liquid outlet (8) and the liquid inlet (10) are staggered and sealed to isolate the reaction chamber from the detection area (12); In the second working position, the liquid outlet (8) is aligned and connected with the liquid inlet (10), so that the amplification product in the reaction chamber enters the detection area (12) through the first channel (7), the liquid outlet (8), the liquid inlet (10) and the second channel (11). In the third working position, the liquid outlet (8) and the liquid inlet (10) are again staggered and sealed, so that the detection area (12) forms a closed detection cavity.

2. The closed nucleic acid amplification and detection device according to claim 1, characterized in that, The amplification unit is a vertical PCR reaction column (1), the reaction chamber is a vertically extending columnar inner cavity, and the detection unit is a detection disk (9) located below the vertical PCR reaction column (1).

3. The closed nucleic acid amplification and detection device according to claim 2, characterized in that, The vertical PCR reaction column (1) is provided with at least one internal partition (4). The internal partition (4) extends along the height direction of the vertical PCR reaction column (1), and the upper end of the internal partition (4) forms a top communication gap (5) with the top wall of the columnar cavity, and the lower end of the internal partition (4) forms a bottom communication gap (6) with the bottom wall of the columnar cavity, so as to guide the reaction liquid to form a thermal convection circulation around the internal partition (4) under the vertical temperature gradient.

4. The closed nucleic acid amplification and detection device according to claim 2, characterized in that, The detection disk (9) is provided with a flow channel, which connects the second channel (11) and the detection area (12). At least part of the inner wall of the flow channel is provided with a hydrophilic flow guiding layer, which is used to assist the amplification product to flow from the second channel (11) to the detection area (12) through capillary action.

5. The closed nucleic acid amplification and detection device according to claim 2, characterized in that, The exhaust structure includes a waste liquid area (13) and an exhaust channel. The waste liquid area (13) is connected to the detection area (12) through the exhaust channel. The waste liquid area (13) is used to receive the discharged air and excess liquid during the filling process of the detection area (12). The waste liquid area (13) is provided with liquid-absorbing material and / or hydrophobic and breathable membrane.

6. The closed nucleic acid amplification and detection device according to claim 2, characterized in that, The detection disk (9) is provided with multiple detection areas (12), and the multiple detection areas (12) are respectively connected to the second channel (11) through branch flow channels. Each detection area (12) is used to pre-set the same or different detection probes to realize parallel detection of single or multiple targets.

7. The closed nucleic acid amplification and detection device according to any one of claims 2 to 6, characterized in that, A sealing contact structure is provided between the lower end of the vertical PCR reaction column (1) and the upper surface of the detection disk (9). The sealing contact structure is any one of a planar sealing surface, an annular boss, an elastic sealing ring, or a compressed sealing surface, used to maintain the seal between the vertical PCR reaction column (1) and the detection disk (9) during the rotation of the detection disk (9).

8. The closed nucleic acid amplification and detection device according to any one of claims 1 to 7, characterized in that, The detection disk (9) is provided with a rotation positioning hole (14) for cooperating with an external rotation drive module to drive the detection disk (9) to rotate.

9. A method for nucleic acid detection using the closed nucleic acid amplification and detection device according to any one of claims 1 to 8, characterized in that, Includes the following steps: Step S1: Place the detection unit in the first working position so that the liquid outlet (8) and the liquid inlet (10) are in a misaligned and sealed state; Step S2: Add nucleic acid sample and PCR reaction system into the reaction chamber through the sample addition port, and exhaust air from the reaction chamber through the exhaust port (3) provided on the amplification unit; Step S3: Seal the sample inlet and the vent (3) to form a closed amplification chamber in the reaction chamber; Step S4: Apply a temperature gradient to the reaction chamber to create thermal convection circulation of the reaction solution within the reaction chamber for PCR amplification; Step S5: After amplification is completed, rotate the detection unit to the second working position so that the liquid outlet (8) and the liquid inlet (10) are aligned and connected; Step S6: Using the driving force generated in the reaction chamber, the amplification product is driven to enter the detection area (12) through the first channel (7), the liquid outlet (8), the liquid inlet (10) and the second channel (11). Step S7: Rotate the detection unit to the third working position so that the liquid outlet (8) and the liquid inlet (10) are misaligned and sealed again, thus sealing the detection area (12). Step S8: Apply detection reaction conditions to the detection area (12) to make the amplification product react specifically with the pre-set detection reagent to generate a detectable signal; Step S9: Read the signal in the detection area (12) through the signal reading module and output the detection result.

10. The nucleic acid detection method according to claim 9, characterized in that, In step S4, a first temperature is applied to the upper part of the amplification unit, and a second temperature higher than the first temperature is applied to its lower part to form the temperature gradient; the first temperature is 55°C to 65°C, and the second temperature is 90°C to 98°C; in step S8, the detection reaction condition is a detection reaction temperature of 50°C to 65°C; in step S6, the driving force includes the thermal expansion pressure in the reaction chamber and / or the capillary force in the detection unit; air and / or excess amplification products in the detection area (12) enter the waste liquid area (13) through the exhaust channel to reduce the residual bubbles in the detection area (12); in step S9, the signal reading module is a fluorescence detection module, which detects the fluorescence intensity, fluorescence color or fluorescence change rate in the detection area (12), and outputs positive, negative or quantitative results according to preset interpretation rules.

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