Nucleic acid detection chip and method based on independent gas-liquid flow circulation path and trace IAT-NALFD

Abstract

The invention discloses a nucleic acid detection chip and method based on an independent gas-liquid flow circulation path and trace IAT-NALFD. The chip internally comprises a water storage cavity containing pure water or a buffer solution, a nucleic acid amplification cavity containing a nucleic acid amplification reaction solution for nucleic acid amplification reaction and a test strip detection cavity containing a test strip; the initial lower water storage cavity and the nucleic acid amplification cavity are located below the test strip detection cavity, the strip-shaped length direction of the test strip detection cavity is horizontally arranged, and the water storage cavity and the nucleic acid amplification cavity are located on the same level; the water storage cavity, the nucleic acid amplification cavity, the first liquid channel and the gas channel form an independent gas-liquid flowing circulation channel, and the nucleic acid amplification cavity, the test strip detection cavity, the second liquid channel and the gas channel form another channel. Integrated micro isothermal amplification and test strip detection can be realized, the detection cost is reduced, chambers do not interfere with one another, flowing is smooth, and the chip is simple in structure, low in cost, simple and efficient to operate and high in efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro diagnosis and biochemical analysis, and relates to nucleic acid amplification technology (IAT) and nucleic acid lateral flow dipstick (NALFD) detection technology, and specifically discloses a nucleic acid detection chip and method based on an independent gas-liquid flow circulation pathway and a trace IAT-NALFD integrated nucleic acid detection method for nucleic acid detection. Background Art

[0002] Nucleic acid amplification is a key step in nucleic acid amplification detection. The principle of raw materials (primers, dNTPs or fuel chains) and base complementary pairing is used to generate amplicons, which greatly increases the number of target sequences. Among the many in vitro nucleic acid amplification technologies, isothermal amplification technology (IAT) has its unique advantages. Its biggest feature is that the entire amplification process only requires constant temperature conditions, so only a constant temperature heating module is required. Compared with traditional PCR technology, there is no need to frequently increase or decrease the temperature on the same temperature control module or 2-3 heating and temperature control modules at different temperatures. Commonly used isothermal amplification technologies include loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), etc. This advantage makes IAT widely used in the field of nucleic acid detection, especially in places with limited conditions such as outside laboratories.

[0003] Nucleic Acid Lateral Flow Dipstick (NALFD) is widely used due to its low cost, small size, portability, simple use, and rapid detection. Unlike immune lateral flow test strips that recognize antibodies, NALFD is specifically used to recognize nucleic acids. There are many applications of nucleic acid detection by first amplifying the target nucleic acid with IAT and then detecting the nucleic acid amplicons with NALFD. However, due to the operation of transferring the amplification solution between the two steps, not only is the operation complicated, but there is also the problem of false positives caused by aerosol contamination. In addition, the test strip test requires 50-100 microliters of amplification solution, which is far more than the amount of amplification in ordinary reaction tubes (20-25 microliters), which also greatly increases the cost of detection. Summary of the invention

[0004] In order to solve the above problems and the problems existing in the background technology, the present invention proposes a nucleic acid detection chip and method based on an independent gas-liquid flow circulation pathway and trace IAT-NALFD for trace isothermal amplification and test strip detection.

[0005] The present invention can realize integrated micro-isothermal amplification and test strip detection. The water storage chamber inside the chip stores a large volume of water or buffer, such as phosphate buffer, for the micro-amplification liquid in the isothermal amplification chamber, which reduces the use of amplification reagents and reduces the detection cost. In addition, there are independent gas-liquid flow circulation paths between the water storage chamber and the amplification chamber, and between the amplification chamber and the test strip chamber, and there is no air pressure difference. The solution does not interfere with each other from the water storage chamber to the amplification chamber and from the amplification chamber to the test strip chamber, and flows smoothly. The device provides a constant reaction temperature for the reaction in the chip.

[0006] The technical solution of the present invention is as follows:

[0007] 1. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and trace IAT-NALFD:

[0008] The chip contains a water storage chamber, a nucleic acid amplification chamber, and a test strip detection chamber:

[0009] It includes a semicircular water storage chamber containing pure water or buffer solution;

[0010] It includes a semicircular nucleic acid amplification chamber, which contains nucleic acid amplification reaction liquid for nucleic acid amplification reaction;

[0011] It includes a strip-shaped test strip detection cavity for test strip detection, in which a lateral flow test strip is pre-placed;

[0012] In the initial state, the water storage chamber and the nucleic acid amplification chamber are located below the test strip detection chamber, the length direction of the test strip detection chamber is arranged horizontally, and the water storage chamber and the nucleic acid amplification chamber are located at the same level.

[0013] The water storage chamber, the nucleic acid amplification chamber and the test strip detection chamber are arranged on the same vertical reference plane and are fixed in relative positions on the reference plane.

[0014] The water storage chamber and the nucleic acid amplification chamber are connected at one end close to each other through an arc-shaped first liquid channel, the middle of the first liquid channel is higher than the two ends, and the radial size of the first liquid channel gradually decreases from the water storage chamber to the nucleic acid amplification chamber;

[0015] The connection between the first liquid channel and the nucleic acid amplification chamber is connected to the end of the water storage chamber away from the nucleic acid amplification chamber through a gas channel, so that the water storage chamber, the nucleic acid amplification chamber, the first liquid channel and the gas channel constitute an independent gas-liquid flow circulation path located inside the chip.

[0016] One end of the nucleic acid amplification chamber away from the water storage chamber is connected to one end of the test strip detection chamber through a second liquid channel, and a triangular capillary valve is provided at the connection between the nucleic acid amplification chamber and the second liquid channel. One end of the nucleic acid amplification chamber close to the water storage chamber is connected to the other end of the test strip detection chamber through a gas channel, so that the nucleic acid amplification chamber, the test strip detection chamber, the second liquid channel and the gas channel constitute an independent gas-liquid flow circulation path located inside the chip.

[0017] The size of the end of the capillary valve close to the nucleic acid amplification chamber is larger than the size of the end away from the nucleic acid amplification chamber.

[0018] The second liquid channel is arc-shaped or S-shaped.

[0019] Specifically, one end of the water storage chamber and one end of the nucleic acid amplification chamber are connected by an arc-shaped first liquid channel only for liquid to pass through. The first liquid channel is horizontal as a whole. The two ends of the first liquid channel are respectively connected to the water storage chamber and the nucleic acid amplification chamber. The height of the middle part of the first liquid channel is higher than the height of the two ends.

[0020] The other end of the nucleic acid amplification chamber and one end of the test strip detection chamber are connected through a second liquid channel that is only used for liquid to pass through. A capillary valve is arranged between the second liquid channel and the nucleic acid amplification chamber. The size of the capillary valve at one end close to the nucleic acid amplification chamber is larger than the size at the end away from the nucleic acid amplification chamber.

[0021] The other end of the water storage chamber is connected to the other end of the test strip detection chamber through a first gas channel only for gas passage, and the middle part of the first gas channel is connected to the nucleic acid amplification chamber through a second gas channel only for gas passage.

[0022] The inner diameter of the first gas channel is greater than the inner diameter of the second gas channel.

[0023] 2. A nucleic acid detection method for a nucleic acid detection chip:

[0024] S1. Place the lateral flow test strip into the test chamber with the front side facing upward and the sample pad facing outward, and seal the front side of the chip with tape;

[0025] S2. When in use, in the initial state, the reference surface of the chip is placed horizontally with the back facing upward, and 15-35μL of nucleic acid amplification reaction solution is added to the sample loading port of the nucleic acid amplification chamber. The nucleic acid amplification reaction solution is prepared by itself as needed, and the marker used should match the test strip. The isothermal nucleic acid amplification method and the detection target are selected according to actual conditions. Then add pure water to the sample loading port of the water storage chamber. The total amount of pure water and reaction solution added meets the use conditions of the test strip of 50-100μL. When the viscosity of the reaction solution is relatively high, appropriate surfactants can be added to the pure water and the reaction solution. After adding the sample, seal the two sample loading ports with tape;

[0026] S3, after adding the sample, vertically insert the chip into the chip slot, with the insertion direction being such that the nucleic acid amplification cavity on the chip contacts the heating plate of the aluminum block, and controlling the heating plate of the aluminum block to heat the nucleic acid amplification cavity;

[0027] Specifically, plug in the device and turn on the switch on the side of the device, then the yellow power-on indicator lights up. Set the heating time (nucleic acid amplification reaction time) and target temperature by pressing the button, and press the blue button to start heating and reaction. The display will show the real-time temperature and the countdown from the start of heating.

[0028] S4. After the amplification reaction is completed, the red light on the device lights up, indicating that the reaction is over. Obtain the amplification solution in the nucleic acid amplification chamber, take the chip out of the chip slot, rotate the chip with the direction perpendicular to the reference plane as the axis, and rotate the sample pad of the lateral flow test strip to the lowest position of the chip; or pinch the chip part on the other side of the sample pad of the test strip and shake the chip;

[0029] At this time, the liquid in the water storage chamber and the nucleic acid amplification chamber flows downward due to gravity or centrifugal force, and the pure water flows through the nucleic acid amplification chamber to continuously impact the amplification liquid in the nucleic acid amplification chamber and mixes with it to form a mixed liquid. The mixed liquid flows into the lowest point of the test strip detection chamber and contacts the lowest point of the lateral flow test strip. The mixed liquid moves laterally from the sample pad of the lateral flow test strip to the water absorbent pad through capillary action, and finally the test result is interpreted according to the color development of the C line and the T line on the lateral flow test strip.

[0030] The beneficial effects of the present invention are:

[0031] 1. The chip and method of the present invention can realize the integrated micro-IAT for NALFD nucleic acid detection. The chip is provided with two chambers for containing pure water and nucleic acid amplification reagents respectively, separated by an arc channel in the middle. Due to the introduction of a large volume of pure water or buffer, compared with the existing large volume IAT-NALFD method, the micro-IAT of the present invention greatly reduces the amount of nucleic acid amplification reagents required and reduces the detection cost.

[0032] 2. The water storage chamber and the nucleic acid amplification chamber are connected through the liquid channel and the gas channel inside the chip; the nucleic acid amplification chamber and the test strip detection chamber are connected through the liquid channel and the gas channel inside the chip, forming two independent gas-liquid flow circulation pathways, so that the air pressure inside the chip is automatically balanced, and the liquid flows smoothly in the chip by its own gravity without the need for external equipment to provide power.

[0033] 3. To complete the test, only two simple steps are required. The first is to drip a certain amount of pure water or buffer solution and reaction solution into the sample ports of the water storage chamber and the reaction solution storage chamber, and then seal them; the second is to rotate or shake the chip after the nucleic acid amplification is completed, and the amplification solution flows into the test strip detection chamber. The entire test process does not require opening the cover, pipetting, or manual mixing, which simplifies the test operation process.

[0034] 4. The isothermal nucleic acid amplification method and the composition of the detection reagent are not restricted. The isothermal amplification method can be selected according to the needs, and the reaction time and reaction temperature on the equipment can be adjusted by the user. The user can add the detection reagent according to the detection target.

[0035] 5. After the reaction solution and pure water are added, the two sample ports are sealed with tape. The entire chip system is airtight, and aerosols cannot be released into the surrounding environment, avoiding false positive results in subsequent test results.

[0036] 6. The stopping and flowing of liquid in the chip are precisely controlled by the chamber and flow channel, without the need for external equipment such as centrifuges and pumps. The wide-bore channel on the right side of the water storage chamber and the capillary valve on the right side of the nucleic acid amplification chamber prevent the liquid from flowing before the reaction is completed. After the reaction is completed, rotating or shaking the chip can make the liquid break through the capillary valve and flow smoothly into the test strip detection chamber.

[0037] The chip structure of the present invention is simple and the cost is low. The device of the present invention is simple in appearance and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The schematic diagram of the structure of the chip of the present invention is as follows: 1. reference surface, 2. water storage chamber, 3. pure water sample port, 4. nucleic acid amplification chamber, 5. reaction liquid sample port, 6. capillary valve, 7 test strip detection chamber, 81, 82 liquid channels, 91, 92, gas channels.

[0039] Figure 2 It is a schematic diagram of the structure of the circuit board of the device of the present invention. a, NTC thermistor interface, b, ceramic heating plate interface, c, button interface, d, display interface, e, status indicator interface, f, 5V relay interface, g, 12V DC power socket, h, single chip computer interface, i, 12V to 5V transformer interface, j, switch interface.

[0040] Figure 3It is a schematic diagram of the structure of the housing of the device of the present invention. A, chip slot, B, aluminum block slot, C, switch slot, D, reaction end indicator slot, E, F, G, H, key slot, I, display slot, J, power-on indicator slot, K, circuit board slot.

[0041] Figure 4 These are three test results of the nucleic acid chromatography test strip of Example 1: a) positive result, b) negative result, and c) invalid test result.

[0042] Figure 5 These are five test results of the dual-index nucleic acid chromatography test strip of Example 2. a) Target 1 and 2 are all positive, b) Target 1 is positive and Target 2 is negative, c) Target 2 is positive and Target 1 is negative, d) Target 1 and 2 are all negative, and e) invalid test result.

[0043] In the figure: a reference surface (1), a water storage chamber (2), a pure water injection port (3), a nucleic acid amplification chamber (4), a reaction liquid injection port (5), a capillary valve (6), a test strip detection chamber (7), a first liquid channel (81), a second liquid channel (82), a first gas channel (91), and a second gas channel (92). DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings and specific implementations.

[0045] like Figure 1 As shown, the chip contains a water storage chamber 2, a nucleic acid amplification chamber 4, a test strip detection chamber 7, and a channel connecting these chambers, which are described in detail as follows:

[0046] It comprises a semicircular water storage chamber 2 containing pure water or phosphate buffer with a pH value of 7.4; the water storage chamber 2 is provided with a pure water injection port 3 for adding pure water, and the pure water injection port 3 is arranged on the back of the chip.

[0047] It comprises a semicircular nucleic acid amplification chamber 4 containing nucleic acid amplification reaction liquid for nucleic acid amplification reaction; the nucleic acid amplification chamber 4 has a reaction liquid injection port 5 for adding amplification reaction liquid, and the position of the reaction liquid injection port 5 is set on the back of the chip.

[0048] It includes a strip-shaped test strip detection chamber 7, which is used for test strip detection, and a lateral flow test strip is pre-placed in the chamber;

[0049] In the initial state, the water storage chamber 2 and the nucleic acid amplification chamber 4 are located below the test strip detection chamber 7 before nucleic acid detection. The length direction of the test strip detection chamber 7 is arranged horizontally, and the water storage chamber 2 and the nucleic acid amplification chamber 4 are located at the same level.

[0050] The water storage chamber 2, nucleic acid amplification chamber 4 and test strip detection chamber 7 are arranged on the same vertical reference plane and fixed in relative position on the reference plane. The water storage chamber 2, nucleic acid amplification chamber 4 and test strip detection chamber 7 are driven to rotate as a whole by rotating the reference plane as a whole.

[0051] The reference plane is defined as the side where the whole picture of each chamber can be seen. The reference plane of the chip is perpendicular to the ground to describe the specific position of each chamber. The water storage chamber is located on the lower left side of the chip. The nucleic acid amplification chamber is located on the right side of the water storage chamber and is flush with the water storage chamber. The test strip detection chamber is located above the nucleic acid amplification chamber.

[0052] The water storage chamber 2 and the nucleic acid amplification chamber 4 are connected at one end close to each other through an arc-shaped first liquid channel 81 for liquid to pass only. The middle of the first liquid channel 81 is higher than the two ends, and the radial dimension of the first liquid channel 81 gradually decreases from the water storage chamber 2 to the nucleic acid amplification chamber 4.

[0053] The connection between the first liquid channel 81 and the nucleic acid amplification chamber 4 is connected to the end of the water storage chamber 2 away from the nucleic acid amplification chamber 4 through a gas channel 91, 92 used only for gas passage, so that the water storage chamber 2, the nucleic acid amplification chamber 4, the first liquid channel 81 and the gas channels 91, 92 constitute an independent gas-liquid flow circulation path located inside the chip.

[0054] The gas channels 91 and 92 are very narrow at the locations where they are connected to the nucleic acid amplification chamber 4 and are arranged away from the reaction liquid injection port 5 on the nucleic acid amplification chamber 4 to prevent the reaction liquid or pure water from flowing into the gas channels.

[0055] One end of the nucleic acid amplification chamber 4 away from the water storage chamber 2 and the end on the same side of the test strip detection chamber 7 are connected through a second liquid channel 82 which is only used for liquid to pass through, and a triangular capillary valve 6 is provided at the connection between the nucleic acid amplification chamber 4 and the second liquid channel 82, and one end of the nucleic acid amplification chamber 4 close to the water storage chamber 2 and the other end on the same side of the test strip detection chamber 7 are connected through another gas channel 91, 92 which is only used for gas to pass through, so that the nucleic acid amplification chamber 4, the test strip detection chamber 7, the second liquid channel 82 and the gas channels 91, 92 constitute an independent gas-liquid flow circulation path located inside the chip.

[0056] The gas channels 91 and 92 are very narrow at the locations where they are connected to the nucleic acid amplification chamber 4 and are arranged away from the reaction liquid injection port 5 on the nucleic acid amplification chamber 4 to prevent the reaction liquid or pure water from flowing into the gas channels.

[0057] In specific implementation, Figure 1As shown, the right side of the water storage chamber 2 and the left side of the nucleic acid amplification chamber 4 are connected through an arc-shaped liquid channel inside the chip, and the left sides of the two are connected through a gas channel, forming an independent gas-liquid flow circulation path inside the chip. Among them, the arc-shaped liquid channel adopts a gradually narrowing gradient. The wide aperture on the left side of the arc can eliminate the capillary effect and prevent pure water from entering the nucleic acid amplification chamber 4 in advance; the narrow aperture on the right side of the arc can reduce the evaporation of the reaction liquid during the heating reaction in the nucleic acid amplification chamber 4. The gas channel on the nucleic acid amplification chamber 4 is very narrow and slightly away from the sample addition port on the nucleic acid amplification chamber 4 to prevent the reaction liquid or pure water from flowing into the gas channel.

[0058] The right side of the nucleic acid amplification chamber 4 and the right side of the test strip detection chamber 7 are connected through the liquid channel inside the chip, and the left sides of the two are connected through the gas channel, which also constitutes an independent gas-liquid flow circulation path inside the chip. Among them, a triangular capillary valve 6 is set on the right side of the nucleic acid amplification chamber 4 to intercept the reaction liquid and prevent the reaction liquid from flowing into the test strip detection chamber 7 in advance.

[0059] These circulation paths automatically balance the air pressure inside the chip, and the liquid flows inside the chip by its own gravity, without the need for external equipment to provide power, and is smaller in size.

[0060] The first gas-liquid flow circulation pathway ensures the smooth flow of pure water from the water storage chamber 2 to the nucleic acid amplification chamber 4, and the second gas-liquid flow circulation pathway ensures the smooth flow of pure water and reaction liquid from the nucleic acid amplification chamber 4 to the test strip detection chamber 7, so that the two circuits realize the integration of nucleic acid amplification and test strip detection and reduce the size of the chip.

[0061] The flow of liquid in the chip is precisely controlled by each chamber and channel. When the chip is horizontally loaded and vertically reacted, pure water and reaction liquid are placed in the water storage chamber 2 and nucleic acid amplification chamber 4 respectively. After the nucleic acid amplification reaction is completed, the chip is rotated 90° clockwise with the direction perpendicular to the reference plane 1 as the axis. At this time, the liquid in the water storage chamber 2 and the nucleic acid amplification chamber 4 flows downward due to gravity and finally flows into the lowest point of the test strip detection chamber 7. Among them, the reaction liquid in the nucleic acid amplification chamber 4 breaks through the capillary valve 6 by its own gravity and the extrusion of the pure water above. In spatial order, the reaction liquid flows into the test strip detection chamber 7 first, and the pure water comes later.

[0062] The advantage of this is that pure water will take away the amplification liquid remaining in the amplification chamber and channel, avoiding the loss of amplicons; and when the pure water enters the test strip detection chamber 7, it continuously impacts the amplification liquid and mixes it, which is beneficial to the test strip detection. The presence of pure water also enables the combination of trace IAT and NALFD, reducing the use of amplification reagents and reducing detection costs.

[0063] The volume of the water storage chamber 2 and the nucleic acid amplification chamber 4 can be adjusted according to the amount of pure water and reaction solution added, and the total volume of pure water and reaction solution should meet the use conditions of the test strip (50-100 μL).

[0064] The advantages of the chip configuration of the present invention are:

[0065] First, the water storage chamber inside the chip stores a large volume of water for the trace amplification solution in the isothermal amplification chamber, which reduces the use of amplification reagents and reduces detection costs.

[0066] Second, the flow of the solution from the water storage chamber to the amplification chamber and then to the test strip chamber does not require external forces such as pumps and valves, but flows through the solution's own gravity.

[0067] Third, there are independent gas-liquid flow circulation paths between the water storage chamber and the amplification chamber, and between the amplification chamber and the test strip chamber. There is no air pressure difference, and the solution flows smoothly from the water storage chamber to the amplification chamber and from the amplification chamber to the test strip chamber without interfering with each other.

[0068] Fourth, the amplification area and the test strip detection area are integrated on the same sealed chip, eliminating pipetting operations and avoiding aerosol contamination.

[0069] The device consists of an internal circuit system and a 3D-printed shell.

[0070] The circuit board of the device is as follows Figure 2As shown, it mainly consists of an NTC thermistor interface a, a ceramic heating plate interface b, a button interface c, a display screen interface d, a status indicator light interface e, a relay interface f, a 12V DC power socket g, a single-chip microcomputer interface h, a 12V to 5V transformer interface i, and a switch interface j. The ceramic heating plate is located inside the aluminum block to provide heat for the aluminum block. The aluminum block is a good conductor of heat. The U-shaped aluminum block transfers the heat of the ceramic heating plate to both sides of the nucleic acid amplification cavity 4 in the chip to provide the required temperature for isothermal nucleic acid amplification reaction. The display screen is used to display parameters such as mode (temperature measurement / heating / timing / setting), real-time temperature, reaction time (positive timing) and heating status (ON / OFF). The NTC thermistor, a 10K constant resistor and a single-chip microcomputer are used to measure the actual temperature of the aluminum block. The principle is: measure the voltage between the NTC thermistor and the constant resistor, use the AD module integrated in the single-chip microcomputer to convert the analog signal of the voltage into a digital signal (analog-to-digital conversion), and then convert the digital signal into a temperature value through the program compiled inside the single-chip microcomputer. The transformer converts the 12V voltage into 5V to meet the use requirements of components such as relays. The colors and functions of the four buttons are as follows: the red button is used to adjust the set temperature "-" and turn off the heating, the yellow button is used to set the heating time (i.e. reaction time), the blue button is used to adjust the set temperature "+" and turn on the heating, and the green button is used to set the target temperature (room temperature - 99°C); the two indicator lights are: the red status indicator, which lights up to indicate the end of the reaction, and the yellow power indicator, which lights up to indicate that the circuit is powered normally; the switch is used to turn on and off the heating device; the relay is used to open and close the circuit.

[0071] The housing of the device is Figure 3 As shown, it is mainly composed of a main body at the bottom and a cover at the top, and the two are fixed by screws located at four corners. There is a large circuit board slot K inside the main body, which is used to place the circuit board and the NTC thermistor, constant resistor, transformer, relay, and single-chip microcomputer connected to the circuit board. The aluminum block slot B inside the main body is used to place the aluminum block. The chip slot A embedded with the aluminum block slot B is used to place the above-mentioned chip, and the two sides of the nucleic acid amplification cavity 4 on the chip are fitted with the two inner sides of the U-shaped aluminum block. There is an opening on each side of the main body that is connected to the outside world, which is a square opening (switch slot C) for placing a switch and a circular opening (jack slot) for placing a DC power jack. There is a large square opening (display slot I) in the middle of the upper cover for embedding the display screen; there is a narrow opening (chip slot A) on one side of the display screen for inserting the chip; there are four small openings (key slots E, F, G, H) on the other side of the display screen, which are used to embed four buttons respectively; there are also two openings for embedding two indicator lights (power-on indicator slot J and reaction end indicator slot D).

[0072] Implementation examples of the present invention:

[0073] 1. RPA amplification and single-index lateral flow test strip detection of Salmonella

[0074] The structure of the chip used in this embodiment is as follows Figure 1 The chip is made of polymethyl methacrylate (PMMA) sheet with a thickness of 4 mm. Figure 1 The structure shown (the dimensions are subject to the text description below).

[0075] The chip contains a water storage chamber 2, a nucleic acid amplification chamber 4, and a test strip detection chamber 7, as well as channels 81, 82, 91, and 92 connecting these chambers. There is a sample injection port 3 on the back of the water storage chamber 2. There is a sample injection port 5 on the back of the nucleic acid amplification chamber 4. The water storage chamber 2 is located on the lower left side of the chip. The nucleic acid amplification chamber 4 is located on the right side of the water storage chamber 2 and is flush with the water storage chamber. The test strip detection chamber 7 is located above the nucleic acid amplification chamber 4. The nucleic acid amplification chamber 4 is located on the left side of the rightmost end of the test strip detection chamber 7.

[0076] The semicircular water storage chamber 2 has a radius of 7mm and a depth of 2mm. The pure water injection port 3 is a square with a side length of 3mm. The semicircular nucleic acid amplification chamber 4 has a radius of 4mm and a depth of 2mm. The reaction liquid injection port 5 is a square with a side length of 3mm. The triangular capillary valve 6 has sides of 2mm, 2mm, and 2.5mm, respectively, and a depth of 0.5mm. The rectangular test strip detection chamber 7 is 4mm wide, 65mm long, and 3mm deep. The liquid channel 81 is an arc with a gradually decreasing diameter, with a wide diameter of 4mm, a narrow diameter of 1mm, and a depth of 2mm. The cross-sectional size of the liquid channel 82 is 0.5x0.5 mm. If it is too thin, the reaction liquid will not flow smoothly, and if it is too thick, more reaction liquid will remain. The gas channel 91 is 0.3mm wide and 0.3mm deep. The gas channel 92 is 0.5mm wide and 0.5mm deep. The length and shape of each channel are set according to actual needs. The corners of the liquid channels 82 and 83 are designed to be rounded to facilitate the flow of the liquid in the channels. If you want the solution to flow faster and more smoothly in the chip, you can add a surfactant to the pure water and the reaction solution.

[0077] This embodiment takes the detection of Salmonella as an example, takes the virulence gene invA in Salmonella as the target, and performs RPA amplification and lateral flow test strip detection.

[0078] Salmonella with a concentration of 1000 cfu / ml was extracted using a nucleic acid extraction kit to obtain a DNA solution. The samples tested in this embodiment were the above DNA solution and ddH20.

[0079] Place the nucleic acid lateral chromatography test strip with the front side facing up and the sample pad facing outward into the test strip detection chamber 7. The test strip is coated with avidin (SA) at the detection line, and the latex microspheres are labeled with anti-FAM antibodies. The nucleic acid amplification products containing both Biotin and FAM labels are detected by the sandwich method. Seal the front of the chip with tape for later use. When in use, place the chip horizontally, and add 25 μL of nucleic acid amplification reaction solution to the sample port 5 of the nucleic acid amplification chamber 4. Add 50 μL of pure water to the sample port 3 of the water storage chamber 2. After adding the sample, seal the two sample ports 3 and 5 with tape.

[0080] The system of RPA reaction solution is as follows:

[0081] Reaction components concentration Enzyme buffer 1x Super ScriptⅣ 20U invA RPA primer F / R 0.42μM / 0.42μM RNase H 1U Salmonella DNA 2ul MgOAc 14mM

[0082] Insert the chip vertically into the chip slot A of the device, with the insertion direction being based on the contact between the nucleic acid amplification cavity on the chip and the aluminum block. Plug in the power supply of the device and turn on the switch on the side of the device. The yellow power-on indicator lights up. Set the heating time (nucleic acid amplification reaction time) to 30 minutes and the target temperature to 37°C by pressing the button, and press the blue button to start heating and reaction. The real-time temperature and the countdown from the start of heating will be displayed on the display.

[0083] After the amplification reaction is completed, the red light on the device lights up, indicating that the reaction is over. The amplification liquid is obtained in the nucleic acid amplification chamber 4. Take out the chip, rotate the chip with the direction perpendicular to the reference plane 1 as the axis, and rotate the sample pad of the test strip to the lowest position; or after taking out the chip, pinch the chip part on the other side of the sample pad of the test strip and shake the chip. At this time, the liquid in the water storage chamber 2 and the nucleic acid amplification chamber 4 flows downward due to gravity or centrifugal force, and finally flows into the lowest point of the test strip detection chamber 7. Pure water continuously impacts the amplification liquid and mixes with it. The mixed liquid moves laterally from the sample pad to the absorbent pad through capillary action. Finally, the test results are interpreted according to the color development of the C line and the T line on the test strip. If both the C line and the T line are colored, it means that the tested sample contains Salmonella ( Figure 4 a). If the C line is colored and the T line is not colored, it means that the sample does not contain Salmonella or the content is lower than the detection limit of the test strip ( Figure 4 b). If both the C line and the T line are colored, it means that the operation is wrong or the test strip has deteriorated and is invalid. In this case, the problem should be eliminated and the test should be repeated ( Figure 4 c).

[0084] 2. Dual LAMP amplification and dual-index test strip detection of hepatitis B virus (HBV) and hepatitis A virus (HCV)

[0085] The structure of this embodiment is as follows Figure 1 As shown, the chip structure and processing method are the same as those in Example 1.

[0086] Among the hepatotropic viruses, hepatitis B virus (HBV) and hepatitis A virus (HCV) are the most contagious. This embodiment takes the detection of HBV and HCV as an example to perform dual LAMP amplification and dual-index test strip detection.

[0087] Place the nucleic acid lateral chromatography test strip with the front side facing up and the sample pad facing outward into the test strip detection chamber 7. The detection line T1 of the dual-index test strip is coated with anti-FITC antibody, and the detection line T2 is coated with anti-Digoxin antibody. Some latex microspheres are labeled with streptavidin, and some latex microspheres are labeled with anti-TAMRA antibody. The nucleic acid amplification products containing the corresponding markers are detected by the sandwich method. Seal the front of the chip with tape for later use. When in use, place the chip horizontally, and add 15 μL of nucleic acid amplification reaction solution to the sample port 5 of the nucleic acid amplification chamber 4. Add 60 μL of phosphate buffer with a pH value of 7.4 to the sample port 3 of the water storage chamber 2. After adding the sample, seal the two sample ports 3 and 5 with tape.

[0088] The LAMP reaction solution system is as follows:

[0089]

[0090] Insert the chip vertically into the chip slot A of the device, with the insertion direction being based on the contact between the nucleic acid amplification cavity on the chip and the aluminum block. Plug in the power supply of the device and turn on the switch on the side of the device. The yellow power-on indicator lights up. Set the heating time (nucleic acid amplification reaction time) to 30 minutes and the target temperature to 65°C by pressing the button, and press the blue button to start heating and reaction. The real-time temperature and the countdown from the start of heating will be displayed on the display.

[0091] After the amplification reaction is completed, the red light on the device lights up, indicating that the reaction is over. The amplification liquid is obtained in the nucleic acid amplification chamber 4. Take out the chip, rotate the chip with the direction perpendicular to the reference plane 1 as the axis, and rotate the sample pad of the test strip to the lowest position; or after taking out the chip, pinch the chip part on the other side of the sample pad of the test strip and shake the chip. At this time, the liquid in the water storage chamber 2 and the nucleic acid amplification chamber 4 flows downward due to gravity or centrifugal force, and finally flows into the lowest point of the test strip detection chamber 7. Pure water continuously impacts the amplification liquid and mixes with it. The mixed liquid moves laterally from the sample pad to the absorbent pad through capillary action. Finally, the test results are interpreted according to the color development of the C line, T1 line and T2 line on the test strip. If the C line, T1 line and T2 line are all colored, it means that the test is valid and the sample tested contains both HBV and HCV viruses ( Figure 5 a). If the C line and T1 line are colored, and the T2 line is not colored, it means that the test is effective and the sample tested contains HBV but not HCV ( Figure 5 b). If the C line and T2 line are colored, and the T1 line is not colored, it means that the test is valid and the sample tested contains HCV but not HBV ( Figure 5c). If the C line is colored, and the T1 and T2 lines are not colored, it means that the test is effective and the sample does not contain HBV and HCV ( Figure 5 d). If the C line does not show color, it means that the operation is wrong or the test strip has deteriorated and is invalid. In this case, the problem should be eliminated and the test should be repeated ( Figure 5 e).

Claims

1. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and a trace amount of IAT-NALFD, characterized in that: The chip contains a water storage chamber (2), a nucleic acid amplification chamber (4), and a test strip detection chamber (7): It comprises a semicircular water storage chamber (2) containing pure water or buffer solution; It comprises a semicircular nucleic acid amplification chamber (4) containing a nucleic acid amplification reaction solution for performing a nucleic acid amplification reaction; It comprises a strip-shaped test strip detection chamber (7) for test strip detection, in which a lateral flow test strip is pre-placed; In the initial state, the water storage chamber (2) and the nucleic acid amplification chamber (4) are located below the test strip detection chamber (7), the length direction of the test strip detection chamber (7) is arranged horizontally, and the water storage chamber (2) and the nucleic acid amplification chamber (4) are located at the same level.

2. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and a trace amount of IAT-NALFD according to claim 1, characterized in that: The water storage chamber (2), the nucleic acid amplification chamber (4) and the test strip detection chamber (7) are arranged on the same vertical reference plane and are fixed in relative positions on the reference plane.

3. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and a trace amount of IAT-NALFD according to claim 1, characterized in that: The water storage chamber (2) and the nucleic acid amplification chamber (4) are connected to each other at one end thereof via an arc-shaped first liquid channel (81); the middle portion of the first liquid channel (81) is higher than the two ends thereof; and the radial dimension of the first liquid channel (81) gradually decreases from the water storage chamber (2) to the nucleic acid amplification chamber (4); The connection point between the first liquid channel (81) and the nucleic acid amplification chamber (4) is connected to the end of the water storage chamber (2) away from the nucleic acid amplification chamber (4) through the gas channels (91, 92), so that the water storage chamber (2), the nucleic acid amplification chamber (4), the first liquid channel (81) and the gas channels (91, 92) form an independent gas-liquid flow circulation path located inside the chip.

4. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and trace IAT-NALFD according to claim 1, characterized in that: The end of the nucleic acid amplification chamber (4) away from the water storage chamber (2) and the end of the test strip detection chamber (7) are connected via a second liquid channel (82), and a triangular capillary valve (6) is provided at the connection between the nucleic acid amplification chamber (4) and the second liquid channel (82), and the end of the nucleic acid amplification chamber (4) close to the water storage chamber (2) and the other end of the test strip detection chamber (7) are connected via gas channels (91, 92), so that the nucleic acid amplification chamber (4), the test strip detection chamber (7), the second liquid channel (82) and the gas channels (91, 92) form an independent gas-liquid flow circulation path located inside the chip.

5. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and a trace amount of IAT-NALFD according to claim 4, characterized in that: The size of the end of the capillary valve (6) close to the nucleic acid amplification chamber (4) is larger than the size of the end away from the nucleic acid amplification chamber (4).

6. A nucleic acid detection chip based on an independent gas-liquid flow circulation pathway and a trace amount of IAT-NALFD according to claim 4, characterized in that: The second liquid channel (82) is arc-shaped or S-shaped.

7. The nucleic acid detection method of the nucleic acid detection chip according to any one of claims 1 to 6, characterized in that: S1. Place the lateral flow test strip with the front side facing upward and the sample pad facing outward into the test strip detection cavity (7), and seal the entire surface of the chip with tape; S2. In the initial state, the reference surface of the chip is placed horizontally, 15-35 μL of nucleic acid amplification reaction solution is added to the sample injection port (5) of the nucleic acid amplification chamber (4), and then pure water is added to the sample injection port (3) of the water storage chamber (2). After adding the sample, the two sample injection ports are sealed with tape; S3, after adding the sample, vertically insert the chip into the chip slot, with the insertion direction being such that the nucleic acid amplification cavity (4) on the chip contacts the heating plate of the aluminum block, and controlling the heating plate of the aluminum block to heat the nucleic acid amplification cavity (4); S4. After the amplification reaction is completed, an amplification solution is obtained in the nucleic acid amplification chamber (4), the chip is taken out, and the chip is rotated with the direction perpendicular to the reference plane (1) as the axis, so that the sample pad of the lateral flow test strip is rotated to the lowest position of the chip; At this time, the liquid in the water storage chamber (2) and the nucleic acid amplification chamber (4) flows downward due to gravity or centrifugal force, and the pure water flows through the nucleic acid amplification chamber (4) to continuously impact the amplification liquid in the nucleic acid amplification chamber (4) and mixes with the amplification liquid to form a mixed liquid. The mixed liquid flows into the lowest point of the test strip detection chamber (7) and contacts the lowest point of the lateral flow test strip. The mixed liquid moves laterally from the sample pad of the lateral flow test strip to the water absorption pad through capillary action. Finally, the detection result is interpreted based on the color development of the C line and the T line on the lateral flow test strip.

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