Nucleic acid test cartridge and nucleic acid test method

Abstract

A nucleic acid test cartridge and a nucleic acid test method. The cartridge is provided with a sample inlet and a sample chamber in communication with the sample inlet. A lysis chamber, an elution chamber, and a reaction chamber, which are sequentially in communication by means of a flow channel, are arranged inside the cartridge. The sample chamber is in communication with the lysis chamber by means of a flow channel. A magnetic bead chamber is further arranged inside the cartridge. The magnetic bead chamber is in communication with the lysis chamber by means of a flow channel. A waste liquid chamber is further arranged inside the cartridge. The waste liquid chamber is in communication with the lysis chamber and the elution chamber by means of flow channels. The cartridge is provided with a first air vent in communication with at least one of the sample chamber and the lysis chamber, a second air vent in communication with the elution chamber, and a reagent inlet in communication with the lysis chamber and the elution chamber.

Description

Nucleic acid test kits and nucleic acid testing methods Technical Field

[0001] This application relates to the field of in vitro medical diagnostic technology, specifically to a nucleic acid detection kit and a nucleic acid detection method. Background Technology

[0002] Nucleic acid testing has a wide range of applications in the biomedical field and is of great clinical significance, especially in in vitro medical diagnostics. Compared with traditional tube-based bioanalyzers, test cartridges have advantages such as smaller reaction volume and reduced reagent consumption.

[0003] Nucleic acid detection technology typically includes steps such as lysis, binding, washing, elution, and subsequent PCR (polymerase chain reaction, also known as in vitro DNA amplification technology) and optical detection.

[0004] Among the existing nucleic acid test kits, some have low integration, requiring manual intervention in many processes, resulting in low testing efficiency, a high probability of misoperation, unstable test results, and unreliable accuracy. Others, while having high integration, have numerous components, complex structures, and high costs. Summary of the Invention

[0005] In view of the above problems, this application provides a nucleic acid detection cartridge and a nucleic acid detection method, which can improve detection efficiency and accuracy of detection results while ensuring that the cartridge is small in size, simple in structure, and low in cost.

[0006] According to one aspect of the embodiments of this application, a nucleic acid detection cartridge is provided. The cartridge has an inlet port and a sample chamber communicating with the inlet port. The cartridge also has a lysis chamber, an elution chamber, and a reaction chamber that are sequentially connected by flow channels with valves. The sample chamber and the lysis chamber are connected by flow channels. The cartridge also has a magnetic bead chamber containing magnetic beads, which is connected to the lysis chamber by flow channels. The cartridge also has a waste liquid chamber that is connected to the lysis chamber and the elution chamber by flow channels with valves. The cartridge has a first vent communicating with at least one of the sample chamber and the lysis chamber. The cartridge also has a second vent communicating with the elution chamber. The cartridge also has reagent inlets communicating with the lysis chamber and the elution chamber.

[0007] According to another aspect of the embodiments of this application, a nucleic acid detection method is provided, applied to the nucleic acid detection cartridge of any of the above claims. The method includes: injecting a sample into a sample chamber through an injection port; keeping all valves closed; introducing pressure through a first vent to allow the sample to flow into a magnetic bead chamber and mix with the magnetic beads to form a first mixed fluid; introducing pressure again through the first vent to allow the first mixed fluid to flow into a lysis chamber; injecting a lysis agent and a binding agent into the lysis chamber through a reagent inlet connected to the lysis chamber, so that the lysis agent and the binding agent mix and react with the first mixed fluid to form a second mixed fluid, wherein the cells and / or viruses in the first mixed fluid undergo lysis under the action of the lysis agent. The nucleic acid is exposed and bound to magnetic beads by a binding agent. The magnetic beads in the second mixed fluid are then adsorbed, fixing the nucleic acid-bound beads within the lysis chamber. The first valve on the flow channel between the lysis chamber and the waste liquid chamber is opened, and positive pressure is introduced through the first vent, causing the liquid in the lysis chamber to drain into the waste liquid chamber. The first valve is then closed, releasing the adsorption on the magnetic beads. Detergent is injected into the lysis chamber through the reagent inlet connected to the lysis chamber to clean the magnetic beads and form a third mixed fluid. The second valve on the flow channel between the lysis chamber and the elution chamber is opened, and positive pressure is introduced through the first vent or negative pressure is introduced through the second vent, causing the third mixed fluid to flow into the elution chamber. In the elution chamber, the second valve is then closed. The magnetic beads in the third mixed fluid are adsorbed and fixed within the elution chamber. The third valve on the flow channel between the elution chamber and the waste liquid chamber is opened, and positive pressure is introduced through the first or second vent to drain the liquid from the elution chamber into the waste liquid chamber. The third valve is then closed, and the adsorption of the magnetic beads is released. Detergent is injected into the elution chamber through the reagent inlet connected to the elution chamber to clean the magnetic beads and form a fourth mixed fluid. The magnetic beads in the fourth mixed fluid are adsorbed and fixed within the elution chamber. The third valve is then opened, and positive pressure is introduced through the first or second vent to drain the liquid from the elution chamber into the waste liquid chamber. Next, close the third valve and release the magnetic beads from adsorption; open the second and / or third valves and dry the magnetic beads in the elution chamber so that the liquid on the surface of the magnetic beads and the liquid in the elution chamber vaporize and enter the lysis chamber and / or waste liquid chamber for condensation; then close the second and / or third valves; inject the eluent into the elution chamber through the reagent inlet connected to the elution chamber to elute the nucleic acid from the magnetic beads; adsorb the magnetic beads in the elution chamber so that the magnetic beads are fixed in the elution chamber; open the fourth valve on the flow channel connecting the elution chamber and the reaction chamber, and introduce positive pressure into the first or second vent to inject the liquid mixed with nucleic acid in the elution chamber into the reaction chamber for detection.

[0008] Based on research findings that lysis and binding can occur simultaneously without interfering with each other, the cartridge provided in this application only includes a lysis chamber to handle nucleic acid lysis and binding with magnetic beads. Furthermore, to prevent residual impurities on the inner wall of the lysis chamber from affecting the detection results, an elution chamber is also included to handle the elution of nucleic acids from the magnetic beads. This ensures the purity of the extracted nucleic acids in the eluted liquid, improves the accuracy of the detection results, and minimizes the cartridge volume and cost. Waste liquid chambers connected to both the lysis and elution chambers are provided to ensure the orderly discharge of waste liquid from these chambers. Moreover, the use of valves on the flow channels between the chambers and vents connecting to some chambers simplifies the detection process and ensures precise control of the internal fluid flow. Meanwhile, by utilizing the temperature difference between the elution chamber and the lysis chamber and / or waste liquid chamber, as well as controlling the valves between them, the vaporized gas can flow into the lysis chamber and / or waste liquid chamber to condense during the drying of the magnetic beads, thus preventing it from flowing back into the elution chamber and further improving the purity of nucleic acid extraction.

[0009] A key factor in ensuring the accuracy of nucleic acid testing is avoiding the mixing of impurities generated during lysis into the eluted solution. This is because the eluted solution needs to be injected into the reaction chamber for PCR amplification. If the eluted solution contains impurities from lysis, it will affect the accuracy of the PCR amplification. For this reason, the cartridge provided in this application uses a separate lysis chamber and elution chamber. The lysis chamber is responsible for lysis, and the elution chamber is responsible for elution. Compared to using the same chamber for both lysis and elution, this prevents impurities from lysis from significantly affecting the elution process. Furthermore, multiple washes effectively prevent impurities from lysis from mixing into the eluted solution.

[0010] Furthermore, the cartridge provided in this application embodiment also features independent vents for lysis and elution, namely a first vent and a second vent. The first vent is connected to at least one of the sample chamber and the lysis chamber, and is mainly responsible for driving the flow of solution between the sample chamber and the lysis chamber. The second vent is connected to the elution chamber and is responsible for driving the flow of solution from the elution chamber to the waste liquid chamber and the reaction chamber. Compared with a single vent that is responsible for driving the solution flow, this method prevents impurities remaining in the lysis chamber from entering the elution chamber during the liquid-driven process, thus avoiding contamination of the extracted product in the elution chamber (i.e., the nucleic acid-containing detection solution obtained after eluting the magnetic beads) and preventing interference with subsequent nucleic acid detection results.

[0011] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0012] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0013] Figure 1 is a three-dimensional structural diagram of the nucleic acid detection card provided in the embodiment of this application from one perspective;

[0014] Figure 2 is a three-dimensional structural diagram of the nucleic acid detection cartridge provided in the embodiment of this application from another perspective;

[0015] Figure 3 is a schematic diagram of the exploded structure of the nucleic acid detection cartridge provided in the embodiment of this application from one perspective;

[0016] Figure 4 is a schematic diagram of the exploded structure of the nucleic acid test kit provided in the embodiment of this application from another perspective;

[0017] Figure 5 is a schematic diagram of the internal perspective structure of the nucleic acid test kit provided in the embodiment of this application from the front view;

[0018] Figure 6 is an enlarged structural diagram of point A in Figure 4;

[0019] Figure 7 is a schematic diagram of the exploded structure of a nucleic acid detection card box provided in another embodiment of this application from one perspective;

[0020] Figure 8 is a schematic diagram of the exploded structure of a nucleic acid test kit provided in another embodiment of this application from another perspective;

[0021] Figure 9 is a schematic diagram of the internal perspective structure of a nucleic acid detection cartridge from a side view according to another embodiment of this application;

[0022] Figure 10 is a structural schematic diagram of the nucleic acid detection card box from another planar perspective according to another embodiment of this application;

[0023] Figure 11 is a structural schematic diagram of the nucleic acid detection card box provided in the embodiment of this application from a bottom view;

[0024] Figure 12 is a flowchart illustrating the nucleic acid detection method provided in the embodiments of this application;

[0025] Figure 13 is a flowchart illustrating a nucleic acid detection method provided in another embodiment of this application.

[0026] The reference numerals in the detailed embodiments are as follows: 100, Card box; 101, Card box body; 102, Cover; 110, Sample inlet; 111, Sealing cap; 120, Sample chamber; 130, Magnetic bead chamber; 140, Pyrolysis chamber; 150, Elution chamber; 160, Reaction chamber; 161, Water-proof and breathable membrane; 162, Second through hole; 163, Shut-off valve; 170, Waste liquid chamber; 171, Water absorption component; 180, Thin sheet structure; 11, First flow channel; 12, Second flow channel; 121, Serpentine flow channel; 13, Third flow channel; 14, Fourth flow channel; 15, Fifth flow channel; 16, Sixth flow channel; 17, Seventh flow channel; 21. First valve; 22. Second valve; 23. Third valve; 24. Fourth valve; 25. First through hole; 26. Stepped surface; 27. Pressing plate; 31. First vent; 32. Second vent; 41. First inlet; 42. Second inlet; 43. Third inlet; 44. Fourth inlet; 51. First reagent pack; 52. Second reagent pack; 53. Third reagent pack; 54. Fourth reagent pack; 60. Pressure relief hole; 71. First waterproof and breathable membrane; 72. Second waterproof and breathable membrane; 73. Third waterproof and breathable membrane; 81. First absorbent and breathable membrane; 82. Second absorbent and breathable membrane; 91. First channel; 92. Second channel; 921. Micro valve. Detailed Implementation

[0027] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0029] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent three cases: A exists, A and B exist simultaneously, and B exists. In addition, the character " / " in this document generally indicates that the related objects before and after it have an "or" relationship.

[0032] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0033] In the description of the embodiments of this application, the technical terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0034] In the description of the embodiments of this application, unless otherwise expressly specified and limited, technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. For those skilled in the art, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0035] Nucleic acid detection cartridges, such as microfluidic cartridges, offer several advantages over traditional tubular bioanalytical methods. These advantages include smaller reaction volumes, reduced reagent consumption, lower contamination, easier high-throughput analysis, lower cost, greater adaptability, smaller size, more flexible design, and faster detection speed. Furthermore, they can break down complex systems into numerous simpler systems containing only a single research object, simplifying the background for quantitative and qualitative analysis.

[0036] For some highly integrated and fully functional card boxes, the internal design generally requires the setting of corresponding chambers and channels for different operation steps. This results in a very large number of integrated chambers and channels in the card box, making the card box large in size and complex in structure.

[0037] To ensure miniaturization of cartridge products, different reactions in the nucleic acid detection process can be carried out in the same chamber to reduce the number of chambers and thus reduce cartridge volume. However, processing samples differently in the same chamber may cause interference between the reactions, resulting in more impurities in the final test solution, which may compromise sample processing capacity and the accuracy of test results.

[0038] In view of the above problems, the inventors of this application, after analyzing the processing characteristics of each step in the nucleic acid detection process, have carried out corresponding structural design inside the cartridge. By designing the communication relationship between the chambers, reagent inlets, pores and flow channels, as well as the position design of the valves that can control the opening and closing of the flow channels, it is possible to ensure that the entire process of lysis, binding, washing, elution and PCR detection can be met, that the processing steps do not affect each other, and that the detection efficiency and accuracy are high, while minimizing the number of chambers and flow channels required, thereby ensuring that the cartridge has a smaller volume and lower processing and manufacturing costs.

[0039] According to one aspect of the embodiments of this application, a nucleic acid detection cartridge is provided. Please refer to Figures 1 to 4 for details. Figures 1 and 2 show the three-dimensional structure of the nucleic acid detection cartridge from two different perspectives, while Figures 3 and 4 show the exploded structure of the nucleic acid detection cartridge from two different perspectives. As shown in the figures, the cartridge 100 has a sample inlet 110. The interior of the cartridge 100 is provided with a sample cavity 120 and a magnetic bead cavity 130 connected sequentially by flow channels. Specifically, as shown in the three-dimensional view in Figure 4 and the planar view in Figure 5, the sample cavity 120 and the magnetic bead cavity 130 are connected through a first flow channel 11. The sample cavity 120 is also connected to the sample inlet 110. The magnetic bead cavity 130 contains magnetic beads (not shown).

[0040] Referring to Figures 3 to 5, the cartridge 100 also contains a pyrolysis chamber 140, an elution chamber 150, and a reaction chamber 160, which are sequentially connected by flow channels with valves. The pyrolysis chamber 140 is also connected to the magnetic bead chamber 130 through a flow channel. Specifically, the magnetic bead chamber 130 is connected to the pyrolysis chamber 140 through a second flow channel 12, the pyrolysis chamber 140 is connected to the elution chamber 150 through a third flow channel 13 with a second valve 22, and the elution chamber 150 is connected to the reaction chamber 160 through a fourth flow channel 14 with a fourth valve 24.

[0041] Furthermore, as shown in Figures 3 to 5, the inside of the cartridge 100 is also provided with a waste liquid chamber 170. The waste liquid chamber 170 is connected to the pyrolysis chamber 140 and the elution chamber 150 respectively through flow channels with valves. Specifically, the pyrolysis chamber 140 is connected to the waste liquid chamber 170 through the fifth flow channel 15 with the first valve 21, and the elution chamber 150 is connected to the waste liquid chamber 170 through the sixth flow channel 16 with the third valve 23.

[0042] As shown in Figure 5, for flow channels with valves, such as the third flow channel 13, the portions located at both ends of the second valve 22 are represented by bright solid lines and dark dashed lines, respectively. The bright solid lines indicate that this portion of the flow channel is located on the front side of the card box 100 shown in Figure 5, while the dark dashed lines indicate that this portion of the flow channel is located on the back side of the card box 100, which is not shown in Figure 5. In other words, the dark dashed lines represent the perspective structure. The flow channels on both sides are interconnected through a through hole penetrating the card box 100, and both flow channels are closed by a cover plate. This part can be seen from the three-dimensional perspective of the two sides shown in Figures 3 and 4. The valve is installed in the through hole to control the opening or closing of the through hole, thereby achieving the opening and closing control of the corresponding flow channel.

[0043] Specifically, please refer to Figure 6, which shows the enlarged structure at point A in Figure 4. It mainly shows the exploded structure at the valve. As shown in the figure, a stepped surface 26 can be set in the first through hole 25, and a movable pressure plate 27 is set on the stepped surface 26 to form a valve. When the pressure plate 27 is separated from the stepped surface 26 or there is a gap, both ends of the first through hole 25 are in the open state, and the flow channels on both sides are interconnected. When the pressure plate 27 is pressed tightly on the stepped surface 26, the first through hole 25 is closed, and the flow channels on both sides are not interconnected.

[0044] The above is only one embodiment provided by this application, and it does not constitute a limitation on the specific structure of this application. For example, in some other embodiments, a flow channel may be entirely set on one side of the card box 100, or formed inside the card box 100. The valve may be a sealing member set in the flow channel. When the card box at the valve position is pressed, the wall of the flow channel deforms and seals against the valve, so that the flow channel is closed. When released, the flow channel is opened.

[0045] Furthermore, the cartridge 100 has vents that communicate with the pyrolysis chamber 140 and the elution chamber 150, respectively. Referring to Figures 3 to 5, in the specific embodiments shown, the cartridge 100 has a first vent 31 and a second vent 32. The first vent 31 communicates with the pyrolysis chamber 140, and the second vent 32 communicates with the elution chamber 150. In other embodiments, the cartridge 100 may have only one vent (i.e., the first vent 31 and the second vent 32 are the same vent), and this vent communicates with the pyrolysis chamber 140 and the elution chamber 150 respectively through two flow channels with valves inside the cartridge 100. Therefore, by controlling the valves on the corresponding flow channels, the vent can communicate with one or both of the pyrolysis chamber 140 and the elution chamber 150 simultaneously.

[0046] Please refer to Figures 3 to 5. The cartridge 100 also has reagent inlets that communicate with the lysis chamber 140 and the elution chamber 150, respectively. In the specific embodiments shown in Figures 3 to 5, the reagent inlets include a first inlet 41 and a second inlet 42 communicating with the lysis chamber 140, and a third inlet 43 and a fourth inlet 44 communicating with the elution chamber 150. The first inlet 41 is used for the lysis agent and binding agent to enter the lysis chamber 140, the second inlet is used for the detergent to enter the lysis chamber, the third inlet 43 is also used for the detergent to enter the elution chamber 150, and the fourth inlet 44 is used for the eluent to enter the elution chamber 150. This arrangement allows the corresponding reagent packs to be placed at the corresponding actual inlets in advance during the preparation of the test, thus eliminating the need for multiple reagent pack replacements during the test and improving the test efficiency. Similar to the pore configuration, the figure only shows one possible arrangement of the reagent inlet provided in this application. In other embodiments, only one reagent inlet may be provided, which is connected to the pyrolysis chamber 140 and the elution chamber 150 respectively through two branch channels with valves. When reagents are injected into the corresponding chambers, the valve in the channel connected to that chamber is opened, and the other valve is closed. Of course, more reagent inlets may be provided, and this is not limited here.

[0047] The above is an introduction to the specific structure of the card box 100. Based on the structure described above, the testing process of the card box 100 will be explained in detail below.

[0048] Before proceeding, it should be noted that the detection process of cartridge 100 can be performed manually or using the accompanying instrument. The following mainly uses the cartridge 100 in the product form shown in Figures 1-5 as an example to illustrate the nucleic acid detection operation using the accompanying instrument. Manual operation means that the actions performed by the instrument are performed manually. For some structural modifications compared to the illustrated embodiment, such as the valve setting method, changes in the number of vents and reagent inlets, the essential detection principle remains unchanged. Only the corresponding details of the operation need to be adapted. For details, please refer to the description of the operation related to the modified structure of valves, vents and reagent inlets above, which will not be repeated below.

[0049] Please refer to Figures 4 and 5. First, place and fix the four reagent packs containing the corresponding reaction reagents (the first reagent pack 51, the second reagent pack 52, the third reagent pack 53 and the fourth reagent pack 54 shown in Figure 4) at the first inlet 41, the second inlet 42, the third inlet 43 and the fourth inlet 44 respectively, and seal the corresponding reagent inlets by the reagent packs themselves or by additional sealing materials.

[0050] Next, the sample is added into the sample chamber 120 through the injection port 110, and then the injection port 110 is closed. Then, the cartridge 100 is placed into the matching detection instrument, so that each valve abuts against the drive rod on the detection instrument, each air hole is connected to the drive pipeline of the detection instrument, and the drive rod of the detection instrument presses the pressure plate 27 on each valve to keep all valves in a closed state.

[0051] Next, negative pressure is introduced through the drive pipe into the first vent 31 to draw the sample from the sample chamber 120 into the magnetic bead chamber 130, mixing the sample with the magnetic beads in the chamber 130. To ensure uniform mixing, positive / negative pressure can be introduced repeatedly through the first vent 31 once or multiple times, causing the sample and magnetic beads to move back and forth in the flow channels of the magnetic bead chamber 130 and its vicinity, achieving uniform mixing. Alternatively, the magnetic beads and sample can be ultrasonically mixed. This step can also be achieved by introducing negative pressure through the first vent 31, or by opening the second valve 22 and introducing negative pressure through the second vent 32. Other similar steps described below are analogous and will not be elaborated further.

[0052] Then, negative pressure is introduced through the first vent 31 to draw the fluid formed by mixing the sample with the magnetic beads into the lysis chamber 140. Squeezing the first reagent pack 51 causes the lysis agent and binding agent inside to flow into the lysis chamber 140 through the first inlet 41. Cells and / or viruses in the sample undergo lysis under the action of the lysis agent, exposing nucleic acids. The exposed nucleic acids then bind to the magnetic beads under the action of the binding agent. Similarly, to ensure sufficient lysis and binding reactions, the lysis chamber 140 can be ultrasonically mixed using the ultrasonic module of the detection instrument. Alternatively, mixing can be achieved by repeatedly shaking the cartridge or repeatedly introducing positive and negative pressure; the specific mixing method is not limited here.

[0053] After lysis and binding are completed, the magnetic beads are first attracted and fixed in the lysis chamber 140 by the magnetic adsorption module of the detection instrument. Then, the first valve 21 is opened and positive pressure is introduced into the first vent 31 to discharge the waste liquid in the lysis chamber 140 to the waste liquid chamber 170. After the liquid is discharged, the pressurization is stopped, the first valve 21 is closed, and the magnetic adsorption module is removed to release the adsorption of the magnetic beads. At this time, the lysis chamber 140 contains magnetic beads bound with nucleic acid as well as some residual liquid and impurities.

[0054] Squeeze the second reagent pack 52 to allow the detergent inside to flow into the lysis chamber 140 through the second inlet 42 to clean the magnetic beads bound with nucleic acid, so as to remove as many other impurities adsorbed on the magnetic beads as possible. This process can also be mixed to ensure thorough cleaning.

[0055] After cleaning, open the second valve 22 and introduce negative pressure through the second vent 32 to draw the fluid from the pyrolysis chamber 140 into the elution chamber 150, then close the second valve 22. The fluid drawn into the elution chamber 150 can then be mixed again. It is understood that in this step, positive pressure can also be introduced through the first vent 31 to push the fluid from the pyrolysis chamber 140 into the elution chamber 150; similar steps are described below and will not be elaborated further.

[0056] Next, the magnetic beads are adsorbed again to fix them in the elution chamber 150. Then, the third valve 23 is opened and positive pressure is introduced into the second vent 32 to discharge the waste liquid in the elution chamber 150 into the waste liquid chamber 170. After the discharge is completed, the positive pressure is stopped, the third valve 23 is closed, and the adsorption of the magnetic beads is released.

[0057] At this point, the magnetic beads may only contain nucleic acid, or they may still contain a small amount of impurities. To ensure the accuracy of the test results, the magnetic beads can be cleaned again. Specifically, the third reagent pack 53 is squeezed to allow the detergent inside to flow into the elution chamber 150 through the third inlet 43, washing away the impurities adsorbed on the magnetic beads in the elution chamber 150. This process can also be made more thorough by mixing.

[0058] After cleaning, the magnetic beads in the elution chamber 150 are adsorbed and fixed. The third valve 23 is opened again, and positive pressure is introduced into the second vent 32 to discharge the waste liquid into the waste liquid chamber 170. Then, the third valve 23 is closed to stop the introduction of positive pressure and release the adsorption of the magnetic beads.

[0059] At this point, the magnetic beads are no longer mostly adsorbed with impurities, but some residual detergent droplets may remain. Next, the magnetic beads are dried. Specifically, the second valve 22 is opened, and the elution chamber 150 is heated. This causes the small droplets on the magnetic beads inside the elution chamber 150 to vaporize. Because the second valve 22 is open, the vaporized droplets expand and flow into the pyrolysis chamber 140 through the third flow channel 13. Since the pyrolysis chamber 140 is at room temperature, the vaporized gas condenses on the inner wall of the pyrolysis chamber 140, ensuring it does not flow back into the elution chamber 150. After drying, heating of the elution chamber 150 is stopped, and the second valve 22 is closed. It should be noted that besides opening the second valve 22 to allow the vaporized gas to enter the pyrolysis chamber 140 for condensation, the third valve 23 can also be opened to allow the vaporized gas to enter the waste liquid chamber 170 for condensation. Alternatively, both the second valve 22 and the third valve 23 can be opened simultaneously.

[0060] After multiple washing and drying processes, only the bound nucleic acid remains on the magnetic beads. Then, the fourth reagent pack 54 is squeezed to allow the eluent inside to flow into the elution chamber 150 through the fourth inlet 44, where the eluent elutes the nucleic acid from the magnetic beads. To ensure thorough elution, mixing can also be performed in this step.

[0061] After elution, the magnetic beads are first adsorbed and fixed in the elution chamber 150. Then, the fourth valve 24 is opened, and positive pressure is introduced into the second vent 32 to inject the liquid containing the eluted nucleic acid in the elution chamber 150 into the reaction chamber 160 through the fourth flow channel 14. In some embodiments, the reaction chamber 160 may be pre-contained with reaction reagents. The liquid containing nucleic acid injected into the reaction chamber 160 will re-dissolve with the reaction reagents. Then, the reaction chamber 160 is subjected to thermal cycling and optical analysis by a detection instrument, thus completing the entire process of nucleic acid detection. To simplify the production process of the cartridge 100, in some other embodiments, the reaction chamber 160 may also be an empty cavity. The reaction reagent is injected through a reagent inlet connected to the lysis chamber 140 or the elution chamber 150 and pressurized to enter the reaction chamber 160. Alternatively, a separate reagent inlet connected to the reaction chamber 160 through a flow channel may be provided to inject the reaction reagent into the reaction chamber 160 through this inlet.

[0062] It should be noted that the design of the number and location of reagent inlets used in the above detection process and the illustrated embodiments is only one specific method provided by this application, and does not constitute a limitation on the specific number and location of reagent inlets.

[0063] To ensure convenient sample injection, the inlet 110 is generally set to be relatively large, and the volume of the sample chamber 120 is also set to be relatively large. Practical testing has shown that a larger volume of sample chamber 120 results in more sample liquid adhering to the inner wall of sample chamber 120, forming small droplets. Based on this, when the first vent 31 is connected to the lysis chamber 140 as shown in Figure 5, during the process of introducing negative pressure through the first vent 31 to draw the sample liquid from sample chamber 120 into magnetic bead chamber 130 and lysis chamber 140, the small droplets adhering to the inner wall of sample chamber 120 cannot be effectively drawn into magnetic bead chamber 130 and lysis chamber 140 due to the characteristics of negative pressure drive. As a result, these small droplets cannot participate in the subsequent sample processing process, thus affecting the sample processing capability of cartridge 100.

[0064] In view of the above problems, in addition to the specific structure shown in Figures 1-6, in other embodiments of the card holder 100, such as the exploded and perspective structures shown in Figures 7 to 10, the first vent 31 can also be configured to communicate with the sample chamber 120. Positive pressure is introduced into the first vent 31 to pump the sample from the sample chamber 120 into the magnetic bead chamber 130, the fluid in the magnetic bead chamber 130 into the pyrolysis chamber 140, and the fluid in the pyrolysis chamber 140 into the waste liquid chamber 170. The direction of gas flow from the first vent 31 to the sample chamber 120 is shown by the dashed arrow in Figure 9, located in the channel between the first vent 31 and the sample chamber 120. Comparing Figures 5 and 9, it can be seen that the specific embodiments shown in the two figures are basically the same except for the chamber communicating with the first vent 31.

[0065] By connecting the first vent 31 to the sample chamber 120 and using positive pressure introduced through the first vent 31 to push the sample liquid from the sample chamber 120 to the magnetic bead chamber 130 and the lysis chamber 140, the positive pressure-driven characteristic allows the gas introduced into the sample chamber 120 to push most of the small liquid droplets suspended on the inner wall of the sample chamber 120 into the magnetic bead chamber 130 and the lysis chamber 140 along the inner wall and flow channel. This ensures that more sample liquid participates in subsequent processing and detection, thereby improving sample processing capacity and detection effect.

[0066] Of course, in some other embodiments, at least two first vents 31 may be provided on the cartridge 100 at the same time, at least one of which is connected to the sample chamber 120 and at least the other is connected to the lysis chamber 140. When driving the sample, positive pressure can be introduced into the first vent 31 connected to the sample chamber 120, while negative pressure can be introduced into the first vent 31 connected to the lysis chamber 140.

[0067] In the cartridge 100, the magnetic bead cavity 130, in addition to being connected to the sample cavity 120 and the lysis cavity 140 via a flow channel as provided in the above embodiments, can also be set independently. Specifically, the sample cavity 120 is directly connected to the lysis cavity 140 via a flow channel, and the magnetic bead cavity 130 is independently set in the cartridge 110, and the magnetic bead cavity 130 is connected to the lysis cavity 140 via another flow channel. Furthermore, in order to enable the magnetic beads to smoothly enter the lysis cavity 140, the magnetic bead cavity 130 can contain a certain amount of solution in addition to containing the magnetic beads, and the cartridge 100 can be additionally provided with vents communicating with the magnetic bead cavity 130. Based on this, during the detection process, the flow of the sample liquid from the sample chamber 120 to the lysis chamber 140 is basically the same as described above, and can be achieved by introducing pressure into the first vent 31. The refusion of the magnetic beads and the sample liquid can be achieved by introducing positive pressure into the vent on the cartridge 100 that is connected to the magnetic bead chamber 130, so that the magnetic beads in the magnetic bead chamber 130 are driven into the lysis chamber 140 along with the solution, thereby allowing the magnetic beads and the sample liquid to refusion in the lysis chamber 140.

[0068] In summary, based on research findings that lysis and binding can be performed simultaneously without mutual interference, the cartridge 100 provided in this application only includes a lysis chamber 140 to handle nucleic acid lysis and binding with magnetic beads. Furthermore, to prevent residual impurities on the inner wall of the lysis chamber 140 from affecting the detection results, an elution chamber 150 is provided to handle the step of eluting nucleic acid from the magnetic beads, ensuring the purity of nucleic acid extraction in the eluted liquid, improving the accuracy of the detection results, and minimizing the volume and cost of the cartridge 100. The orderly discharge of waste liquid from the lysis chamber 140 and elution chamber 150 is ensured by providing waste liquid chambers 140 and 150 respectively. Moreover, the valves on the flow channels between the chambers and the vents communicating with some chambers simplify the detection process and ensure precise control of the internal fluid flow direction. Meanwhile, by utilizing the temperature difference between the elution chamber 150 and the lysis chamber 140 and / or the waste liquid chamber 170, and by controlling the valves between them, the vaporized gas can flow into the lysis chamber 140 and / or the waste liquid chamber 170 to condense during the drying of the magnetic beads, thus preventing it from flowing back into the elution chamber 150, thereby further improving the purity of nucleic acid extraction.

[0069] It is particularly important to emphasize that a key factor in ensuring the accuracy of nucleic acid testing is avoiding the mixing of impurities generated during lysis into the eluted solution. This is because the eluted solution needs to be injected into the reaction chamber 160 for PCR amplification. If the eluted solution contains impurities generated during lysis, it will affect the accuracy of the PCR amplification. For this reason, the cartridge 100 provided in this embodiment employs an independent configuration of the lysis chamber 140 and the elution chamber 150. The lysis chamber 140 is responsible for lysis, and the elution chamber 150 is responsible for elution. Compared to using the same chamber for both lysis and elution, this prevents impurities generated during lysis from significantly affecting the elution process. Furthermore, through multiple washes, the mixing of impurities generated during lysis into the eluted solution can be effectively prevented.

[0070] Furthermore, the cartridge 100 provided in this embodiment of the application also has independent vents for lysis and elution, namely a first vent 31 and a second vent 32. The first vent 31 is connected to at least one of the sample chamber 120 and the lysis chamber 140, and is mainly responsible for driving the flow of the solution between the sample chamber 120 and the lysis chamber 140. The second vent 32 is connected to the elution chamber 150 and is responsible for driving the flow of the solution in the elution chamber 150 to the waste liquid chamber and the reaction chamber. Compared with the method of setting only one vent and having the same vent responsible for driving the solution flow, this can prevent impurities remaining in the lysis chamber 140 from mixing into the elution chamber 150 during the liquid driving process, so as to avoid contaminating the extraction product in the elution chamber 150 (i.e., the detection solution containing nucleic acid obtained after eluting the magnetic beads) and prevent subsequent nucleic acid detection results from being interfered with.

[0071] To improve the uniformity of mixing between the magnetic beads and the sample, this application further proposes an implementation method, as shown in Figure 5. As shown, the second flow channel 12 includes a serpentine flow channel 121. The serpentine flow channel 121 increases the flow distance between the magnetic bead cavity 130 and the lysis cavity 140, allowing the magnetic beads to mix thoroughly with the sample during flow. Furthermore, its curved corners also ensure thorough mixing between the solid magnetic beads and the liquid sample.

[0072] Furthermore, by introducing positive / negative pressure through the first air hole 31 and allowing the fluid formed by the sample and magnetic beads to flow back and forth in the serpentine channel 121, the mixing effect between the magnetic beads and the sample can be optimized.

[0073] Considering that the pressure in the reaction chamber 160 will increase during the process of injecting the liquid containing nucleic acid from the elution chamber 150 into the reaction chamber 160, which may affect the stability of the structure of the cartridge 100 at the reaction chamber 160, as shown in FIG5, in some embodiments of this application, the reaction chamber 160 is also connected to the waste liquid chamber 170 through a seventh flow channel 17 with a water-proof and breathable membrane 161, so that when the liquid in the elution chamber 150 enters the reaction chamber 160, the gas in the reaction chamber 160 is discharged to the waste liquid chamber 170, while the liquid is blocked inside the reaction chamber 160. At the same time, the water-proof and breathable membrane 161 can prevent the aerosol generated in the reaction chamber 160 from leaking to the outside and contaminating the detection environment.

[0074] In the specific embodiments shown in Figures 4 and 5, similar to the valve arrangement, the seventh flow channel 17 includes two parts respectively opened on both sides of the cartridge 100. These two parts are connected by a second through hole 162 penetrating the cartridge 100. A water-proof and breathable membrane 161 covers the second through hole 162 to allow gas to pass through while blocking liquid. Of course, in some other embodiments, the seventh flow channel 17 may also be formed only on one side of the cartridge 100 or injection molded inside the cartridge 100, and the water-proof and breathable membrane 161 may be set to cover the entire cross-section of the seventh flow channel 17.

[0075] By setting a seventh flow channel 17 to connect the reaction chamber 160 and the waste liquid chamber 170, and setting a water-proof and breathable membrane 161 in the seventh flow channel 17, the gas in the reaction chamber 160 can be normally discharged to the waste liquid chamber 170 when liquid is injected into the reaction chamber 160, thus ensuring the stable pressure in the reaction chamber 160.

[0076] In addition to ensuring pressure stability in the reaction chamber 160 by setting the seventh flow channel 17 and the water-proof and breathable membrane 161, the reaction chamber 160 can also be vacuumed during the manufacturing process of the cartridge 100 to ensure smooth liquid injection into the reaction chamber 160 and stable internal pressure. Alternatively, the cartridge 100 can be made of a material with good structural strength and high pressure resistance to ensure good stability even when the internal pressure of the reaction chamber 160 increases.

[0077] To improve the accuracy of detection, this application further proposes an implementation method, as shown in Figure 5 again. As shown in the figure, both the fourth flow channel 14 and the seventh flow channel 17 are provided with a shut-off valve 163, which is configured to close after the liquid is injected into the reaction chamber 160.

[0078] Specifically, the shut-off valve 163 can be a pressure plate type as described in the above structural description of each valve, or it can be a diaphragm valve as shown in the embodiment of Figure 5. Specifically, by compressing the diaphragm to seal the holes on the corresponding flow channel, the two sections of the corresponding flow channel are isolated. After the liquid is injected into the reaction chamber 160, the shut-off valve 163 closes to seal the reaction chamber 160. This ensures that the reaction chamber 160 maintains a certain volume for thermal cycling and detection, thus ensuring the accuracy of the detection results.

[0079] To reduce detection errors and ensure the accuracy of test results, in some embodiments, as shown in Figure 5, there are multiple reaction chambers 160, and these multiple reaction chambers 160 are arranged in parallel. Specifically, the parallel arrangement of multiple reaction chambers 160 means that each reaction chamber 160 is independently connected to other chambers through corresponding flow channels and valves. During the liquid injection process into the reaction chamber 160, the liquid containing nucleic acid in the elution chamber 150 will fill each reaction chamber 160 respectively. In subsequent testing, the test liquid in each reaction chamber 160 is tested separately to meet the testing requirements of different items and improve sample testing capabilities.

[0080] To improve the efficiency of detecting the detection liquid in the reaction chamber 160, this application further proposes an embodiment, as shown in Figure 11. The figure shows the bottom view of the cartridge 100. As shown in the figure, a thin sheet structure 180 is formed on one side of the cartridge 100, and the reaction chamber 160 is disposed inside the thin sheet structure 180. The wall thickness of the thin sheet structure 180 is less than the wall thickness of the rest of the cartridge 100.

[0081] In this embodiment, by placing the reaction chamber 160 within a thin sheet structure 180 with a thinner wall thickness, the heat loss when passing through the wall of the reaction chamber 160 during PCR detection of the detection solution in the reaction chamber 160 can be reduced, thereby improving thermal conductivity, shortening amplification time, and ultimately achieving the goal of improving detection efficiency.

[0082] For reagent injection, in addition to placing existing reagent packs at the reagent inlet on cartridge 100 for injection, reagent packs can also be directly integrated onto cartridge 100. Please refer to Figures 1 and 4 for details. As shown in the figures, the reagent inlet is sealed with reagent packs (first reagent pack 51, second reagent pack 52, third reagent pack 53, and fourth reagent pack 54 in the figures; the specific number of reagent packs is not limited), and the reagent packs contain reagents.

[0083] The reagent pack can be a flexible vesicle and can be made by means of film hot pressing, etc. The specific method is not limited here. After the cartridge 100 and the reagent pack are manufactured separately, the reagent pack is assembled and fixed to the reagent inlet on the cartridge 100 by means of adhesive bonding, hot welding or other methods, and the reagent inlet is sealed to prevent the aerosol in the internal space of the cartridge 100 and the vaporized reagent from leaking through the reagent inlet and causing environmental pollution.

[0084] Furthermore, in order to facilitate the rupture of the reagent pack for liquid injection, in some embodiments, a puncture structure is provided at the reagent inlet, which is used to squeeze and puncture the reagent pack under force, so that the reagent inside can enter the cartridge 100 through the reagent inlet.

[0085] Specifically, the puncture structure can be a conical protrusion, a needle, or other similar structure. By setting up a puncture structure, on the one hand, the corresponding reagent pack can be easily punctured when reagent needs to be injected; on the other hand, the position of the puncture structure can be controlled to prevent the reagent pack from breaking due to excessive pressure, which could damage the seal between the reagent inlet and the external environment and cause environmental pollution.

[0086] Considering that the pressure in the waste liquid chamber 170 will increase after the liquid is discharged into the waste liquid chamber 170, which may affect the structural stability of the cartridge 100, this application further proposes an implementation method. Please refer to Figures 3 and 5 again for details. As shown in the figures, the cartridge 100 is also provided with a pressure relief hole 60 that communicates with the waste liquid chamber 170. The pressure relief hole 60 is used to release the gas in the waste liquid chamber 170 during the liquid discharge process to ensure the pressure in the waste liquid chamber 170 is stable.

[0087] Similar to the gas released from the reaction chamber 160 during liquid injection mentioned in the above embodiments, the waste liquid chamber 170 can also be stabilized by using a pressure relief hole 60 to ensure pressure stability, or by using a vacuum method, or by using materials with good structural strength and high pressure resistance to ensure the structural stability of the waste liquid chamber 170.

[0088] To further reduce pollution to the testing environment, this application proposes an embodiment in which at least one of the sample chamber 120 and the lysis chamber 130 is covered with a waterproof and breathable membrane between itself and the first vent 31, between the elution chamber 150 and the second vent 32, and between the pressure relief hole 60 and the waste liquid chamber 170. The waterproof and breathable membrane is used to prevent leakage of aerosols and vaporized reagents inside the cartridge 100. In the specific embodiment shown in Figure 3, a first waterproof and breathable membrane 71 is covered between the first vent 31 and the lysis chamber 140, a second waterproof and breathable membrane 72 is covered between the second vent 32 and the elution chamber 150, and a third waterproof and breathable membrane 73 is covered between the pressure relief hole 60 and the waste liquid chamber 170. In the specific embodiment shown in Figure 8, the first waterproof and breathable membrane 71 is located between the first vent 31 and the sample chamber 120, and the positions of the second and third waterproof and breathable membranes 72 and 73 are the same as in the embodiment shown in Figure 3.

[0089] Specifically, the first waterproof and breathable membrane 71 can cover the opening of the first pore 31 as shown in Figures 3 and 8, or it can cover the flow channel between the first pore 31 and the lysis chamber 140 or the sample chamber 120. The second waterproof and breathable membrane 72 and the third waterproof and breathable membrane 73 are similar, and will not be described in detail here.

[0090] In this embodiment, by sealing the channels connecting the internal and external spaces of the cartridge 100, such as the vents and pressure relief holes 60, with a waterproof and breathable membrane, it is possible to effectively prevent internal aerosol contamination of the detection environment.

[0091] In some embodiments, a water-absorbing and breathable membrane is provided between at least one of the sample chamber 120 and the lysis chamber 140 and the first vent 31, and between the elution chamber 150 and the second vent 32. The water-absorbing and breathable membrane is used to prevent moisture from entering the cartridge 100 and affecting the accuracy of the test results when positive pressure is introduced through the vent. Furthermore, air bubbles will burst upon contact with the water-absorbing and breathable membrane, thus eliminating air bubbles. In addition, the water-absorbing and breathable membrane also has the function of absorbing water vapor evaporated during the drying process of the magnetic beads. In the specific embodiments shown in Figures 4 and 5, a first water-absorbing and breathable membrane 81 is provided between the first vent 31 and the lysis chamber 140, and a second water-absorbing and breathable membrane 82 is provided between the second vent 32 and the elution chamber 150. In the specific embodiments shown in Figures 8 and 9, the first water-absorbing and breathable membrane 81 is located between the first vent 31 and the sample chamber 120, and the position of the second water-absorbing and breathable membrane 82 is the same as in the embodiments shown in Figures 4 and 5.

[0092] Considering that the air permeability of some absorbent and breathable membranes may decrease after absorbing a large amount of liquid, and that liquid splashing may occur inside the lysis chamber 140 and elution chamber 150 during ultrasonic vibration mixing, as shown in Figures 4 and 5, a significant amount of liquid may pass through the first channel 91 between the lysis chamber 140 and the first absorbent and breathable membrane 81 to reach and be absorbed by the first absorbent and breathable membrane 81. Similarly, a significant amount of liquid may pass through the elution chamber 150. The second channel 92 between the first absorbent and breathable membrane 81 and the second absorbent and breathable membrane 82 reaches the second absorbent and breathable membrane 82 and is absorbed by the second absorbent and breathable membrane 82, causing a decrease in the air permeability of the first absorbent and breathable membrane 81 and the second absorbent and breathable membrane 82. Consequently, when air pressure is introduced into the first pore 31 and the second pore 32, the permeability of the gas at the first absorbent and breathable membrane 81 and the second absorbent and breathable membrane 82 decreases, and the driving ability of the internal liquid decreases. In severe cases, it may even lead to the inability to drive the sample to flow normally between the internal chambers and channels.

[0093] Comparing Figures 9 and 5, it can be seen that, compared to the embodiment shown in Figure 5, the embodiment shown in Figure 9 has an advantage in that the first absorbent and breathable membrane 81 is located between the first pore 31 and the sample cavity 120. Therefore, during ultrasonic mixing in the lysis chamber 140, the longer and more tortuous flow channel between the first absorbent and breathable membrane 81 and the lysis chamber 140 prevents splashed liquid from reaching and being absorbed by the first absorbent and breathable membrane 81. This ensures the breathability of the first absorbent and breathable membrane 81 and guarantees the driving effect of positive pressure on the liquid when the first pore 31 is applied. Furthermore, even if some liquid splashes into the flow channel between the lysis chamber 140 and the sample 120, or into the sample cavity 120, positive pressure can be introduced through the first pore 31 to push this portion back into the lysis chamber 140 to participate in subsequent reactions, ensuring the sample processing capacity.

[0094] As shown in Figure 9, for the second absorbent breathable membrane 82 between the second pore 32 and the elution chamber 150, since the second waterproof breathable membrane 72 at the second pore 32 shown in Figure 10 is prone to failure when encountering large air bubbles, a micro valve 921 is added to the second channel 92 between the second absorbent breathable membrane 82 and the elution chamber 150. The cross-sectional area of ​​the second channel 92 at the micro valve 921 is reduced and covered with an elastic pad. The function of the micro valve 921 is to eliminate or transform large air bubbles that splash and enter the second channel 92 into small air bubbles when the sample in the elution chamber 150 is ultrasonically mixed, under the impact of the elastic pad and when entering the smaller gap section from a larger cross section. This allows the small air bubbles to be quickly absorbed when they reach the second absorbent breathable membrane 82, preventing large air bubbles from passing through the second channel 92 and penetrating the second absorbent breathable membrane 82 and reaching the second waterproof breathable membrane 72, causing the second waterproof breathable membrane 72 to fail.

[0095] Of course, the microvalve 921 can also be set as a flow control valve. Before the sample in the elution chamber 150 is ultrasonically mixed, the microvalve 921 can be turned off in advance to ensure that the liquid will not splash onto the second absorbent and breathable membrane 82 and affect the breathability of the second absorbent and breathable membrane 82, thus ensuring the driving ability of the sample when pressure is introduced into the second pore 32.

[0096] Furthermore, in the embodiments shown in Figures 9 and 10, the first absorbent breathable membrane 81 and the second absorbent breathable membrane 82 are disposed at the edge or corner of the card holder 100. This is because the middle area of ​​the card holder 100 needs to integrate more cavities and channels. In order to ensure that the first absorbent breathable membrane 81 and the second absorbent breathable membrane 82 have a large area so as not to cause blockage when absorbing more liquid, the first absorbent breathable membrane 81 and the second absorbent breathable membrane 82 are disposed at the edge or corner of the card holder 100 to ensure that the area of ​​the first absorbent breathable membrane 81 and the second absorbent breathable membrane 82 can be set as large as possible.

[0097] Considering the complexity and high cost of the one-piece microchannel molding method inside the cartridge 100, this application proposes a manufacturing method that facilitates mass production of the cartridge 100 and effectively reduces costs. Referring to Figures 1 to 4, the cartridge 100 includes a cartridge body 101 and a cover 102. The sample inlet 110, vent, and reagent inlet are all located on the cartridge body 101. The sample chamber 120, magnetic bead chamber 130, lysis chamber 140, elution chamber 150, reaction chamber 160, waste liquid chamber 170, and the channels connecting them are all formed by grooves on the surface of the cartridge body 101. The cover 102 seals and covers these grooves.

[0098] In the specific embodiment shown in the figure, grooves forming flow channels are formed on both sides of the card holder body 101, and correspondingly, cover members 102 are covered on both sides of the grooves of the card holder body 101. In the specific production process, in order to ensure structural strength, the card holder body 101 can be made of rigid material, while in order to ensure sealing performance, the cover member 102 can be made of flexible film material. The two can be assembled and fixed by means of bonding, heat fusion or other methods.

[0099] Compared to the method of integrally molding the flow channel and cavity inside, the method provided in this embodiment, which involves opening a groove in the card holder body 101 and sealing the groove with a cover 102, is more convenient for the production and manufacturing of the card holder 100, and is conducive to improving production efficiency and reducing production costs.

[0100] Please refer to Figure 4. In some embodiments of this application, a water-absorbing component 171 is provided in the waste liquid chamber 170. The water-absorbing component 171 is used to absorb the liquid discharged into the waste liquid chamber 170, which on the one hand prevents waste liquid leakage, and on the other hand facilitates subsequent treatment of waste liquid.

[0101] To ensure smooth sample flow, as shown in Figures 4 and 5, in some embodiments, the first flow channel 11 is a siphon channel, so that the siphon principle can be used to ensure smooth sample flow during the process of drawing the sample from the sample cavity 120 into the magnetic bead cavity 130.

[0102] To facilitate sealing of the injection port 110 after injection, as shown in Figure 1, a detachable sealing cap 111 is provided at the injection port 110. Specifically, the sealing cap 111 can seal the injection port 110 by means of threaded connection, compression interference fit, or other methods.

[0103] Based on the cartridge 100 provided in the above embodiments, according to another aspect of the embodiments of this application, a nucleic acid detection method is also provided, which is applied to the cartridge 100 in any of the above embodiments. Please refer to Figure 5 for details, and further refer to Figure 12, which illustrates the flow of the nucleic acid detection method. As shown in the figures, the method includes the following steps:

[0104] Step 210: Inject the sample into the sample chamber 120 through the injection port 110;

[0105] Step 230: Keep all valves closed;

[0106] Step 251: Introduce negative pressure into the first vent 31 to draw the sample into the magnetic bead cavity 130 and mix it with the magnetic beads to form a first mixed fluid;

[0107] Step 271: Introduce negative pressure again into the first vent 31 to draw the first mixed fluid into the pyrolysis chamber 140;

[0108] When this method is applied to the cartridge 100 shown in Figures 7 to 10, steps 251 and 271 are replaced by introducing positive pressure into the first vent 31 in steps 252 and 272 as shown in Figure 13. The driving direction and effect on the sample and the first mixed fluid are the same as in steps 251 and 271. However, when this method is applied to an embodiment in which multiple first vents 31 are connected to the sample chamber 120 and the lysis chamber 140 respectively, steps 251 and 271 are replaced by introducing positive pressure into the first vent 31 connected to the sample chamber 120 and negative pressure into the first vent 31 connected to the lysis chamber 140. The driving direction and effect on the sample and the first mixed fluid are also the same. Similar steps in the following text are similar and will not be described in detail.

[0109] Step 290: Inject the lysing agent and binding agent into the lysing chamber 140 through the reagent inlet (first inlet 41 in Figure 5) connected to the lysing chamber 140, so that the lysing agent and binding agent are mixed with the first mixed fluid and react to form a second mixed fluid. In the first mixed fluid, the cells and / or viruses in the lysing agent are lysed to expose the nucleic acid, and the nucleic acid is bound to the magnetic beads under the action of the binding agent.

[0110] Step 310: Adsorb the magnetic beads in the second mixed fluid so that the magnetic beads bound with nucleic acid are fixed in the lysis chamber 140. Open the first valve 21 on the flow channel (the fifth flow channel 15 in Figure 5) between the lysis chamber 140 and the waste liquid chamber 170, and introduce positive pressure into the first vent 31 so that the liquid in the lysis chamber 140 is discharged into the waste liquid chamber 170. Then close the first valve 21 and release the adsorption of the magnetic beads.

[0111] Step 330: Inject detergent into the pyrolysis chamber 140 through the reagent inlet (second inlet 42 in Figure 5) connected to the pyrolysis chamber 140 to clean the magnetic beads and form a third mixed fluid;

[0112] Step 351: Open the second valve 22 on the flow channel (third flow channel 13 in Figure 5) between the pyrolysis chamber 140 and the elution chamber 150, and introduce negative pressure into the second vent 32 communicating with the elution chamber 150 to draw the third mixed fluid into the elution chamber 150. Then close the second valve 22. Of course, this step can also be replaced by step 352 shown in Figure 13, that is, to achieve the same driving effect on the third mixed fluid by introducing positive pressure into the first vent 31. Similar steps in the following text are the same and will not be described in detail.

[0113] Step 370: Adsorb the magnetic beads in the third mixed fluid so that the magnetic beads are fixed in the elution chamber 150. Open the third valve 23 on the flow channel (sixth flow channel 16 in Figure 5) between the elution chamber 150 and the waste liquid chamber 170, and introduce positive pressure into the second vent 32 so that the liquid in the elution chamber 150 is discharged to the waste liquid chamber 170. Then close the third valve 23 and release the adsorption of the magnetic beads.

[0114] Step 390: Inject detergent into elution chamber 150 through reagent inlet (third inlet 43 in Figure 5) connected to elution chamber 150 to clean the magnetic beads and form a fourth mixed fluid;

[0115] Step 410: Adsorb the magnetic beads in the fourth mixed fluid so that the magnetic beads are fixed in the elution chamber 150. Open the third valve 23 and introduce positive pressure into the second vent 32 so that the liquid in the elution chamber 150 is discharged to the waste liquid chamber 170. Then close the third valve 23 and release the adsorption of the magnetic beads.

[0116] Step 430: Open the second valve 22 and / or the third valve 23, and dry the magnetic beads in the elution chamber 150 so that the liquid on the surface of the magnetic beads and the liquid in the elution chamber 150 are vaporized and enter the pyrolysis chamber 140 and / or the waste liquid chamber 170 for condensation. Then close the second valve 22 and / or the third valve 23.

[0117] Step 450: Inject the eluent into the elution chamber 150 through the reagent inlet (fourth inlet 44 in Figure 5) which is connected to the elution chamber 150, so as to elute the nucleic acid from the magnetic beads;

[0118] Step 470: Adsorb the magnetic beads in the elution chamber 150 so that the magnetic beads are fixed in the elution chamber 150. Open the fourth valve 24 on the flow channel (fourth flow channel 14 in Figure 5) that connects the elution chamber 150 and the reaction chamber 160, and introduce positive pressure into the second vent 32 to inject the liquid mixed with nucleic acid in the elution chamber 150 into the reaction chamber 160 for detection.

[0119] It should be noted that if there are residual lysis solvent or other impurity solutions in the elution chamber 150, they will contaminate the subsequently injected elution solvent, resulting in impurities in the liquid mixed with nucleic acid injected from the elution chamber 150 into the reaction chamber 160, which will affect PCR amplification and detection.

[0120] Therefore, in the nucleic acid detection method provided in this application embodiment, after the lysis agent and impurities are initially discharged into the waste liquid chamber 170 in step 310, the detergent is first injected into the lysis chamber 140 in step 330 to clean the residual lysis agent and other impurities therein to form a third mixed fluid. Then, the third mixed fluid is driven into the elution chamber 150 in step 351, and the impurities in the third mixed fluid (including residual lysis agent, detergent, etc.) are discharged from the elution chamber 150 to the waste liquid chamber 170 in step 370, completing the first washing and drainage. Then, the detergent is injected into the elution chamber 150 again in step 390 to clean the impurities that may still remain, and the impurities in the elution chamber 150 (including detergent and the remaining lysis agent, etc.) are discharged into the waste liquid chamber 170 in step 390, completing the second washing and drainage. Thus, by washing twice, impurities in the elution chamber 150 are thoroughly removed, and the magnetic beads in the elution chamber 150 are further dried in step 450 to evaporate and discharge any residual liquid droplets in the elution chamber 150. This ensures the purity of the solution in the elution chamber 150 after the eluent is injected into the elution chamber 150 in step 450, thereby improving the accuracy of nucleic acid detection in the reaction chamber in step 470.

[0121] Specifically, in step 470, reaction reagents can be pre-placed in reaction chamber 160. A liquid mixture containing nucleic acids, injected from elution chamber 150 into reaction chamber 160, reacts with the reaction reagents to form a detection solution for PCR testing. Alternatively, the reaction reagents can be injected into the reaction chamber through a reagent inlet connected to elution chamber 150 or lysis chamber 140.

[0122] The procedure for the above nucleic acid testing method is the same as the description of the nucleic acid testing procedure in the above-mentioned nucleic acid testing card example, and will not be repeated here.

[0123] The nucleic acid detection method provided in this application, by using the cartridge 100 provided in the above embodiment, can ensure the purity of nucleic acid extraction in the eluted liquid, improve the accuracy of the detection results, and the entire detection process is simple to operate, and can achieve precise control of the fluid inside the cartridge 100.

[0124] Furthermore, for the cartridge 100 in which the magnetic bead cavity 130 and the lysis cavity 140 are connected by the serpentine flow channel 121, step 251 in the nucleic acid detection method may include:

[0125] Positive / negative pressure is introduced n times through the first vent 31 connected to the pyrolysis chamber 140, so that the sample and the magnetic beads flow back and forth in the magnetic bead chamber 130 and the serpentine flow channel 121 and mix to form a first mixed fluid, where n≥1 and n is an integer.

[0126] To ensure sufficient reaction in each step, the fluid is mixed after step 290, and / or after step 330, and / or after step 351, and / or after step 390, and / or after step 450. Mixing methods include, but are not limited to, ultrasonic mixing, shaking mixing, etc.

[0127] When this method is applied to the cartridge 100 shown in Figures 7 to 10, the microvalve 921 can be opened before pressure is introduced into the second vent 32 to ensure that the airflow enters the elution chamber 150 normally and drives the internal fluid flow. Before mixing the internal fluid, such as before ultrasonic mixing, the microvalve 921 can be closed to ensure that splashed fluid does not reach the second absorbent membrane 82 and be absorbed by it.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way.

Claims

1. A nucleic acid testing cartridge, characterized by, The cartridge is provided with a sample inlet, and a sample chamber is arranged in the cartridge and communicates with the sample inlet; The cartridge is further provided with a lysis chamber, an elution chamber and a reaction chamber which are sequentially communicated through flow channels with valves, and the sample chamber communicates with the lysis chamber through a flow channel; The cartridge is further provided with a magnetic bead chamber, and the magnetic bead chamber contains magnetic beads, and the magnetic bead chamber communicates with the lysis chamber through a flow channel; The cartridge is further provided with a waste liquid chamber, and the waste liquid chamber communicates with the lysis chamber and the elution chamber through flow channels with valves, respectively; The cartridge is provided with a first air hole which communicates with at least one of the sample chamber and the lysis chamber; The cartridge is further provided with a second air hole which communicates with the elution chamber; The cartridge is further provided with a reagent inlet which respectively communicates with the lysis chamber and the elution chamber.

2. The nucleic acid testing cartridge of claim 1, wherein, The magnetic bead chamber is communicated between the sample chamber and the lysis chamber through a flow channel, so that the sample chamber communicates with the lysis chamber through a flow channel and the magnetic bead chamber.

3. The nucleic acid testing cartridge of claim 2, wherein, The first air hole communicates with the sample chamber, and the first air hole is used to drive the sample from the sample chamber into the magnetic bead chamber and from the magnetic bead chamber into the lysis chamber when a positive pressure is introduced.

4. The nucleic acid testing cartridge of claim 1, wherein, The reaction chamber further communicates with the waste liquid chamber through a flow channel with a water-proof and air-permeable membrane, so that in the process of liquid in the elution chamber entering the reaction chamber, the gas in the reaction chamber is discharged to the waste liquid chamber, and the liquid is blocked in the interior of the reaction chamber, while preventing the leakage of aerosol in the reaction chamber.

5. The nucleic acid testing cartridge of claim 4, wherein, A blocking valve is arranged in the flow channel between the reaction chamber and the elution chamber and the flow channel between the reaction chamber and the waste liquid chamber, and the blocking valve is configured to be closed after the liquid injection in the reaction chamber is completed.

6. The nucleic acid testing cartridge of claim 1, wherein, The reaction chamber contains a reaction reagent, and the reaction reagent is used to react with the liquid injected into the reaction chamber from the elution chamber.

7. The nucleic acid testing cartridge of claim 1, wherein, One side of the cartridge is formed with a sheet structure, the reaction chamber is arranged in the interior of the sheet structure, and the wall thickness of the sheet structure is smaller than that of the remaining part of the cartridge.

8. The nucleic acid testing cartridge of claim 1, wherein, The reaction chamber is a plurality of chambers, and the plurality of chambers are arranged in parallel.

9. The nucleic acid testing cartridge of claim 1, wherein, The reagent inlet includes a first inlet and a second inlet which respectively communicate with the lysis chamber, the first inlet is used for introducing a lysis reagent and a binding reagent into the lysis chamber, and the second inlet is used for introducing a washing reagent into the lysis chamber; The reagent inlet further includes a third inlet and a fourth inlet, the third inlet is used for introducing a washing reagent into the elution chamber, and the fourth inlet is used for introducing an elution reagent into the elution chamber.

10. The nucleic acid testing cartridge of claim 1, wherein, The cartridge is further provided with a pressure relief hole which communicates with the waste liquid chamber.

11. The nucleic acid testing cartridge of claim 10, wherein, At least one of the sample chamber and the lysis chamber, the elution chamber, the pressure relief hole and the waste liquid chamber are covered with a water-proof and air-permeable membrane, and the water-proof and air-permeable membrane is used to prevent the leakage of aerosol and vaporized reagent in the interior.

12. A method of detecting a nucleic acid, characterized by, The nucleic acid detection cartridge of any one of claims 2-11 is applied to the method, and the method comprises: injecting a sample into the sample chamber through the sample inlet; keeping all valves closed; introducing pressure into the first air hole to make the sample flow into the magnetic bead cavity and mix with the magnetic beads to form a first mixed fluid; introducing pressure into the first air hole again to make the first mixed fluid flow into the lysis cavity; injecting a lysis agent and a binding agent into the lysis cavity through a reagent inlet connected with the lysis cavity to make the lysis agent and the binding agent mix and react with the first mixed fluid to form a second mixed fluid, wherein the cells and / or viruses in the first mixed fluid are lysed by the lysis agent to expose nucleic acid, and the nucleic acid is bound to the magnetic beads by the binding agent; adsorbing the magnetic beads in the second mixed fluid to fix the magnetic beads with the nucleic acid in the lysis cavity, opening a first valve on a flow channel between the lysis cavity and the waste liquid cavity, introducing positive pressure into the first air hole to make the liquid in the lysis cavity flow into the waste liquid cavity, then closing the first valve and releasing the adsorption of the magnetic beads; injecting a washing agent into the lysis cavity through the reagent inlet connected with the lysis cavity to clean the magnetic beads and form a third mixed fluid; opening a second valve on a flow channel between the lysis cavity and the elution cavity, introducing positive pressure into the first air hole or negative pressure into the second air hole to make the third mixed fluid flow into the elution cavity, then closing the second valve; adsorbing the magnetic beads in the third mixed fluid to fix the magnetic beads in the elution cavity, opening a third valve on a flow channel between the elution cavity and the waste liquid cavity, introducing positive pressure into the first air hole or the second air hole to make the liquid in the elution cavity flow into the waste liquid cavity, then closing the third valve and releasing the adsorption of the magnetic beads; injecting a washing agent into the elution cavity through the reagent inlet connected with the elution cavity to clean the magnetic beads and form a fourth mixed fluid; adsorbing the magnetic beads in the fourth mixed fluid to fix the magnetic beads in the elution cavity, opening the third valve, introducing positive pressure into the first air hole or the second air hole to make the liquid in the elution cavity flow into the waste liquid cavity, then closing the third valve and releasing the adsorption of the magnetic beads; opening the second valve and / or the third valve and drying the magnetic beads in the elution cavity to make the liquid on the surface of the magnetic beads and the liquid in the elution cavity vaporize and then condense in the lysis cavity and / or the waste liquid cavity, then closing the second valve and / or the third valve; injecting an elution agent into the elution cavity through the reagent inlet connected with the elution cavity to elute the nucleic acid from the magnetic beads; adsorbing the magnetic beads in the elution cavity to fix the magnetic beads in the elution cavity, opening a fourth valve on a flow channel between the elution cavity and the reaction cavity, and introducing positive pressure into the first air hole or the second air hole to inject the liquid mixed with the nucleic acid in the elution cavity into the reaction cavity for detection.

13. The nucleic acid detection method according to claim 12, wherein After the step of injecting a lysis reagent and a binding reagent into the lysis chamber through a reagent inlet in communication with the lysis chamber, mixing and reacting the lysis reagent and the binding reagent with the first mixed fluid to form a second mixed fluid, and / or, after the step of injecting a washing reagent into the lysis chamber through a reagent inlet in communication with the lysis chamber to wash the magnetic beads and form a third mixed fluid, and / or, after the step of opening a second valve on a flow path between the lysis chamber and the elution chamber, and introducing a positive pressure through the first air hole or a negative pressure through the second air hole to allow the third mixed fluid to flow to the elution chamber, and then closing the second valve, and / or, after the step of injecting a washing reagent into the elution chamber through a reagent inlet in communication with the elution chamber to wash the magnetic beads and form a fourth mixed fluid, and / or, after the step of injecting an elution reagent into the elution chamber through a reagent inlet in communication with the elution chamber to elute the nucleic acid from the magnetic beads, the method further comprises the step of: mixing the fluid.

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