Nucleic acid detection kit and test method

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection kit and a test method. The nucleic acid detection kit comprises a plurality of liquid storage cavities, a micro-valve assembly, a sample assembly, a mixing pool, a freeze-drying reagent cavity, a reaction unit and a plunger pump, the plurality of liquid storage cavities are respectively filled with a lysis solution, a combination solution, a cleaning solution and an eluent, a first micro-valve of the micro-valve assembly is connected with the liquid storage cavity filled with the eluent, a second micro-valve is connected with the liquid storage cavity filled with the cleaning solution, a third micro-valve is connected with the liquid storage cavity filled with the combination solution, a fourth micro-valve is connected with the liquid storage cavity filled with the lysis solution, and a fifth micro-valve is connected with the liquid storage cavity filled with the eluent. The fifth micro valve is used for controlling connection and disconnection between the sample assembly and the mixing pool, and connection and disconnection between the sixth micro valve and the first micro valve are achieved through a valve body of the sixth micro valve. The invention provides a nucleic acid detection kit and a test method, which can safely complete nucleic acid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a nucleic acid detection kit and a testing method. Background Art

[0002] Nucleic acid detection is a technology for detecting biological genetic material, which is widely used in the fields of pathogenic microorganism detection and infectious disease diagnosis. In order to quickly and conveniently detect nucleic acids, the present invention uses microfluidic technology to develop a nucleic acid detection kit to detect samples to be tested, wherein the flow control of each liquid reagent stored in the kit is achieved by microfluidic technology. Specifically, microfluidic technology can control the release, flow, and distribution of fluids in the chip, and with the cooperation of external instruments, ultimately achieve nucleic acid detection.

[0003] In the related art, the driving source for realizing fluid driving comes from an external device, such as a pump, and the external pump may cause the risk of cross infection to the test kit. Summary of the invention

[0004] The present invention provides a nucleic acid detection kit and a testing method, which can safely complete nucleic acid detection.

[0005] In a first aspect, the present invention provides a nucleic acid detection kit, comprising a plurality of liquid storage chambers, a microvalve assembly, a sample assembly, a mixing pool, a freeze-dried reagent chamber, a reaction unit and a plunger pump;

[0006] The plurality of liquid storage chambers are respectively filled with lysate, binding liquid, cleaning liquid and eluent, and the microvalve assembly includes a first microvalve, a second microvalve, a third microvalve, a fourth microvalve, a fifth microvalve, a sixth microvalve, a seventh microvalve, an eighth microvalve and a ninth microvalve, the first microvalve is connected to the liquid storage chamber containing the eluent, the second microvalve is connected to the liquid storage chamber containing the cleaning liquid, the third microvalve is connected to the liquid storage chamber containing the binding liquid, the fourth microvalve is connected to the liquid storage chamber containing the lysate, the first microvalve, the second microvalve, the third microvalve and the fourth microvalve are connected in sequence, the first microvalve, the second microvalve, the third microvalve and the fourth microvalve are connected to the liquid storage chambers connected to them respectively by opening and closing the valves, and the fifth microvalve is connected to the liquid storage chamber containing the cleaning liquid. Used to control the on-off between the sample component and the mixing pool, the sixth microvalve and the first microvalve are connected and disconnected through the valve of the sixth microvalve, the sixth microvalve is connected to the freeze-dried reagent chamber, the freeze-dried reagent chamber is connected to the reaction unit, the reaction unit is connected to the air permeable component, the seventh microvalve is connected to the plunger pump, the sample component and the eighth microvalve, the seventh microvalve is opened and closed to control the connection between it and the sample component, the eighth microvalve is connected to the mixing pool and the seventh microvalve, the eighth microvalve is used to control the on-off between it and the seventh microvalve, the ninth microvalve is used to cooperate with the air permeable component to make the air path unobstructed when opened, and is used to seal the reaction unit when closed.

[0007] In a possible design, each microvalve of the microvalve assembly is composed of a valve seat, a connecting structure and a sealing membrane, and the valve is switched on and off by controlling the sealing membrane to block the connecting structure.

[0008] In a possible design, a phase change material is also included, and the phase change material is used to seal the freeze-dried reagent chamber and the channel of the reaction unit and the air outlet of the air permeable component respectively through a process of heating, melting, flowing and re-solidifying and sealing, thereby achieving sealing of the reaction unit.

[0009] In a possible design, it also includes a main body and a puncture device;

[0010] The main body comprises an adjacent sealing cavity and a limiting bin, the sealing cavity comprises a first opening and a second opening whose opening directions are perpendicular to each other, the second opening is in communication with the limiting bin, the first opening and the second opening are sealed with a sealing film, the puncture device comprises a first puncture member and a second puncture member, the first puncture member faces the first opening, the second puncture member penetrates into the limiting bin, and a limiting inclined surface is arranged on the inner wall of the limiting bin relative to the second opening;

[0011] When the fluid in the sealed cavity needs to be released, the puncture device is driven to move toward the main body so that the first puncture member punctures the sealing membrane at the first opening, and the second puncture member in the limiting bin is bent toward the second opening under the restriction of the limiting slope to puncture the sealing membrane at the second opening, so that the fluid flows into the liquid storage cavity at the lower part of the limiting bin.

[0012] In a possible design, the second puncture member includes a guide portion and a puncture portion connected in sequence, a guide groove is provided in the limiting bin, the guide portion passes through the guide groove, and the guide groove is used to limit the second puncture member from moving in a preset direction.

[0013] In a possible design, a plate-shaped force-bearing plate is provided at a portion of the guide portion close to the puncture portion, and the force-bearing plate is used to cooperate with the limiting slope to control the deformation direction of the puncture portion to puncture the sealing membrane on the second opening.

[0014] In a possible design, the puncture device further includes a buckle, a clamping position is provided in the limit bin, and the buckle cooperates with the clamping position to limit the position of the puncture device;

[0015] The locking position includes a first locking position and a second locking position. When the buckle is located at the first locking position, the puncture device does not puncture the sealing film on the first opening and the sealing film on the second opening. When the buckle is located at the second locking position, the puncture device punctures the sealing film on the first opening and the sealing film on the second opening.

[0016] In a possible design, the puncture device also includes a force-driven member, which includes a mounting plate and a driving surface obliquely arranged on the mounting plate, the first puncture member and the second puncture member are respectively installed on the side of the mounting plate away from the driving surface, a plunger pump and a driving member are arranged on one side of the driving surface, and the driving member is arranged on a reciprocating rod of the plunger pump, the reciprocating rod is parallel to the mounting plate, and moves along its axial direction through the reciprocating rod, and the driving member, the driving surface, the guide portion and the guide groove on the reciprocating rod cooperate to drive the puncture device to move along the preset direction; wherein, the plunger pump is connected to the microfluidic chip to provide it with power for fluid control.

[0017] In a second aspect, the present invention provides a nucleic acid detection method, based on any of the nucleic acid detection kits described above, the method comprising:

[0018] Close the first microvalve, the second microvalve, the third microvalve, the fourth microvalve, the fifth microvalve and the seventh microvalve, and open the remaining microvalve, and use the plunger pump to discharge the gas in each channel;

[0019] The fourth microvalve, the fifth microvalve, the seventh microvalve and the ninth microvalve are opened, and the other microvalve are closed, and the lysate in the liquid storage chamber connected to the fourth microvalve and the sample in the sample assembly are pumped into the mixing pool by the plunger pump, wherein the mixing pool includes a heating device and a rotor, and the sample and the lysate in the mixing pool are mixed and lysed by the heating device and the rotor to obtain the target nucleic acid;

[0020] The third microvalve, the fifth microvalve, the seventh microvalve and the ninth microvalve are opened, and the rest are closed, and the binding liquid in the liquid storage chamber connected to the third microvalve is pumped into the mixing pool by the plunger pump, and under the action of the binding liquid, the target nucleic acid is adsorbed on the magnetic beads in the mixing pool, and the fourth microvalve, the fifth microvalve and the seventh microvalve are opened, and the rest are closed, and the waste liquid in the mixing pool is pumped into the liquid storage chamber connected to the fourth microvalve by the plunger pump;

[0021] The second microvalve, the fifth microvalve, the seventh microvalve and the ninth microvalve are opened, and the rest are closed, and the cleaning liquid in the liquid storage chamber connected to the second microvalve is pumped into the mixing pool by the plunger pump to clean the residual reagents other than the target nucleic acid in the mixing pool, and the third microvalve, the fifth microvalve and the seventh microvalve are opened, and the rest are closed, and the waste liquid in the mixing pool is pumped into the liquid storage chamber connected to the third microvalve by the plunger pump;

[0022] The first microvalve, the fifth microvalve, the seventh microvalve and the ninth microvalve are opened, and the rest are closed; the eluate in the liquid storage chamber connected to the first microvalve is pumped into the mixing pool by the plunger pump to clean the target nucleic acid on the magnetic beads in the mixing pool; the sixth microvalve, the fifth microvalve, the seventh microvalve and the ninth microvalve are opened, and the rest are closed; the eluate with the target nucleic acid in the mixing pool is pumped into the reaction unit by the plunger pump to complete nucleic acid detection; when the reaction unit performs an amplification reaction, the ninth microvalve is closed.

[0023] In one possible design, after the reaction is completed, the phase change material is heated to melt, allowing it to flow to the channel connecting the freeze-dried reagent chamber and the reaction unit, and to the channel connecting the air permeable component with the outside world, and then the reaction unit is physically sealed after cooling.

[0024] In a possible design, the side surface or bottom surface of the reaction unit is transparent to allow the interior of the reaction unit to receive external optical signals.

[0025] In a possible design, the reaction unit is provided with a heat conducting surface, and the heat conducting surface is used to contact with the heating control unit.

[0026] In a possible design, the heat conducting surface is formed by a film with a thickness of less than 0.1 mm and a thermal conductivity of more than 1 W / mK.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] In the present invention, through the opening and closing coordination of multiple microvalves of the microvalve assembly, different liquids, such as lysis solution, binding solution, cleaning solution and elution solution, are released into the mixing pool in sequence, thereby realizing the extraction of target nucleic acid from the sample. Finally, through the coordination of multiple microvalves of the microvalve assembly, the liquid carrying the target nucleic acid is transported from the mixing pool through the freeze-drying reagent chamber to the reaction unit to complete the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is a schematic diagram of the structure of a nucleic acid test kit provided by an embodiment of the present invention;

[0031] Figure 2 It is a schematic diagram of the connection structure of a nucleic acid test kit provided by an embodiment of the present invention;

[0032] Figure 3a This is a schematic diagram of the microvalve opening state structure of a nucleic acid test kit provided by an embodiment of the present invention;

[0033] Figure 3b It is a schematic structural diagram of a microvalve closed state of a nucleic acid test kit provided by an embodiment of the present invention;

[0034] Figure 4 It is a partial structural schematic diagram of a nucleic acid test kit provided by an embodiment of the present invention;

[0035] Figure 5 is a schematic structural diagram of a piercing device provided by an embodiment of the present invention;

[0036] Figure 6 is a schematic diagram of a fluid structure in a sealed state of a sealed cavity provided by an embodiment of the present invention;

[0037] Figure 7 It is a schematic diagram of a fluid structure in a sealed cavity punctured state provided by an embodiment of the present invention;

[0038] Figure 8is a schematic diagram of a flow chart of releasing fluid provided by an embodiment of the present invention;

[0039] Fig. 9 It is a schematic structural diagram of a piercing portion of a nucleic acid test kit provided by an embodiment of the present invention;

[0040] Fig.10 It is a structural schematic diagram of a mounting plate and accessories of a nucleic acid test kit provided by an embodiment of the present invention;

[0041] Fig.11a It is a schematic structural diagram from a first angle of another nucleic acid test kit structure provided by an embodiment of the present invention;

[0042] Fig.11b is a second angle structural schematic diagram of another nucleic acid test kit structure provided by an embodiment of the present invention;

[0043] Fig.12 is a side view of the piercing portion structure of a nucleic acid test kit provided by an embodiment of the present invention;

[0044] Fig.13 is a schematic diagram of fluid flow in a preparation process of a nucleic acid test kit provided by an embodiment of the present invention;

[0045] Fig.14 It is a schematic diagram of fluid flow in the lysis process of a nucleic acid test kit provided by an embodiment of the present invention;

[0046] Fig.15 It is a schematic diagram of fluid flow in the binding process of a nucleic acid test kit provided by an embodiment of the present invention;

[0047] Fig.16 It is a schematic diagram of fluid flow in a cleaning process of a nucleic acid test kit provided by an embodiment of the present invention;

[0048] Fig.17 It is a schematic diagram of fluid flow in the elution process of a nucleic acid test kit provided by an embodiment of the present invention;

[0049] Fig.18 It is a schematic diagram of fluid flow in a phase change sealing process of a nucleic acid test kit provided by an embodiment of the present invention;

[0050] Fig.19 It is a schematic diagram of a detection method of a nucleic acid test kit provided by an embodiment of the present invention;

[0051] Fig. 20 It is a schematic diagram of another detection method of a nucleic acid test kit provided in an embodiment of the present invention.

[0052] In the figure:

[0053] 100-microvalve assembly;

[0054] 101-first microvalve;

[0055] 102- second microvalve;

[0056] 103- the third microvalve;

[0057] 104- fourth microvalve;

[0058] 105-fifth microvalve;

[0059] 106-sixth microvalve;

[0060] 107-seventh microvalve;

[0061] 108-eighth microvalve;

[0062] 109-9th microvalve;

[0063] 200 - sample assembly;

[0064] 300-mixing pool;

[0065] 400-lyophilized reagent chamber;

[0066] 500-reaction unit;

[0067] 600-phase change material;

[0068] 700-ventilation component;

[0069] 1- Sealed cavity;

[0070] 11- First opening;

[0071] 12- second opening;

[0072] 2-Limited bin;

[0073] 21- limiting inclined plane;

[0074] 22-guide groove;

[0075] 23-First card position;

[0076] 24-Second card position;

[0077] 3-first piercing member;

[0078] 4- second piercing member;

[0079] 41- guide part;

[0080] 411-stress plate;

[0081] 42-puncture part;

[0082] 5- Buckle;

[0083] 6-Mounting plate;

[0084] 61- air guide groove;

[0085] 62-support body;

[0086] 7- driving surface;

[0087] 8- plunger pump;

[0088] 81 - reciprocating rod;

[0089] 82-driving member;

[0090] 9-liquid storage chamber;

[0091] 10-Guide warehouse. DETAILED DESCRIPTION

[0092] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0093] In the description of the embodiments of the present invention, unless otherwise clearly specified and limited, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance; unless otherwise specified or explained, the term "multiple" refers to two or more; the terms "connected" and "fixed" should be understood in a broad sense, for example, "connected" can be a fixed connection, a detachable connection, an integral connection, or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0094] In the description of this specification, it should be understood that the directional words such as "upper" and "lower" described in the embodiments of the present invention are described at the angles shown in the drawings and should not be understood as limiting the embodiments of the present invention. In addition, in the context, it should also be understood that when it is mentioned that an element is connected to another element "upper" or "lower", it can not only be directly connected to another element "upper" or "lower", but also indirectly connected to another element "upper" or "lower" through an intermediate element.

[0095] like Figure 1 To Figure 3 (wherein Figure 3 includes Figure 3a and Figure 3b), an embodiment of the present invention provides a nucleic acid detection kit, comprising a plurality of liquid storage chambers 9, a microvalve assembly 100, a sample assembly 200, a mixing pool 300, a freeze-dried reagent chamber 400, a reaction unit 500 and a plunger pump 8;

[0096] A plurality of liquid storage chambers 9 are respectively filled with lysate, binding liquid, cleaning liquid and eluent, and the microvalve assembly 100 includes a first microvalve 101, a second microvalve 102, a third microvalve 103, a fourth microvalve 104, a fifth microvalve 105, a sixth microvalve 106, a seventh microvalve 107, an eighth microvalve 108 and a ninth microvalve 109. The first microvalve 101 is connected to the liquid storage chamber 9 containing the eluent, the second microvalve 102 is connected to the liquid storage chamber 9 containing the cleaning liquid, the third microvalve 103 is connected to the liquid storage chamber 9 containing the binding liquid, and the fourth microvalve 104 is connected to the liquid storage chamber 9 containing the lysate. The first microvalve 101, the second microvalve 102, the third microvalve 103, and the fourth microvalve 104 are connected in sequence, and the first microvalve 101, the second microvalve 102, the third microvalve 103, and the fourth microvalve 104 are connected to the liquid storage chamber 9 connected to each other by opening and closing the valves. Connect and disconnect, the fifth microvalve 105 is used to control the connection and disconnection between the sample component 200 and the mixing pool 300, the sixth microvalve 106 and the first microvalve 101 are connected and disconnected through the valve of the sixth microvalve 106, the sixth microvalve 106 is connected to the freeze-dried reagent chamber 400, the freeze-dried reagent chamber 400 is connected to the reaction unit 500, the reaction unit 500 is connected to the air permeable component 700, the seventh microvalve 107 is connected to the plunger pump 8, the sample component 200 and the eighth microvalve 108, the seventh microvalve 107 is opened and closed to control the connection between it and the sample component 200, the eighth microvalve 108 is connected to the mixing pool 300 and the seventh microvalve 107, the eighth microvalve 108 is used to control the connection and disconnection between it and the seventh microvalve 107, the ninth microvalve 109 is used to cooperate with the air permeable component to make the air path unblocked when it is opened, and is used to seal the reaction unit when it is closed.

[0097] In the present invention, through the opening and closing coordination of multiple microvalves of the microvalve assembly 100, different liquids, such as lysis solution, binding solution, cleaning solution and elution solution, are released into the mixing pool 300 in sequence, thereby realizing the extraction of target nucleic acid from the sample. Finally, through the coordination of multiple microvalves of the microvalve assembly 100, the liquid carrying the target nucleic acid is transported from the mixing pool 300 through the freeze-drying reagent chamber 400 to the reaction unit 500 to complete the reaction.

[0098] In some embodiments of the present invention, each microvalve of the microvalve assembly 100 is composed of a valve seat, a communication structure and a sealing membrane, and the valve is switched by controlling the sealing membrane to block the communication structure.

[0099] The microvalve is mainly composed of a valve seat, a connecting structure, and a valve body sealing membrane; the valve seat is part of the substrate, and the connecting structure includes a through hole and a flow channel. This structure can be understood as a three-way valve, but unlike the common three-way valve, in order to achieve the switching of two flow paths, other microvalves are required to be used together.

[0100] In some embodiments of the present invention, a phase change material 600 is further included. The phase change material 600 is used to seal the freeze-dried reagent chamber 400 and the channel of the reaction unit 500 and the air outlet of the air permeable component 700 respectively through a process of heating, melting, flowing and re-solidifying and sealing, thereby achieving the sealing of the reaction unit 500.

[0101] In this embodiment, after the liquid carrying the target nucleic acid enters the reaction unit 500, an amplification reaction needs to be performed on the reaction unit 500. After the reaction is completed, the phase change material 600 can be heated, for example, it can be paraffin, and after the phase change material 600 melts, it flows into a preset channel, and the preset channel connects the freeze-dried reagent chamber 400 and the channel of the reaction unit 500 and the gas outlet of the gas permeable component 700. After the phase change material 600 solidifies again, a part of the solid phase change material 600 blocks the freeze-dried reagent chamber 400 and the channel of the reaction unit 500, that is, blocks the inlet of the reaction unit 500, and another part of the solid phase change material 600 blocks the outlet of the reaction unit 500, so as to achieve the sealing of the reaction unit 500 and prevent the risk of aerosol contamination.

[0102] Please refer to Figures 4 to 8 , in some embodiments of the present invention, further comprising a main body and a puncture device;

[0103] The main body includes an adjacent sealed cavity 1 and a limiting chamber 2, the sealed cavity 1 includes a first opening 11 and a second opening 12 whose opening directions are perpendicular to each other, the second opening 12 is connected to the limiting chamber 2, the first opening 11 and the second opening 12 are sealed with a sealing film, the puncture device includes a first puncture member 3 and a second puncture member 4, the first puncture member 3 faces the first opening 11, the second puncture member 4 penetrates into the limiting chamber 2, and a limiting inclined surface 21 is provided on the inner wall of the limiting chamber 2 relative to the second opening 12;

[0104] When the fluid in the sealed cavity 1 needs to be released, the puncture device is driven to move toward the main body so that the first puncture member 3 punctures the sealing membrane at the first opening 11, and the second puncture member 4 in the limiting chamber 2 is bent toward the second opening 12 under the restriction of the limiting slope 21 to puncture the sealing membrane at the second opening 12, so that the fluid flows into the liquid storage cavity at the lower part of the limiting chamber 2.

[0105] In this embodiment, the sealing chamber 1 further comprises a first opening 11 and a second opening 12, and a sealing film is disposed at each of the first opening 11 and the second opening 12 to seal and preserve the fluid in the sealing chamber 1. The limiting chamber 2 is disposed back to back with the sealing chamber 1, and the limiting chamber 2 and the sealing chamber 1 are connected through the second opening 12. The first piercing member of the puncture device is disposed above the first opening 11, and the second piercing member is disposed inside the limiting chamber 2. When the fluid needs to be released, the puncture device can be driven to move, and the first piercing member and the second piercing member move downward, and the first piercing member directly pierces the sealing film at the first opening 11 during the movement. At the same time, the second piercing member inside the limiting chamber 2 moves toward the position where the second opening 12 is located, and when the second piercing member approaches the second opening 12, it will contact the limiting inclined surface 21 inside the limiting chamber 2. The second piercing member continues to move, and the limiting inclined surface 21 forces the second piercing member to bend along its inclined surface, that is, bend toward the second opening 12, and then the second piercing member pierces the sealing film at the second opening 12. After the sealing films of the first opening 11 and the second opening 12 of the sealed cavity 1 are all punctured, the fluid in the sealed cavity 1 flows out through the second opening 12 into the limiting chamber 2 to release the fluid. The bottom of the limiting chamber 2 can be directly or indirectly connected to the fluid control unit of the microfluidic chip to control the fluid released from the sealed cavity 1.

[0106] In this embodiment, the sealing and storage function can be achieved only by the cooperation of the sealing cavity 1 with the first opening 11 and the second opening 12 and the sealing film. When the fluid needs to be released, it is only necessary to puncture the sealing film at the first opening 11 and the second opening 12. The entire storage and release mechanism has a simple structure and convenient release operation.

[0107] It should be noted that the fluid can flow out freely only after the first opening 11 and the second opening 12 of the sealed cavity 1 are punctured at the same time. If only the second opening 12 at the bottom is punctured, the fluid in the sealed cavity 1 will be hindered by the atmospheric pressure and will be difficult to flow out.

[0108] In some embodiments of the present invention, the second puncture member 4 includes a guide portion 41 and a puncture portion 42 connected in sequence, a guide groove 22 is provided in the limiting bin 2, the guide portion 41 passes through the guide groove 22, and the guide groove 22 is used to limit the second puncture member 4 to move along a preset direction.

[0109] In this embodiment, in order to enable the second piercing member 4 to move accurately to the second opening 12 of the sealing cavity 1, a guide groove 22 is provided in the limiting chamber 2, and the guide portion 41 of the second piercing member 4 moves along a preset direction under the action of the guide groove 22 so that the piercing portion 42 of the second piercing member 4 can accurately reach the second opening 12.

[0110] It should be noted that the second puncture member 4 is a part of the puncture device, that is, the cooperation between the guide groove 22 and the guide portion 41 can enable the entire puncture device to move along a preset direction.

[0111] In some embodiments of the present invention, a plate-shaped force-bearing plate 411 is provided at a position of the guide portion 41 close to the puncture portion 42 . The force-bearing plate 411 is used to cooperate with the limiting slope 21 to control the deformation direction of the puncture portion 42 to puncture the sealing film on the second opening 12 .

[0112] In this embodiment, the force-bearing plate 411 has a larger contact area with the limiting inclined surface 21 than other thinner parts of the guide portion 41 , which is more conducive to controlling its bending direction, ie, bending toward the second opening 12 .

[0113] Please refer to Figures 9 to 12 In some embodiments of the present invention, the puncture device further includes a buckle 5, a position is provided in the limit bin 2, and the buckle 5 and the position cooperate to limit the position of the puncture device;

[0114] The locking positions include a first locking position 23 and a second locking position 24. When the buckle 5 is located on the first locking position 23, the puncture device does not puncture the sealing film on the first opening 11 and the sealing film on the second opening 12. When the buckle 5 is located on the second locking position 24, the puncture device punctures the sealing film on the first opening 11 and the sealing film on the second opening 12.

[0115] In this embodiment, the puncture device is further provided with a buckle 5, which can cooperate with the clamping position in the limiting bin 2 to fix the puncture device at different positions.

[0116] In this embodiment, when the buckle 5 is opened in the first position 23, the puncture device is located in the upper part of the sealing cavity 1 and the limiting chamber 2, and the first opening 11 and the second opening 12 are not punctured. When it is necessary to puncture the sealing cavity 1, the puncture device is driven to move, and the buckle 5 is disengaged from the first position 23 and moves downward to the second position 24. During the movement, the first puncture member 3 and the second puncture member 4 puncture the sealing film at the first opening 11 and the sealing film at the second opening 12 respectively. After completing the puncture task, the buckle 5 is locked in the second position 24 so that the puncture device is still in a fixed state.

[0117] It is understandable that, in order to prevent the puncture device from being separated from the second clamping position 24, the second clamping position 24 can be set as an angular clamping position, and the buckle 5 is provided with a barb structure.

[0118] In some embodiments of the present invention, the puncture device also includes a force-driven member 82, which includes a mounting plate 6 and a driving surface 7 obliquely arranged on the mounting plate 6. The first puncture member 3 and the second puncture member 4 are respectively installed on the side of the mounting plate 6 away from the driving surface 7. A plunger pump 8 and a driving member 82 are arranged on one side of the driving surface 7. The driving member 82 is arranged on the reciprocating rod 81 of the plunger pump 8. The reciprocating rod 81 is parallel to the mounting plate 6 and moves along its axial direction through the reciprocating rod 81. The driving member 82, the driving surface 7, the guide portion 41 and the guide groove 22 on the reciprocating rod 81 cooperate to drive the puncture device to move in a preset direction; wherein, the plunger pump 8 is connected to the microfluidic chip to provide it with power for fluid control.

[0119] In this embodiment, the driving member 82 on the plunger pump 8 can not only drive the puncture device to move to puncture the first opening 11 and the second opening 12 of the sealed cavity 1, but the plunger pump 8 itself can also provide power for fluid control for the microfluidic chip.

[0120] In this embodiment, in order to achieve a precise pumping function, the reciprocating rod 81 may be a screw rod.

[0121] It should be noted that after the driving device drives the puncture device to perform the puncture operation, the puncture device is fixed in the puncture state due to the cooperation between the second clamping position 24 and the buckle 5. At this time, the reciprocating rod 81 of the plunger pump 8 can still reciprocate to provide power.

[0122] The embodiment of the present invention further provides a nucleic acid detection method, based on any of the above nucleic acid detection kits, the method comprising:

[0123] Step 1, close the first microvalve 101, the second microvalve 102, the third microvalve 103, the fourth microvalve 104, the fifth microvalve 105 and the seventh microvalve 107, and open the rest of the microvalve, and use the plunger pump 8 to discharge the gas in each channel;

[0124] Step 2, open the fourth microvalve 104, the fifth microvalve 105, the seventh microvalve 107 and the ninth microvalve 109, and close the rest, and use the plunger pump 8 to pump the lysate in the liquid storage chamber 9 connected to the fourth microvalve 104 and the sample in the sample assembly 200 into the mixing pool 300, the mixing pool 300 includes a heating device and a rotor, and the sample and the lysate in the mixing pool 300 are mixed and lysed by the heating device and the rotor to obtain the target nucleic acid;

[0125] Step 3, open the third microvalve 103, the fifth microvalve 105, the seventh microvalve 107 and the ninth microvalve 109, and close the rest, use the plunger pump 8 to pump the binding liquid in the liquid storage chamber 9 connected to the third microvalve 103 into the mixing pool 300, under the action of the binding liquid, the target nucleic acid is adsorbed on the magnetic beads in the mixing pool 300, open the fourth microvalve 104, the fifth microvalve 105 and the seventh microvalve 107, and close the rest, and use the plunger pump 8 to pump the waste liquid in the mixing pool 300 into the liquid storage chamber 9 connected to the fourth microvalve 104;

[0126] Step 4, open the second microvalve 102, the fifth microvalve 105, the seventh microvalve 107 and the ninth microvalve 109, and close the rest, use the plunger pump 8 to pump the cleaning liquid in the liquid storage chamber 9 connected to the second microvalve 102 into the mixing pool 300 to clean the residual reagents other than the target nucleic acid in the mixing pool 300, open the third microvalve 103, the fifth microvalve 105 and the seventh microvalve 107, and close the rest, and use the plunger pump 8 to pump the waste liquid in the mixing pool 300 into the liquid storage chamber 9 connected to the third microvalve 103;

[0127] Step 5, open the first microvalve 101, the fifth microvalve 105, the seventh microvalve 107 and the ninth microvalve 109, and close the rest, use the plunger pump 8 to pump the eluate in the liquid storage chamber 9 connected to the first microvalve 101 into the mixing pool 300 to clean the target nucleic acid on the magnetic beads in the mixing pool 300, open the sixth microvalve 106, the fifth microvalve 105, the seventh microvalve 107 and the ninth microvalve 109, and close the rest, use the plunger pump 8 to pump the eluate containing the target nucleic acid in the mixing pool 300 into the reaction unit 500 to complete the nucleic acid detection, and close the ninth microvalve 109 when the reaction unit 500 performs an amplification reaction.

[0128] Please refer to Fig.13 , step 1 is the preparation process, specifically, adding the sample to be tested into the sample assembly, and fastening the sealing plug to complete the sealing of the card box;

[0129] The external instrument rotates the reciprocating rod 81 and moves the driving member 82 to the right. At this time, some microvalves remain closed, and the driving member 82 pushes the puncture slider to release the liquid reagent in each sealed cavity 1; because the first to fourth microvalves are in the closed state, the above reagents will not contaminate the flow channel in advance;

[0130] At the same time, the plunger pump 8 pushes the generated gas, which passes through the waterproof breathable membrane and is discharged from the substrate in the direction shown in the figure;

[0131] Then the reciprocating rod 81 rotates in the reverse direction and moves to the left, and the plunger pump 8 completes the inflation and is ready to provide power for the subsequent reaction;

[0132] For the sake of brevity, the rotation direction of the reciprocating rod 81 will not be analyzed in detail below, and the plunger pump 8 will only be functionally described using "push air / exhale air" or similar terms.

[0133] Please refer to Fig.14 , step 2 is the lysis process, specifically, the plunger pump 8 pushes / extracts air to push the sample and the lysis solution into the mixing pool 300 according to the directions shown in the figure;

[0134] On the external instrument, at the corresponding position of the mixing tank 300, there are: a motor that can rotate the magnet to control the rotor that is also composed of magnetic elements; a heating unit. The above two components are not shown in the drawings;

[0135] Under the combined action of the heating unit and the rotor, the sample fully reacts with the lysis reagent to release the target nucleic acid to be tested from the cells / viruses.

[0136] Please refer to Fig.15 , step 3 is the combining process, specifically, the plunger pump 8 pumps air and sucks the combined liquid into the mixing tank 300 in the direction shown in the figure;

[0137] The motor of the rotating magnet drives the rotor to rotate, and in the presence of the binding solution, the released target nucleic acid is fully mixed with the magnetic beads, and finally the nucleic acid is adsorbed on the magnetic beads;

[0138] The magnet stops rotating, and under the action of the magnetic field, the magnetic beads are gradually adsorbed in the chamber.

[0139] The plunger pump 8 pushes air to push the liquid in the chamber to the chamber where the lysate was originally located. At this time, the chamber acts as a waste liquid pool.

[0140] Please refer to Fig.16 , step 4 is a cleaning process, specifically, the plunger pump 8 draws air and sucks the cleaning liquid into the mixing tank 300 in the direction shown in the figure;

[0141] The motor of the rotating magnet drives the rotor to rotate, and in the presence of the cleaning solution, the pool is fully stirred so that the magnetic beads and the cleaning solution are fully mixed, and the reagents remaining on the magnetic beads in the previous process are cleaned, such as samples, lysate, and binding solution;

[0142] The magnet stops rotating, and under the action of the magnetic field, the magnetic beads are gradually adsorbed in the chamber.

[0143] The plunger pump 8 pushes the air, pushing the liquid in the chamber to the chamber where it originally was, and the chamber now acts as a waste liquid pool;

[0144] According to the experimental requirements, the above steps can be performed once or repeated multiple times to finally complete the magnetic bead cleaning process.

[0145] Please refer to Fig.17, step 5 is the elution process, specifically, the plunger pump 8 draws air and sucks the elution into the mixing tank 300 in the direction shown in the figure;

[0146] The motor of the rotating magnet drives the rotor to rotate, and in the presence of the elution solution, the pool is fully stirred so that the magnetic beads and the elution solution are fully mixed, and the nucleic acid is eluted from the magnetic beads;

[0147] The magnet stops rotating, and under the action of the magnetic field, the magnetic beads are gradually adsorbed in the chamber.

[0148] The plunger pump 8 pushes air to push the liquid in the chamber to the reaction unit 500;

[0149] When the liquid is pushed to the reaction unit 500, the liquid will first flow through the freeze-dried reagent chamber 400 to re-dissolve the freeze-dried amplification reagent pre-stored therein;

[0150] Finally, each independent reaction unit 500 contains samples, amplification reagents, specific primers, fluorescent probes and other substances, and has the basic conditions for performing an amplification reaction.

[0151] Please refer to Fig.18 In some embodiments of the present invention, after the reaction is completed, the phase change material 600 is heated to melt, so that it flows to the channel connecting the freeze-dried reagent chamber 400 and the reaction unit 500, and flows to the channel connecting the air permeable component 700 and the outside world, and then the reaction unit 500 is physically sealed after cooling.

[0152] In this embodiment, when the reaction is carried out, the corresponding microvalve is closed, the reaction chamber is in a closed state, and the heating and optical collection modules of the external instrument start working to synchronously carry out the amplification reaction;

[0153] Generally speaking, PCR amplification reaction requires 40-45 cycles, and LAMP amplification reaction takes about 40 minutes. The instrument collects fluorescence signals once per cycle or per minute, and the final output is an amplification curve.

[0154] After the reaction is completed, the heating module of the external instrument is turned on to melt the paraffin particles in the phase change material 600 component, and the two ends of the reaction chamber are physically and permanently closed through the overflow structure, thereby avoiding the risk of aerosol contamination;

[0155] Each microvalves is opened, and the cartridge is removed from the instrument to complete the entire reaction.

[0156] Please refer to Fig.19 and Fig. 20 In some embodiments of the present invention, the side or bottom of the reaction unit 500 is transparent to allow the reaction unit 500 to receive external optical signals.

[0157] In this embodiment, detection can be performed from the transparent bottom and side surfaces.

[0158] In some embodiments of the present invention, the reaction unit 500 is provided with a heat-conducting surface, and the heat-conducting surface is used to contact with the heating control unit.

[0159] In some embodiments of the present invention, the heat-conducting surface is formed by a thin film with a thickness of less than 0.1 mm and a thermal conductivity of more than 1 W / mK.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid detection kit, characterized in that: It comprises a plurality of liquid storage chambers (9), a microvalve assembly (100), a sample assembly (200), a mixing pool (300), a freeze-dried reagent chamber (400), a reaction unit (500) and a plunger pump (8); The plurality of liquid storage chambers (9) are respectively filled with a lysate, a binding solution, a cleaning solution and an eluent. The microvalve assembly (100) includes a first microvalve (101), a second microvalve (102), a third microvalve (103), a fourth microvalve (104), a fifth microvalve (105), a sixth microvalve (106), a seventh microvalve (107), an eighth microvalve (108) and a ninth microvalve (109). The first microvalve (101) is connected to the liquid storage chamber (9) containing the eluent, the second microvalve (102) is connected to the liquid storage chamber (9) containing the cleaning solution, The first microvalve (101), the second microvalve (102), the third microvalve (103), the fourth microvalve (104) are connected to the liquid storage chamber (9) containing the lysate, the first microvalve (101), the second microvalve (102), the third microvalve (103), and the fourth microvalve (104) are connected in sequence, and the first microvalve (101), the second microvalve (102), the third microvalve (103), and the fourth microvalve (104) are connected to the liquid storage chamber (9) respectively connected thereto by opening and closing the valves, and the fifth microvalve (104) is connected to the liquid storage chamber (9) containing the lysate. The microvalve (105) is used to control the connection and disconnection between the sample component (200) and the mixing pool (300); the sixth microvalve (106) and the first microvalve (101) are connected and disconnected through the valve of the sixth microvalve (106); the sixth microvalve (106) and the freeze-dried reagent chamber (400) are connected; the freeze-dried reagent chamber (400) and the reaction unit (500) are connected; the reaction unit (500) and the air permeable component (700) are connected; the seventh microvalve (107) and the plunger pump (8 ), the sample component (200) is connected to the eighth microvalve (108), the seventh microvalve (107) is opened and closed to control the communication between it and the sample component (200), the eighth microvalve (108) is connected to the mixing tank (300) and the seventh microvalve (107), the eighth microvalve (108) is used to control the connection between it and the seventh microvalve (107), and the ninth microvalve (109) is used to cooperate with the air permeable component to make the air path unobstructed when it is opened, and is used to seal the reaction unit (500) when it is closed.

2. The nucleic acid detection kit according to claim 1, characterized in that: Each microvalve of the microvalve assembly (100) is composed of a valve seat, a communication structure and a sealing film, and the valve is switched on and off by controlling the sealing film to block the communication structure.

3. The nucleic acid detection kit according to claim 1, characterized in that: It also includes a phase change material (600), which is used to seal the freeze-dried reagent chamber (400) and the channel of the reaction unit (500) and the air outlet of the air permeable component (700) respectively through a process of heating, melting, flowing and re-solidifying and sealing, thereby achieving sealing of the reaction unit (500).

4. The nucleic acid detection kit according to claim 1, characterized in that: Also included is a main body and a piercing device; The main body comprises adjacent sealed cavities (1) and limiting chambers (2); the sealed cavities (1) comprise a first opening (11) and a second opening (12) whose opening directions are perpendicular to each other; the second opening (12) is in communication with the limiting chamber (2); the first opening (11) and the second opening (12) are sealed with a sealing film; the puncturing device comprises a first puncturing member (3) and a second puncturing member (4); the first puncturing member (3) faces the first opening (11); the second puncturing member (4) penetrates into the limiting chamber (2); a limiting inclined surface (21) is provided on the inner wall of the limiting chamber (2) relative to the second opening (12); When it is necessary to release the fluid in the sealed cavity (1), the puncture device is driven to move toward the main body so that the first puncture member (3) punctures the sealing membrane at the first opening (11), and the second puncture member (4) in the limiting chamber (2) is bent toward the second opening (12) under the restriction of the limiting slope (21) to puncture the sealing membrane at the second opening (12), so that the fluid flows into the liquid storage cavity at the lower part of the limiting chamber (2).

5. The nucleic acid detection kit according to claim 4, characterized in that: The second piercing member (4) comprises a guide portion (41) and a piercing portion (42) which are connected in sequence, a guide groove (22) is provided in the limiting chamber (2), the guide portion (41) passes through the guide groove (22), and the guide groove (22) is used to limit the movement of the second piercing member (4) along a preset direction.

6. The nucleic acid detection kit according to claim 5, characterized in that: A plate-shaped force-bearing plate (411) is provided at a portion of the guide portion (41) close to the puncture portion (42); the force-bearing plate (411) is used to cooperate with the limiting inclined surface (21) to control the direction in which the puncture portion (42) is deformed so as to puncture the sealing membrane on the second opening (12).

7. The nucleic acid detection kit according to claim 4, characterized in that: The puncture device further comprises a buckle (5), a buckle position is provided in the limit bin (2), and the buckle (5) cooperates with the buckle position to limit the position of the puncture device; The locking position comprises a first locking position (23) and a second locking position (24); when the buckle (5) is located at the first locking position (23), the puncturing device does not puncture the sealing film on the first opening (11) and the sealing film on the second opening (12); when the buckle (5) is located at the second locking position (24), the puncturing device punctures the sealing film on the first opening (11) and the sealing film on the second opening (12).

8. The nucleic acid detection kit according to claim 5, characterized in that: The puncture device further comprises a force-driven member (82), the force-driven member (82) comprising a mounting plate (6) and a driving surface (7) obliquely arranged on the mounting plate (6); the first puncture member (3) and the second puncture member (4) are respectively installed on the side of the mounting plate (6) away from the driving surface (7); a plunger pump (8) and a driving member (82) are arranged on one side of the driving surface (7); the driving member (82) is arranged on a reciprocating rod (81) of the plunger pump (8); the reciprocating rod (81) is parallel to the mounting plate (6) and moves along its axial direction through the reciprocating rod (81); the driving member (82), the driving surface (7), the guide portion (41) and the guide groove (22) on the reciprocating rod (81) cooperate to drive the puncture device to move along the preset direction; wherein the plunger pump (8) is connected to the microfluidic chip to provide it with power for fluid control.

9. A nucleic acid detection method, characterized in that: Based on the nucleic acid detection kit according to any one of claims 1 to 8, the method comprises: The first microvalve (101), the second microvalve (102), the third microvalve (103), the fourth microvalve (104), the fifth microvalve (105) and the seventh microvalve (107) are closed, and the remaining microvalve are opened, and the gas in each channel is discharged by using the plunger pump (8); The fourth microvalve (104), the fifth microvalve (105), the seventh microvalve (107) and the ninth microvalve (109) are opened, and the rest are closed, and the lysate in the liquid storage chamber (9) connected to the fourth microvalve (104) and the sample in the sample assembly (200) are pumped into the mixing pool (300) by the plunger pump (8), wherein the mixing pool (300) includes a heating device and a rotor, and the sample and the lysate in the mixing pool (300) are mixed and lysed by the heating device and the rotor to obtain the target nucleic acid; The third microvalve (103), the fifth microvalve (105), the seventh microvalve (107) and the ninth microvalve (109) are opened, and the rest are closed; the binding liquid in the liquid storage chamber (9) connected to the third microvalve (103) is pumped into the mixing pool (300) by using the plunger pump (8); under the action of the binding liquid, the target nucleic acid is adsorbed on the magnetic beads in the mixing pool (300); the fourth microvalve (104), the fifth microvalve (105) and the seventh microvalve (107) are opened, and the rest are closed; the waste liquid in the mixing pool (300) is pumped into the liquid storage chamber (9) connected to the fourth microvalve (104) by using the plunger pump (8); The second microvalve (102), the fifth microvalve (105), the seventh microvalve (107) and the ninth microvalve (109) are opened, and the rest are closed; the cleaning liquid in the liquid storage chamber (9) connected to the second microvalve (102) is pumped into the mixing pool (300) by using the plunger pump (8) to clean the residual reagents other than the target nucleic acid in the mixing pool (300); the third microvalve (103), the fifth microvalve (105) and the seventh microvalve (107) are opened, and the rest are closed; the waste liquid in the mixing pool (300) is pumped into the liquid storage chamber (9) connected to the third microvalve (103) by using the plunger pump (8); The first microvalve (101), the fifth microvalve (105), the seventh microvalve (107) and the ninth microvalve (109) are opened, and the rest are closed. The eluent in the liquid storage chamber (9) connected to the first microvalve (101) is pumped into the mixing pool (300) by using the plunger pump (8) to clean the target nucleic acid on the magnetic beads in the mixing pool (300). The sixth microvalve (106), the fifth microvalve (105), the seventh microvalve (107) and the ninth microvalve (109) are opened, and the rest are closed. The eluent containing the target nucleic acid in the mixing pool (300) is pumped into the reaction unit (500) by using the plunger pump (8) to complete the nucleic acid detection. When the reaction unit (500) performs an amplification reaction, the ninth microvalve (109) is closed.

10. The nucleic acid detection method according to claim 9, characterized in that: After the reaction is completed, the phase change material (600) is heated until it melts, allowing it to flow to the channel connecting the freeze-drying reagent chamber (400) and the reaction unit (500), and to the channel connecting the air permeable component (700) and the outside world, and then the reaction unit (500) is physically sealed after cooling.

11. The nucleic acid detection method according to claim 9, characterized in that: The side surface or bottom surface of the reaction unit (500) is transparent to allow the interior of the reaction unit (500) to receive external optical signals.

12. The nucleic acid detection method according to claim 9, characterized in that: The reaction unit (500) is provided with a heat-conducting surface, and the heat-conducting surface is used for contacting with a heating control unit.

13. The nucleic acid detection method according to claim 12, characterized in that: The heat-conducting surface is composed of a film with a thickness of less than 0.1 mm and a thermal conductivity of more than 1 W / mK.

Images