A multi-channel nucleic acid amplification chip and a multi-channel nucleic acid amplification detection method

Abstract

The present application belongs to the field of medical detection technology, and relates to a multi-channel nucleic acid amplification chip, including: an inlet, a plurality of dissolution chambers, a plurality of amplification chambers, a plurality of waste liquid chambers and an air intake port, wherein a freeze-dried ball is arranged in the dissolution chamber, and the freeze-dried ball is used to fuse the biological sample liquid flowing into the dissolution chamber to form a nucleic acid test liquid. The present application also relates to a multi-channel nucleic acid amplification detection method. The technical solution provided by the present application enables the nucleic acid amplification chip to be provided with an air intake port and realize the fluid control function by generating a pressure difference between the air intake port and the inlet through external power. At the same time, the freeze-dried ball reagent pre-packaging technology and unique flow channel design of the present application realize the staged liquid feeding of samples / reagents and further fluid control, breaking through the technical bottleneck of only single-chip and single-channel detection in complex nucleic acid purification and detection processes, and realizing efficient, rapid and highly sensitive nucleic acid amplification detection.

Description

Technical Field

[0001] The present application relates to the field of medical detection technology, and more specifically, to a multi-channel nucleic acid amplification chip and a multi-channel nucleic acid amplification detection method using the multi-channel nucleic acid amplification chip. Background Art

[0002] Microfluidics is a technology that can detect trace samples with high sensitivity. However, the use of microfluidics chips in nucleic acid amplification currently faces the following problems:

[0003] 1) Chips often need to be matched with high-precision large instruments to achieve sample / reagent addition and fluid control;

[0004] 2) Some complex purification processes can only be achieved through single-chip single-channel detection;

[0005] 3) Due to the volume of the chip, the reagents often need to be matched to the chip, which limits the types of reagents. The above problems limit the development of nucleic acid amplification chips in terms of portability, popularization and home use. Summary of the invention

[0006] The technical problem to be solved by the embodiments of the present application is that the existing technology limits the development of portability, popularization and household use of nucleic acid amplification chips.

[0007] In order to solve the above technical problems, the present application embodiment provides a multi-channel nucleic acid amplification chip, which adopts the following technical solution:

[0008] A multi-channel nucleic acid amplification chip, comprising:

[0009] An inlet port, used for allowing the biological sample liquid to enter;

[0010] A plurality of dissolution chambers are connected to the sample inlet, wherein freeze-dried balls are arranged in the dissolution chambers, and the freeze-dried balls are used to fuse the biological sample liquid flowing into the dissolution chambers to form a nucleic acid test liquid;

[0011] A plurality of amplification chambers are connected to the dissolution chamber; a retaining element is provided in the amplification chamber, and the retaining element is used to filter the nucleic acid test liquid so that the nucleic acid test liquid is separated into a nucleic acid concentrate and a waste liquid;

[0012] A plurality of waste liquid chambers, connected to the amplification chamber, for storing the waste liquid;

[0013] The air inlet is communicated with the waste liquid chamber.

[0014] Further, the amplification chamber includes a first amplification zone and a second amplification zone connected to the first amplification zone, the first amplification zone is connected to the dissolution chamber, the second amplification zone is connected to the waste liquid chamber, and the interception element is arranged in the first amplification zone and / or the second amplification zone.

[0015] Further, the second amplification region is arranged on the periphery of the first amplification region, and the second amplification region is in an "Ω" shape; or,

[0016] The first amplification region is disposed on the periphery of the second amplification region, and the first amplification region is in an inverted "Ω" shape; or,

[0017] The amplification chamber is in one of the following shapes: funnel-shaped, round-shaped, square-shaped, and gourd-shaped.

[0018] Further, the depth of the first amplification zone is greater than or equal to the depth of the second amplification zone, and the interception element extends toward the second amplification zone.

[0019] Furthermore, the depth of the second amplification zone is greater than or equal to the depth of the first amplification zone, and the interception element extends toward the first amplification zone.

[0020] Furthermore, the intercepting element is configured as a microporous filter membrane, and the microporous filter membrane includes one or more of filter paper, glass fiber, fiber fabric, polyethersulfone, non-woven fabric, and synthetic fiber.

[0021] Furthermore, the intercepting element is configured as a nucleic acid adsorbent filler, and the nucleic acid adsorbent filler includes one or more of a silica gel membrane, a glass fiber, a nitrocellulose membrane, and a nylon membrane.

[0022] Furthermore, the volume of the dissolution chamber is greater than the sum of the volumes of the amplification chamber and the waste liquid chamber.

[0023] Furthermore, the air inlet is connected to an external power source via an interface, and the interface is configured as an air valve or as a groove disposed in the multi-channel nucleic acid amplification chip.

[0024] In order to solve the above technical problems, the present application also provides a multi-channel nucleic acid amplification detection method, which adopts the following technical solution:

[0025] A multi-channel nucleic acid amplification detection method comprises the following steps:

[0026] Providing a chip: providing the multi-channel nucleic acid amplification chip;

[0027] Adding biological sample liquid: closing the air inlet and adding the biological sample liquid to the injection port;

[0028] Liquid separation and volume determination: the sample liquid flows into the dissolution chamber and dissolves the freeze-dried ball to form a nucleic acid test liquid;

[0029] Concentration and drainage: when the pressure of the air inlet is lower than the pressure of the sample inlet, the nucleic acid test liquid in the dissolution chamber moves to the amplification chamber under the action of pressure, and the interception member separates the nucleic acid test liquid into nucleic acid concentrated liquid and waste liquid, so that the nucleic acid concentrated liquid remains in the amplification chamber, and the waste liquid moves from the amplification chamber to the waste liquid chamber under the action of pressure;

[0030] Amplification: The injection port is closed, and the concentrated nucleic acid solution remaining in the amplification chamber is amplified.

[0031] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0032] Compared with the prior art, the nucleic acid amplification chip of the present application is provided with the air inlet and generates a pressure difference between the air inlet and the injection port through external power to realize the fluid control function. At the same time, the freeze-dried ball reagent pre-packaging technology and unique flow channel design of the present application realize the staged liquid feeding of samples / reagents and further fluid control, breaking through the technical bottleneck of only single-chip and single-channel detection in complex nucleic acid purification and detection processes, solving the problem of chip volume limiting reagent matching, and simplifying the operation steps, improving the integration of the card box, and optimizing the reagent pre-volume compatibility and detection sensitivity. The multi-channel nucleic acid amplification chip realizes efficient, rapid and highly sensitive nucleic acid amplification detection with the characteristics of high integration, portable operation, simple structure, low cost, low risk of liquid crosstalk and diversified nucleic acid concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the scheme of the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 is an exploded view of the multi-channel nucleic acid amplification chip of Example 1 of the present application;

[0035] Figure 2 It is a structural schematic diagram of the front side of the main body in the first embodiment of the present application;

[0036] Figure 3 It is a structural schematic diagram of the back side of the main body in the first embodiment of the present application;

[0037] Figure 4 is a perspective view of the main body in the first embodiment of the present application;

[0038] Figure 5 It is a structural schematic diagram of the back side of the main body in the second embodiment of the present application;

[0039] Figure 6 is a perspective view of the main body in the second embodiment of the present application;

[0040] Figure 7 is a flow chart of a multi-channel nucleic acid amplification detection method;

[0041] Figure 8 is a schematic structural diagram of an amplification chamber embodiment 1;

[0042] Fig. 9 is a schematic structural diagram of a second embodiment of the amplification chamber;

[0043] Fig.10 Schematic diagram of the structure of the amplification chamber embodiment 3.

[0044] Figure numerals: 1. injection port; 11. injection channel; 12. liquid separation channel; 13. first air hole; 14. exhaust channel; 15. second hydrophobic breathable filler; 2. dissolution chamber; 20. dissolution channel; 23. vacuum channel; 24. connecting piece; 3. amplification chamber; 30. interception piece; 31. first amplification zone; 32. second amplification zone; 33. waste liquid channel; 34. first connecting hole; 4. waste liquid chamber; 40. power channel; 41. second connecting hole; 5. air inlet; 50. first hydrophobic breathable filler; 51. air valve; 52. groove; 6. main body; 7. front sealing film; 8. back sealing film. DETAILED DESCRIPTION

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0046] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0047] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0048] Embodiment 1

[0049] See also Figures 1 to 4 As shown, a multi-channel nucleic acid amplification chip comprises:

[0050] Inlet 1, used for the biological sample liquid to enter;

[0051] A plurality of dissolution chambers 2 are connected to the sample inlet 1, wherein freeze-dried balls are arranged in the dissolution chambers 2, and the freeze-dried balls are used to fuse the biological sample solution flowing into the dissolution chambers 2 to form a nucleic acid test solution;

[0052] A plurality of amplification chambers 3 are connected to the dissolution chamber 2; a retaining element 30 is provided in the amplification chamber 3, and the retaining element 30 is used to filter the nucleic acid test liquid so that the nucleic acid test liquid is separated into a nucleic acid concentrate and a waste liquid;

[0053] A plurality of waste liquid chambers 4, connected to the amplification chamber 3, for storing the waste liquid;

[0054] The air inlet 5 is communicated with the waste liquid chamber 4 .

[0055] The nucleic acid amplification chip described in the present application is provided with the air inlet 5 and generates a pressure difference between the air inlet 5 and the sample inlet 1 through external power to realize the fluid control function. At the same time, the freeze-dried ball reagent pre-packaging technology and unique flow channel design of the present application realize the staged liquid feeding of samples / reagents and further fluid control, breaking through the technical bottleneck of only single-chip and single-channel detection in complex nucleic acid purification and detection processes, solving the problem of chip volume limiting reagent matching, and simplifying the operation steps, improving the integration of the card box, and optimizing the reagent pre-volume compatibility and detection sensitivity. The multi-channel nucleic acid amplification chip realizes efficient, rapid and highly sensitive nucleic acid amplification detection with the characteristics of high integration, portable operation, simple structure, low cost, low risk of liquid cross-flow and diversified nucleic acid concentration.

[0056] Further, the amplification chamber 3 includes a first amplification zone 31 and a second amplification zone 32 communicating with the first amplification zone 31, the first amplification zone 31 is communicated with the dissolution chamber 2, the second amplification zone 32 is communicated with the waste liquid chamber 4, and the interception element 30 is arranged in the first amplification zone 31 and / or the second amplification zone 32. After the interception element 30 separates the nucleic acid test liquid into a nucleic acid concentrated liquid and a waste liquid, and under the action of the pressure difference, the nucleic acid concentrated liquid remains in the first amplification zone 31, and the waste liquid moves to the waste liquid chamber 4 through the second amplification zone 32.

[0057] Specifically, the first amplification zone 31 retains the liquid to be amplified, i.e., the reagent and the concentrated nucleic acid solution, and the second amplification zone 32 retains the reaction storage liquid, i.e., the sample liquid remaining after the reagent and the concentrated nucleic acid solution are retained by the retaining component 30. Of course, the waste liquid is also the sample liquid remaining after the reagent and the concentrated nucleic acid solution are retained by the retaining component 30.

[0058] Furthermore, the multi-channel nucleic acid amplification chip also includes a main body 6, a front sealing film 7 and a back sealing film 8 arranged on the main body 6, the dissolution chamber 2, the amplification chamber 3 and the waste liquid chamber 4 are arranged on the main body 6, and the front sealing film 7 and the back sealing film 8 are used to seal the flow channel and the dissolution chamber 2, the amplification chamber 3 and the waste liquid chamber 4; the front sealing film 7 and the back sealing film 8 are fixed to the multi-channel nucleic acid amplification chip by any one of heat welding, ultrasonic welding, laser welding, and adhesive bonding.

[0059] Furthermore, the injection port 1 is connected to the dissolution chamber 2 through the injection channel 11 and a plurality of liquid separation channels 12, the dissolution chamber 2 is connected to the amplification chamber 3 through the dissolution channel 20, wherein the dissolution channel 20 is connected to the first amplification area 31, the amplification chamber 3 is connected to the waste liquid chamber 4 through the waste liquid channel 33, the waste liquid channel 33 is connected to the second amplification area 32, and the air intake port 5 is connected to the waste liquid chamber 4 through the power channel 40. Since the amplification chamber 3 is on the front of the main body 6 and the waste liquid channel 33 is on the back of the main body 6, the main body 6 is provided with a first connecting hole 34 that penetrates the main body 6 and is used to connect the second amplification area 32 and the waste liquid channel 33.

[0060] Further, the second amplification region 32 is disposed on the periphery of the first amplification region 31, and the second amplification region 32 is in an "Ω" shape; or,

[0061] The first amplification region 31 is disposed on the periphery of the second amplification region 32, and the first amplification region 31 is in an inverted "Ω" shape; or,

[0062] The amplification chamber 3 is in a funnel shape, a round shape, a square shape, or a gourd shape.

[0063] The “Ω” shape, funnel shape, round shape, square shape, and gourd shape increase the connection area between the first amplification zone 31 and the second amplification zone 32, making it easier for waste liquid to quickly pass through the interception element 30 and improving the detection rate.

[0064] See also Figures 1 to 6 and Figure 8 As shown, in the first embodiment of the amplification chamber, the second amplification region 32 is in an “Ω” shape, and the retaining element 30 is placed in the first amplification region 31 .

[0065] See also Fig. 9 As shown, in the second embodiment of the amplification chamber, the first amplification zone 31 is in an inverted “Ω” shape, and the retaining element 30 is placed in the second amplification zone 32.

[0066] See also Fig.10 As shown, in the third embodiment of the amplification chamber, the amplification chamber 3 is funnel-shaped, the upper part of the funnel is the second amplification zone 32, which is circular; the lower part of the funnel is the first amplification zone 31, which is wide at the top and narrow at the bottom. At this time, the retaining element 30 is placed in the second amplification zone 32 at the upper part.

[0067] Furthermore, the interception member 30 can also be arranged between the first amplification zone 31 and the second amplification zone 32, or the interception member 30 can also directly divide the amplification chamber 3 into the first amplification zone 31 and the second amplification zone 32 communicating with the first amplification zone 31. For example, when the amplification chamber 3 is in a circular shape, a square shape or a gourd shape, the interception member 30 is arranged in the amplification chamber 3 to divide the amplification chamber 3 into two amplification zones. Of course, the interception member 30 is sealed and connected to the inner wall of the amplification chamber 3, so that under the action of the pressure difference, the interception member 30 separates the nucleic acid test liquid in the amplification chamber 3 into a nucleic acid concentrate and a waste liquid, that is, the liquid can only enter the first connecting hole 34 after being filtered or adsorbed by the interception member 30, thereby reducing the number of nucleic acid molecules that pass through the interception member 30 to reach the waste liquid chamber 4, and increasing the nucleic acid molecule concentration of the nucleic acid concentrate.

[0068] Further, the depth of the first amplification zone 31 is greater than or equal to the depth of the second amplification zone 32, and the interception member 30 extends toward the second amplification zone 32. The larger the area of ​​the interception member 30, the lower the probability of nucleic acid molecules passing through, the higher the nucleic acid interception rate, and the more nucleic acid is intercepted, so the interception member 30 extends toward the second amplification zone 32 to ensure that the amount of nucleic acid intercepted in the first amplification zone 31 is greater.

[0069] Furthermore, the retention element 30 extends toward the second amplification zone 32 and / or the first amplification zone 31 according to the requirements of the reagent reaction system, and the characteristics and area of ​​the retention element 30 are also determined according to the requirements of the reagent reaction system; the retention element 30 includes but is not limited to biochemically modified or unmodified microporous filter membranes and / or nucleic acid adsorbent fillers.

[0070] Furthermore, the reagent reaction system is related to the characteristics and area of ​​the interception member 30. For a specific reaction system, the characteristics and area of ​​the interception member 30 are different. In addition to the characteristics of hindering the passage of nucleic acids and / or retaining nucleic acids, the characteristics of the interception member 30 also depend on the composition of the freeze-dried ball and the detection principle. First, the composition of the interception member 30 cannot inhibit the reaction of the freeze-dried ball reagent. Secondly, the interception member 30 itself cannot affect the detection. For example, when using the fluorescence method, the interception member 30 cannot have substances that interfere with fluorescence, and when using the color development method, there must be no color that interferes with observation. The area of ​​the interception member 30 depends on the volume of the reagent.

[0071] Further, the depth of the second amplification zone 32 is greater than or equal to the depth of the first amplification zone 31, and the interception member 30 extends toward the first amplification zone 31. The larger the area of ​​the interception member 30, the lower the probability of nucleic acid molecules passing through, the higher the nucleic acid interception rate, and the more nucleic acid is intercepted, so the interception member 30 extends toward the first amplification zone 31 to ensure that the amount of nucleic acid intercepted in the first amplification zone 31 is greater.

[0072] Further, the interception member 30 is configured as a microporous filter membrane, and the microporous filter membrane includes one or more of filter paper, glass fiber, fiber fabric, polyethersulfone, non-woven fabric, and synthetic fiber. The microporous filter membrane can provide a uniform pore size, ensure high filtering accuracy, effectively intercept the target nucleic acid molecules, and allow small molecules such as salts and solvents to pass through, that is, waste liquid, to achieve separation of nucleic acid and waste liquid.

[0073] Furthermore, when the retaining element 30 is configured as a microporous filter membrane, the retaining element 30 is disposed in the first amplification zone 31 and / or the second amplification zone 32 or between the first amplification zone 31 and the second amplification zone 32 , and is usually placed in the second amplification zone 32 .

[0074] Furthermore, the interception element 30 is configured as a nucleic acid adsorbent filler, and the nucleic acid adsorbent filler includes one or more of a silica gel membrane, a glass fiber, a nitrocellulose membrane, and a nylon membrane. The nucleic acid adsorbent filler can efficiently extract nucleic acids from biological samples, simplify the sample preparation procedure, and speed up the operation steps in molecular biology and biomedical research.

[0075] Furthermore, when the retaining element 30 is configured as a nucleic acid adsorbent filler, the retaining element 30 is disposed in the first amplification zone 31 and / or the second amplification zone 32 , and is usually placed in the first amplification zone 31 .

[0076] Furthermore, according to different reagent systems, the material of the interception element 30 may also be mixed with ion interception materials and / or materials having reaction product characterization properties to increase the concentration of the reaction system and / or improve the sensitivity of detection.

[0077] Furthermore, the volume of the dissolution chamber 2 is greater than the sum of the volumes of the amplification chamber 3 and the waste liquid chamber 4. This ensures that the vacuum source can evacuate the dissolution chamber 2 as much as possible during the concentration and drainage process to reduce the risk of crosstalk between chambers caused by temperature and volume changes during the amplification stage.

[0078] Furthermore, in this embodiment, the sum of the volumes of the waste liquid chamber 4 and its corresponding dissolution channel 20, the amplification chamber 3, the waste liquid channel 33 and the power channel 40 is less than or equal to the sum of the volumes of its corresponding liquid separation channel 12 and the dissolution chamber 2. In this embodiment, the sum of the volumes of the waste liquid chamber 4 and its corresponding dissolution channel 20, the amplification chamber 3, the waste liquid channel 33 and the power channel 40 is slightly smaller than the sum of the volumes of its corresponding liquid separation channel 12 and the dissolution chamber 2, so that after the waste liquid chamber 4 is filled with water, there is still sufficient reaction storage liquid left in the second amplification zone 32. During amplification, the reaction storage liquid can replenish the evaporated water of the amplified liquid in the first amplification zone 31, thereby ensuring the stability of the reaction system volume when the volume of the reagent system is too small.

[0079] Furthermore, the air inlet 5 is connected to an external power source through an interface, and the interface is configured as an air valve 51 or configured as a groove 52 arranged in the multi-channel nucleic acid amplification chip. The multi-channel nucleic acid amplification chip can realize the fluid control function only through simple external power source control, which simplifies the operation steps of the instrument, is portable, and does not require a large fluid control instrument.

[0080] Furthermore, the external power source is a vacuum pump, etc., which is connected to the air valve 51 through a first hydrophobic breathable membrane to provide power for the chip, so that a pressure difference is generated between the air intake port 5 and the injection port 1, that is, the pressure of the air intake port 5 is less than the pressure of the injection port 1, thereby realizing the fluid control function, that is, after the interception member 30 separates the nucleic acid test liquid into nucleic acid concentrate and waste liquid, the waste liquid moves from the amplification chamber 3 to the waste liquid chamber 4 under the action of the negative pressure formed by the vacuum pump; the first hydrophobic breathable membrane is arranged between the joint of the vacuum pump and the air valve 51, and the first hydrophobic breathable membrane is installed by ultrasonic welding.

[0081] In this embodiment, the interface is an air valve fixed on the back sealing film 8 and connected to the air inlet 5; the fixing method of the air valve includes but is not limited to ultrasonic welding, adhesive bonding, and pressurization of interference fit of external components. The air inlet 5 is an air hole arranged on the second hydrophobic breathable membrane; the second hydrophobic breathable membrane is placed on the back of the chip.

[0082] Furthermore, the air inlet 5 is an air hole with a hydrophobic breathable membrane and / or a first hydrophobic breathable filler 50; the air inlet 5 is connected to the air inlet 5 through the hydrophobic breathable membrane and / or the first hydrophobic breathable filler 50 to provide power for the chip; the installation methods of the hydrophobic breathable membrane and / or the first hydrophobic breathable filler 50 include but are not limited to ultrasonic welding, laser welding, adhesive bonding and interference filling. The first hydrophobic breathable filler 50 is a material that prevents liquid from passing through but allows gas to pass through. When it contacts liquid, the liquid will directly block the air hole and make the first hydrophobic breathable filler 50 lose its air permeability, thereby closing the air inlet 5 to prevent liquid from rushing into the vacuum pump, and achieving constant volume of the amplification chamber 3 and the waste liquid chamber 4.

[0083] Furthermore, the main body 6 is provided with a first air hole 13 located above the dissolution chamber 2, and the dissolution chamber 2 and the first air hole 13 are connected through an exhaust channel 14; the first air hole 13 is located on the back of the chip, and the dissolution chamber 2 has built-in freeze-dried balls, which are freeze-dried reagents, usually containing reaction buffer, fluorescent dye, Mg2+, dNTP and DNA / RNA polymerase. The freeze-dried ball pre-packaging technology is used instead of on-site liquid reagent preparation, which simplifies the reagent addition steps and reduces the requirements of the card box for storage and transportation conditions; a hydrophobic breathable membrane and / or a second hydrophobic breathable filler 15 are provided in the first air hole 13; the installation methods of the hydrophobic breathable membrane and / or the second hydrophobic breathable filler 15 include but are not limited to ultrasonic welding, laser welding, adhesive taping and interference filling.

[0084] Furthermore, the first air hole 13 is provided with a second hydrophobic breathable filler 15; the second hydrophobic breathable filler 15 is installed by interference filling. The first air hole 13 is provided with a hydrophobic breathable membrane; the hydrophobic breathable membrane is installed by ultrasonic welding. The second hydrophobic breathable filler 15 is a material that prevents liquid from passing through but allows gas to pass through. When it contacts liquid, the liquid will directly block the air hole and make the second hydrophobic breathable filler 15 lose its breathability, thereby closing the first air hole 13, preventing liquid from rushing into the vacuum pump, and achieving constant volume of the amplification chamber 3 and the waste liquid chamber 4.

[0085] Furthermore, the waste liquid flow channel 33 is located on the back side of the chip; the power flow channel 40 includes a power flow channel back section and a power flow channel front section; the power flow channel back section is located on the back side of the chip and connected to the waste liquid chamber 4; the power flow channel front section is located on the front side of the chip and connected to the air intake port 5, and the power flow channel back section and the power flow channel front section are connected through a second connecting hole 41.

[0086] Embodiment 2

[0087] See also Figures 5 and 6 As shown, the second embodiment is substantially the same as the first embodiment, except that the interface is configured as a groove 52 disposed in the multi-channel nucleic acid amplification chip.

[0088] The groove 52 is connected to an external power source with a valve, ie, a vacuum pump, through a vacuum channel 23 and a connector 24 .

[0089] The multi-channel nucleic acid amplification chip of the second embodiment is smaller in size than that of the first embodiment, has higher compatibility with detection instruments, and has higher requirements on chip material strength and injection molding accuracy.

[0090] See also Figure 7 As shown, based on the above-mentioned multi-channel nucleic acid amplification chip, the embodiment of the present application also provides a multi-channel nucleic acid amplification detection method, which adopts the following technical solution: the multi-channel nucleic acid amplification detection method comprises the following steps:

[0091] S100, providing a chip: providing the multi-channel nucleic acid amplification chip;

[0092] S200, adding biological sample liquid: closing the air inlet 5 and adding the biological sample liquid to the injection port 1;

[0093] S300, liquid separation and volume determination: the sample liquid flows into the dissolution chamber 2 and dissolves the freeze-dried ball to form a nucleic acid test liquid;

[0094] S400, concentration and drainage: when the pressure of the air inlet 5 is lower than the pressure of the sample inlet 1, the nucleic acid test liquid in the dissolution chamber 2 moves into the amplification chamber 3 under the action of pressure, and the interception member 30 separates the nucleic acid test liquid into nucleic acid concentrated liquid and waste liquid, so that the nucleic acid concentrated liquid remains in the amplification chamber 3, and the waste liquid moves from the amplification chamber 3 to the waste liquid chamber 4 under the action of pressure;

[0095] S500 , amplification: the sample inlet 1 is closed, and the concentrated nucleic acid solution remaining in the amplification chamber 3 is amplified.

[0096] The nucleic acid amplification chip described in the present application is provided with the air inlet 5 and generates a pressure difference between the air inlet 5 and the sample inlet 1 through external power to realize the fluid control function. At the same time, the freeze-dried ball reagent pre-packaging technology and unique flow channel design of the present application realize the staged liquid feeding of samples / reagents and further fluid control, and combine the liquid separation and volume determination method with the nucleic acid concentration method, breaking through the technical bottleneck of only single-chip and single-channel detection in the complex nucleic acid purification and detection process, solving the problem of chip volume limitation reagent matching, and simplifying the operation steps, improving the integration of the card box, and optimizing the reagent pre-volume compatibility and detection sensitivity. The multi-channel nucleic acid amplification chip realizes efficient, rapid and highly sensitive nucleic acid amplification detection with the characteristics of high integration, portable operation, simple structure, low cost, low risk of liquid cross-flow and diversified nucleic acid concentration.

[0097] Furthermore, when the multi-channel nucleic acid amplification chip is in operation, it is necessary to first place the multi-channel nucleic acid amplification chip upright with its front side facing forward, that is, the injection port 1 is placed upward; before the multi-channel nucleic acid amplification chip is in operation, the air intake port 5 is in a closed state; from the start of the operation of the multi-channel nucleic acid amplification chip to the amplification stage, it is necessary to ensure that the injection port 1 is connected to the atmosphere or a closed space area that can provide positive pressure.

[0098] During detection, a biological sample liquid is added to the injection port 1, and the biological sample liquid will be diverted into a corresponding number of sample diversion liquids by the injection channel 11 and a plurality of liquid diversion channels 12 under the action of gravity and fill the corresponding dissolution chamber 2; the sample diversion liquid will quickly dissolve the freeze-dried ball in the dissolution chamber 2 and be fixed to a corresponding volume of the liquid to be reacted.

[0099] Furthermore, in an embodiment, the dissolution chamber 2 can accommodate 1 to 100 μl of liquid. Taking one of the freeze-dried balls as an example, the capacity of the dissolution chamber 2 is 50 μl. After liquid separation and volume determination, 10 μl of the liquid to be amplified remains in the first amplification zone 31, 20 μl of the reaction storage liquid remains in the second amplification zone 32, and 20 μl of the waste liquid absorbed into the waste liquid chamber 4.

[0100] It can be understood that the dissolution chamber 2 can contain a liquid volume of 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, 23 μl, 24 μl. l, 25μl, 26μl, 27μl, 28μl, 29μl, 30μl, 31μl, 32μl, 33μl, 34μl, 35μl, 36μl, 37μl , 38μl, 39μl, 40μl, 41μl, 42μl, 43μl, 44μl, 45μl, 46μl, 47μl, 48μl, 49μl, 50μl, 5 1μl, 52μl, 53μl, 54μl, 55μl, 56μl, 57μl, 58μl, 59μl, 60μl, 61μl, 62μl, 63μl, 64 μl, 65μl, 66μl, 67μl, 68μl, 69μl, 70μl, 71μl, 72μl, 73μl, 74μl, 75μl, 76μl, 77μl , 78μl, 79μl, 80μl, 81μl, 82μl, 83μl, 84μl, 85μl, 86μl, 87μl, 88μl, 89μl, 90μl, 91μl, 92μl, 93μl, 94μl, 95μl, 96μl, 97μl, 98μl, 99μl, and the range formed by any two of the following values.

[0101] During liquid separation and volume determination, since the air valve connected to the air inlet 5 is in a closed state, the air in the dissolution channel 20, the amplification chamber 3, the waste liquid channel 33, the waste liquid chamber 4, the power channel 40 and the air inlet 5 is in a static state; under the premise of ensuring that the gravity and contact angle conditions of the nucleic acid test liquid cannot break through the capillary action of the dissolution channel 20, that is, the gravity as the driving force cannot exceed the surface tension as the resistance, the biological sample liquid and its split liquid can only flow in the injection port 1, the injection channel 11, the liquid separation channel 12 and the dissolution chamber 2 area. At the same time, due to the high The biological sample liquid first flows to the dissolution chamber 2 at the bottom, and waits for the first air permeable hole 13 connected thereto to be blocked by the second hydrophobic air-permeable filler 15, and the dissolution chamber 2 stops taking liquid in until the dissolution chamber 2 at the bottom is completely filled with liquid and blocks the second hydrophobic air-permeable fillers 15 in all the first air permeable holes 13 connected thereto, and the biological sample liquid then flows to the dissolution chamber 2 at the top, and liquid separation and volume determination are achieved by gravity. The liquid separation and volume determination technology is adopted to avoid the problem of uneven liquid separation caused by the precision limitation of injection molding parts, thereby greatly improving the accuracy of nucleic acid detection.

[0102] Furthermore, in a non-constant volume system, liquid separation is achieved by utilizing the mechanical properties of the fluid in the flow channel, but the precision of the injection molding parts will affect the friction of the flow channel; whereas in the present application, the volume of the chamber is used for reverse liquid volume determination, and the amplification chamber 3 fixes the amount after liquid separation, without considering the mechanical properties of the fluid in the flow channel, thereby avoiding the problem of uneven liquid separation caused by the precision limitation of the injection molding parts, and greatly improving the accuracy of nucleic acid detection.

[0103] During the concentration and discharge, the vacuum pump is connected to the air inlet 5 through the interface, and the nucleic acid test liquid will forcefully break through the capillary action of the dissolution flow channel 20 under negative pressure and enter the amplification chamber 3; at the same time, under the action of negative pressure, the nucleic acid test liquid can be filtered by the interception member 30 in the amplification chamber 3, wherein the nucleic acid concentrate is retained in the first amplification area 31 or the second amplification area 32; one of the liquids retained in the first amplification area 31 and the second amplification area 32 must be used as the amplification liquid and the other as the reaction storage liquid; the remaining liquid after filtration and / or adsorption, i.e., the waste liquid, will continue to enter the waste liquid chamber 4 under negative pressure until it blocks the first hydrophobic and breathable filler 50 in the air inlet 5. The multi-channel nucleic acid amplification chip uses a nucleic acid concentration method to improve the sensitivity of detection and solve the volume difference problem of different reagent systems, thereby expanding the reagent matching library of the chip.

[0104] During amplification, after closing the sample inlet 1 and the air inlet 5 to prevent amplification contamination, the amplification chamber 3 can be subjected to nucleic acid amplification reaction and subsequent detection operations. During the amplification reaction stage, most of the liquid in the dissolution chamber 2 has been extracted, reducing the risk of liquid leakage in the multi-channel microfluidic chip of the prior art.

[0105] In this stage, the nucleic acid amplification reaction is carried out by controlling the temperature of the amplification chamber 3. During this process, the evaporated water in the amplified liquid in the first amplification zone 31 will be replenished by the reaction storage liquid in the second amplification zone 32, ensuring the stability of the reaction system volume when the reagent system volume is too small.

[0106] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A multi-channel nucleic acid amplification chip, characterized in that: include: An inlet (1) for allowing a biological sample liquid to enter; A plurality of dissolution chambers (2) are connected to the sample inlet (1), wherein freeze-drying balls are arranged in the dissolution chambers (2), and the freeze-drying balls are used to fuse the biological sample liquid flowing into the dissolution chambers (2) to form a nucleic acid test liquid; A plurality of amplification chambers (3) are connected to the dissolution chamber (2); a retaining element (30) is provided in the amplification chamber (3), and the retaining element (30) is used to filter the nucleic acid test liquid so that the nucleic acid test liquid is separated into a nucleic acid concentrated liquid and a waste liquid; A plurality of waste liquid chambers (4), connected to the amplification chamber (3) and used for storing the waste liquid; The air inlet (5) is communicated with the waste liquid chamber (4).

2. The multi-channel nucleic acid amplification chip according to claim 1, characterized in that: The amplification chamber (3) includes a first amplification zone (31) and a second amplification zone (32) communicating with the first amplification zone (31); the first amplification zone (31) is communicated with the dissolution chamber (2); the second amplification zone (32) is communicated with the waste liquid chamber (4); and the retaining element (30) is arranged in the first amplification zone (31) and / or the second amplification zone (32).

3. The multi-channel nucleic acid amplification chip according to claim 2, characterized in that: The second amplification region (32) is arranged on the periphery of the first amplification region (31), and the second amplification region (32) is in an "Ω" shape; or, The first amplification region (31) is arranged on the periphery of the second amplification region (32), and the first amplification region (31) is in an inverted "Ω" shape; or, The amplification chamber (3) is in the shape of a funnel, a circle, a square, or a gourd.

4. The multi-channel nucleic acid amplification chip according to claim 2, characterized in that: The depth of the first amplification zone (31) is greater than or equal to the depth of the second amplification zone (32), and the retaining element (30) extends toward the second amplification zone (32).

5. The multi-channel nucleic acid amplification chip according to claim 2, characterized in that: The depth of the second amplification zone (32) is greater than or equal to the depth of the first amplification zone (31), and the retaining element (30) extends toward the first amplification zone (31).

6. The multi-channel nucleic acid amplification chip according to claim 1, characterized in that: The intercepting element (30) is configured as a microporous filter membrane, and the microporous filter membrane includes one or more of filter paper, glass fiber, fiber fabric, polyethersulfone, non-woven fabric, and synthetic fiber.

7. The multi-channel nucleic acid amplification chip according to claim 1, characterized in that: The retaining element (30) is configured as a nucleic acid adsorbent filler, and the nucleic acid adsorbent filler includes one or more of a silica gel membrane, a glass fiber, a nitrocellulose membrane, and a nylon membrane.

8. The multi-channel nucleic acid amplification chip according to claim 1, characterized in that: The volume of the dissolution chamber (2) is greater than the sum of the volumes of the amplification chamber (3) and the waste liquid chamber (4).

9. The multi-channel nucleic acid amplification chip according to any one of claims 1 to 8, characterized in that: The air inlet (5) is connected to an external power source via an interface, and the interface is configured as an air valve (51) or configured as a groove (52) arranged in the multi-channel nucleic acid amplification chip.

10. A multi-channel nucleic acid amplification detection method, characterized in that: The following steps are involved: Providing a chip: providing a multi-channel nucleic acid amplification chip as described in any one of claims 1 to 9; Adding biological sample liquid: closing the air inlet (5) and adding the biological sample liquid to the injection port (1); Liquid separation and volume determination: the biological sample liquid flows into the dissolution chamber (2) and dissolves the freeze-dried ball to form a nucleic acid test liquid; Concentration and drainage: when the pressure of the air inlet (5) is lower than the pressure of the sample inlet (1), the nucleic acid test liquid in the dissolution chamber (2) moves into the amplification chamber (3) under the action of pressure, and the interception member (30) separates the nucleic acid test liquid into nucleic acid concentrated liquid and waste liquid, so that the nucleic acid concentrated liquid remains in the amplification chamber (3), and the waste liquid moves from the amplification chamber (3) to the waste liquid chamber (4) under the action of pressure; Amplification: The sample inlet (1) is closed, and the concentrated nucleic acid solution remaining in the amplification chamber (3) is amplified.

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