Reagent cartridge and nucleic acid extraction, purification and amplification integrated method

Abstract

The application discloses a reagent card box and a nucleic acid extraction, purification and amplification integrated method. The reagent card box is provided with a center pool, a plurality of reagent cavities, a plurality of liquid storage pools, a plurality of ventilation channels and a plurality of first liquid channels. By controlling the air source to apply positive pressure through the ventilation hole, the liquid in the reagent cavity or the liquid storage pool sequentially passes through the corresponding first liquid channel, the rotating assembly and the membrane placement groove into the center pool. By controlling the air source to apply negative pressure through the ventilation hole, the liquid in the center pool sequentially passes through the membrane placement groove, the rotating assembly and the first liquid channel to return to the reagent cavity or the liquid storage pool, and the silica gel membrane is contacted twice in each step in the forward and reverse directions. The application can integrate a plurality of steps required for detection and analysis on a chip to complete sample pretreatment, manual sample addition, reagent mixing, optical detection and other complex operations, and can be used one time, has low cost and reduces false positive results and infection risks.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular diagnostic technology, in particular to a reagent cartridge and a nucleic acid extraction, purification and amplification integrated method. BACKGROUND

[0002] Molecular diagnostic technology refers to a technology that uses nucleic acid as diagnostic material, detects the presence, defects or abnormal expression of genes by using molecular biology techniques, and makes diagnosis on human body state and diseases. The basic principle is to detect whether the structure of nucleic acid changes, the amount and whether the expression function is abnormal, so as to determine whether the abnormal changes of the subject at the gene level. For nucleic acid detection of samples, it is generally divided into three steps of nucleic acid extraction, nucleic acid amplification and nucleic acid detection. The mainstream nucleic acid diagnostic equipment on the market is mostly to complete each step of molecular diagnosis independently. Each step needs to be completed by independent equipment. Multiple equipment is needed in a nucleic acid detection process, and the equipment occupies a large space. After the previous steps are completed, the sample needs to be moved to the subsequent equipment, which is complicated and time-consuming, and requires a higher environment and personnel. At the same time, the non-integrated reagent product is easy to be contaminated by the external environment or contaminate the detection environment during the switching from the previous step to the subsequent step. SUMMARY

[0003] It is necessary to provide a reagent cartridge. The reagent cartridge of the present application integrates the column membrane method nucleic acid extraction into a cartridge type microfluidic chip. The cartridge design is compact, small in size and easy to operate. The nucleic acid extraction and purification can be completed automatically in a short time, and the nucleic acid extraction and purification efficiency is high.

[0004] At least one embodiment of the present application provides a reagent cartridge.

[0005] A reagent cartridge is provided, which is provided with a center pool, a plurality of reagent cavities, a plurality of liquid storage pools, a plurality of ventilation channels and a plurality of first liquid channels. Each of the reagent cavities and each of the liquid storage pools is respectively connected to one end of one of the first liquid channels. Each of the reagent cavities and each of the liquid storage pools is respectively connected to one end of one of the ventilation channels.

[0006] The reagent cartridge is further provided with a rotating assembly, which has a rotating channel. The rotating channel can be connected to the other end of the first liquid channel by rotating the rotating assembly. A nucleic acid adsorption silica gel membrane is arranged in the path between the center pool and the rotating assembly. The rotating channel can also be connected to an amplification chip. The center pool can be connected to a negative pressure source. The liquid in the reagent cavities and the liquid in the liquid storage pools can pass through the nucleic acid adsorption silica gel membrane and enter the center pool. The liquid in the center pool can also flow back. The nucleic acid adsorption silica gel membrane is used to adsorb the nucleic acid in the lysis solution.

[0007] In some embodiments, the reagent cartridge is provided with a sample adding hole, a ventilation hole and an air outlet hole;

[0008] The reagent cartridge is further provided with a gas buffer pool, each of the reagent cavities and each of the liquid storage pools is communicated with one end of the ventilation channel, the other end of the ventilation channel is communicated with the air outlet hole, and the ventilation hole is communicated with the central pool;

[0009] The reagent cartridge is further provided with a valve placing groove, a membrane placing groove and a chip inserting groove, the valve placing groove is provided with a rotating assembly, the rotating assembly has a rotating channel, the rotating channel can be communicated with the other end of the first liquid channel by rotating the rotating assembly, the membrane placing groove is provided with a nucleic acid adsorption silica gel membrane, the membrane placing groove is communicated with the central pool, the membrane placing groove is also communicated with the valve placing groove, and the chip inserting groove is used for inserting an amplification chip.

[0010] In some embodiments, the reagent cartridge further comprises an amplification chip, the amplification chip can be detachably inserted into the chip inserting groove and communicated with the rotating channel for amplifying nucleic acid.

[0011] In some embodiments, the reagent cartridge is further provided with an aerosol adsorption column storage cavity, the aerosol adsorption column storage cavity is used for placing an aerosol adsorption column, and the ventilation hole is communicated with the central pool through the aerosol adsorption column storage cavity;

[0012] The reagent cartridge is further provided with a gas buffer pool, the other end of the ventilation channel is communicated with the gas buffer pool, and the gas buffer pool is communicated with the air outlet hole.

[0013] In some embodiments, the reagent cartridge comprises an upper cover body, a nucleic acid extraction chip, a lower cover body and an amplification chip, the upper cover body, the nucleic acid extraction chip and the lower cover body are sequentially overlapped and connected from top to bottom;

[0014] The upper cover body is provided with the sample adding hole, the ventilation hole, the air outlet hole and a plurality of ventilation channels;

[0015] The nucleic acid extraction chip is provided with the central pool, the gas buffer pool, the aerosol adsorption column storage cavity, a plurality of reagent cavities, a plurality of liquid storage pools and a plurality of first liquid channels;

[0016] The lower cover body is provided with the valve placing groove, the membrane placing groove and the chip inserting groove.

[0017] In some embodiments, the upper cover body comprises an upper cover plate and a first single-sided film, the upper cover plate has a cover plate first surface connected with the first single-sided film and a cover plate second surface connected with the nucleic acid extraction chip, a plurality of ventilation grooves are arranged on the cover plate first surface, the groove bottoms of both ends of the plurality of ventilation grooves respectively penetrate the upper cover plate, and the first single-sided film covers the cover plate first surface and seals each ventilation groove to form a plurality of ventilation channels.

[0018] In some embodiments, the cover plate first surface is further provided with a first communication groove, the first single-sided film covers the cover plate first surface and seals the first communication groove to form a first communication channel, one end of the first communication channel extends to communicate with the aerosol adsorption column storage cavity, and the other end extends to communicate with the central pool.

[0019] In some embodiments, the number of aerosol adsorption column storage cavities is a plurality, the plurality of aerosol adsorption column storage cavities are connected in series, and the plurality of aerosol adsorption column storage cavities are connected in series in a meandering manner, wherein the first aerosol adsorption column storage cavity communicates with the ventilation hole, and the last aerosol adsorption column storage cavity communicates with the first communication channel.

[0020] In some embodiments, the nucleic acid extraction chip has a chip first surface matched with the upper cover body and a chip second surface matched with the lower cover body, the chip second surface is provided with a plurality of first liquid channels, the chip first surface is provided with the central pool, the gas buffer pool, the aerosol adsorption column storage cavity, a plurality of reagent cavities, and a plurality of liquid storage pools, the bottom surface of the aerosol adsorption column storage cavity, the bottom surface of the plurality of reagent cavities, and the bottom surface of the plurality of liquid storage pools all penetrate the chip second surface, the bottom surface of the plurality of reagent cavities and the bottom surface of the plurality of liquid storage pools respectively communicate with one end of one of the first liquid channels after penetrating the chip second surface, the other end of the first liquid channel extends to a first preset position and communicates with the valve placement groove, and the first preset position corresponds to the valve placement groove.

[0021] In some embodiments, the chip second surface is further provided with a second liquid channel, one end of the second liquid channel extends to the first preset position and communicates with the valve placement groove, and the other end of the second liquid channel communicates with the membrane placement groove.

[0022] In some embodiments, the gas buffer pool, the aerosol adsorption column storage cavity, the reagent cavity, and the liquid storage pool all have a funnel structure.

[0023] In some embodiments, the lower cover body has a lower base plate and a second single-sided film, the lower base plate has a base plate first surface matched with the nucleic acid extraction chip and a base plate second surface sealed with the second single-sided film, the lower base plate has the valve placement slot and the film placement slot penetrating through the base plate first surface and the base plate second surface, the base plate second surface has the chip insertion slot, and the base plate first surface is provided with a communication hole communicating with the chip insertion slot, and the chip insertion slot communicates with one of the liquid storage pools through the communication hole.

[0024] In some embodiments, the base plate second surface is further provided with a third liquid channel, one end of the third liquid channel communicates with the film placement slot on the base plate second surface, and the other end of the third liquid channel extends to the base plate first surface and communicates with the valve placement slot.

[0025] In some embodiments, the lower cover body further has a sealing gasket connected to the base plate first surface, the sealing gasket is used to seal the base plate first surface and the nucleic acid extraction chip, the sealing gasket is provided with a central hole communicating with the third liquid channel, a first clearance hole communicating with the film placement slot, and a second clearance hole communicating with the valve placement slot, the central pool communicates with the film placement slot through the first clearance hole, and the valve placement slot communicates with the end of the plurality of first liquid channels through the second clearance hole.

[0026] In some embodiments, the sealing gasket is further provided with a third clearance hole, a plurality of fourth clearance holes, and a plurality of fifth clearance holes, the third clearance hole communicates with the aerosol adsorption column storage cavity, the plurality of fourth clearance holes respectively communicate with the ends of the plurality of first liquid channels one by one, and the plurality of fifth clearance holes respectively communicate with the plurality of liquid storage pools one by one.

[0027] In some embodiments, the rotating assembly has a valve first surface facing the nucleic acid extraction chip and a valve second surface facing the valve placement slot, and the rotating assembly has the rotating channel penetrating through the valve first surface and the valve second surface.

[0028] In some embodiments, the reagent card box further comprises at least one of the following technical features:

[0029] The valve second surface is provided with an alignment station, and correspondingly, the inner wall of the valve placement slot has a plurality of rotation alignment mark grooves matched with the alignment station;

[0030] The valve second surface is further provided with an interface, and the interface is used to match with an external driving motor.

[0031] In some embodiments, a sealing ring is further connected to the rotating assembly, and the sealing ring is located on the second surface of the valve and is used for sealing cooperation between the second surface of the valve and the bottom surface of the valve placing groove.

[0032] In some embodiments, the amplification chip comprises a chip body, an upper cover film, a lower cover film, and a waterproof and breathable film. The chip body is provided with a chip flow channel and an amplification detection cavity. One end of the chip flow channel is communicated with the rotating channel, and the other end is communicated with the amplification detection cavity. The upper cover film is sealingly connected to the upper surface of the chip body. The lower cover film is sealingly connected to the lower surface of the chip body. The waterproof and breathable film covers the amplification detection cavity.

[0033] In some embodiments, the amplification chip further comprises a matching clamping member connected to the upper surface of the chip body. The matching clamping member can be embedded and matched in the chip insertion slot. The matching clamping member is provided with a clamping member through hole communicated with the rotating channel, and the clamping member through hole is further communicated with the chip flow channel.

[0034] In some embodiments, the number of amplification detection cavities is multiple, and multiple amplification detection cavities are connected in parallel to the chip flow channel.

[0035] In some embodiments, the amplification chip further comprises an annular protruding member connected to the upper surface of the chip body. Multiple amplification detection cavities are located in the annular protruding member, and the waterproof and breathable film is sealingly connected to the annular protruding member.

[0036] In some embodiments, the reagent cartridge further comprises an aerosol adsorption column, and the aerosol adsorption column is arranged in the storage cavity of the aerosol adsorption column.

[0037] Another embodiment of the present application further provides a nucleic acid extraction, purification and amplification integrated method.

[0038] A nucleic acid extraction, purification and amplification integrated method comprises the following steps:

[0039] The reagent cartridge comprises a plurality of reagent cavities and a plurality of liquid storage pools. The reagent cavities are respectively filled with lysis solution, washing solution and elution solution.

[0040] The rotating assembly is rotated to be communicated with the reagent cavity filled with the lysis solution. Negative pressure is applied through the air hole, and the lysis solution is adsorbed to the central pool through the silica gel membrane in the membrane placing groove. The silica gel membrane can adsorb nucleic acid in the lysis solution. Positive pressure is applied through the air hole, so that the liquid in the central pool flows back.

[0041] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with a reagent cavity containing a washing solution, applying negative pressure through the air hole to adsorb the washing solution on the silica gel membrane in the membrane placement groove to the center pool to wash the silica gel membrane;

[0042] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with a reagent cavity containing a washing solution, applying negative pressure through the air hole to adsorb the washing solution on the silica gel membrane in the membrane placement groove to the center pool to wash the silica gel membrane;

[0043] rotating the rotating assembly to be communicated with an amplification chip, applying positive pressure through the air hole to transfer the elution solution in the center pool to the amplification chip; and

[0044] controlling the amplification chip to complete nucleic acid amplification and detection.

[0045] Another embodiment of the present application also provides a nucleic acid extraction, purification and amplification integrated method.

[0046] A nucleic acid extraction, purification and amplification integrated method, comprising the following steps: 600 μL of a lysis solution, 500 μL of a washing solution I, 700 μL of a washing solution II, 700 μL of a washing solution III and 200 μL of an elution solution are respectively packaged in a first reagent cavity, a second reagent cavity, a third reagent cavity, a fourth reagent cavity and a first liquid pool on a nucleic acid extraction chip;

[0047] different reaction reagents are respectively packaged in a plurality of amplification and detection cavities of an amplification chip to realize multiple detection of nucleic acids;

[0048] a sample to be detected is added into the reagent cavity containing the lysis solution through a sample adding hole to form a lysis solution;

[0049] rotating the rotating assembly to be communicated with a first liquid channel connected with the first reagent cavity, controlling external air pressure to apply negative pressure through the air hole to adsorb the lysis solution on the silica gel membrane in the membrane placement groove to the center pool, the silica gel membrane being capable of adsorbing nucleic acids in the lysis solution; controlling external air pressure to apply positive pressure through the air hole to transfer the liquid in the center pool to the first reagent cavity;

[0050] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with a reagent cavity containing a washing solution, applying negative pressure through the air hole to adsorb the washing solution on the silica gel membrane in the membrane placement groove to the center pool to wash the silica gel membrane;

[0051] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with the first liquid channel connected with the third reagent cavity, controlling the external air pressure to apply negative pressure through the air hole, adsorbing the cleaning liquid II through the silica gel membrane in the membrane placing groove to the center pool to clean the silica gel membrane; controlling the external air pressure to apply positive pressure through the air hole, transferring the liquid in the center pool to the third reagent cavity;

[0052] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with the first liquid channel connected with the fourth reagent cavity, controlling the external air pressure to apply negative pressure through the air hole, adsorbing the cleaning liquid III through the silica gel membrane in the membrane placing groove to the center pool to clean the silica gel membrane; controlling the external air pressure to apply positive pressure through the air hole, transferring the liquid in the center pool to the fourth reagent cavity;

[0053] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with the first liquid channel connected with the second liquid storage pool, controlling the external air pressure to apply positive pressure through the air hole to dry the silica gel membrane;

[0054] controlling the positive pressure to stop and rotating the rotating assembly to be communicated with the first liquid channel connected with the first liquid storage pool, controlling the external air pressure to apply negative pressure through the air hole, adsorbing the elution liquid through the silica gel membrane in the membrane placing groove to the center pool to elute the silica gel membrane, stopping the negative pressure to keep still, so that the silica gel membrane is fully contacted with the elution liquid, and the nucleic acid is fully eluted for 5-10 min;

[0055] rotating the rotating assembly to be communicated with the amplification chip, controlling the external air pressure to apply positive pressure through the air hole, transferring the elution liquid in the center pool to the amplification chip; and

[0056] controlling the amplification chip to complete nucleic acid amplification and detection under the cooperation and action of the external instrument heating module and the fluorescence detection module.

[0057] In some embodiments, the positive pressure is 0-20 kPa, and the negative pressure is 0-10 kPa.

[0058] The reagent card kit can quickly, sensitively and accurately detect pathogenic microorganisms on site, and is crucial for preventing infectious diseases and controlling disease outbreaks. The reagent card kit can integrate various steps required for detection and analysis on a chip, realize complex operations such as sample pretreatment, manual sample addition, reagent mixing and optical detection, and can provide a closed environment required for diagnosis, can be made into a disposable chip, has low cost, reduces false positive results and infection risks caused by repeated use. Compared with the traditional detection method, the on-site detection of the present application has the following advantages: faster detection speed, sensitivity and specificity comparable to conventional methods, lower cost, higher efficiency, rapid detection at any location, and non-professionals can complete the operation in a short time.

[0059] In summary, compared with the prior art, the reagent card kit of the present application has the following beneficial effects:

[0060] 1. The column membrane method nucleic acid extraction is integrated into a card box type microfluidic chip, the card box is compact in design, small in size and few in operation steps, and can complete automatic nucleic acid extraction and purification in a short time.

[0061] 2. The nucleic acid detection reagent is integrated into a card box type microfluidic chip, which reduces the errors and pollution caused by manual operation on nucleic acid detection, and reduces false positives and inaccurate detection results.

[0062] 3. The present application realizes the whole detection process of nucleic acid extraction, purification, amplification and detection in a closed system, avoiding the aerosol pollution caused by nucleic acid amplification and the possible false positive results caused by external aerosol;

[0063] 4. The present application can realize multiple nucleic acid detection in a chip system, improve the nucleic acid detection throughput, and reduce the sample amount required for detection.

[0064] 5. The present application realizes automatic nucleic acid detection with external control instrument, reduces personnel time and uncertainty, improves the accuracy and stability of nucleic acid detection, and can avoid reagent pollution, environmental pollution and other problems in the nucleic acid detection process. BRIEF DESCRIPTION OF DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0066] For a more complete understanding of the present application and the advantages thereof, reference is now made to the following descriptions taken in connection with the accompanying drawings in which like parts are marked with like numbers throughout the figures.

[0067] Figure 1 The schematic diagram of the reagent cartridge according to an embodiment of the present application;

[0068] Figure 2 The schematic diagram of the upper cover of the reagent cartridge according to an embodiment of the present application;

[0069] Figure 3 The schematic diagram of the upper cover plate of the upper cover of the reagent cartridge according to an embodiment of the present application;

[0070] Figure 4 The schematic diagram of the nucleic acid extraction chip of the reagent cartridge according to an embodiment of the present application;

[0071] Figure 5 The schematic diagram of the first surface of the nucleic acid extraction chip of the reagent cartridge according to an embodiment of the present application;

[0072] Figure 6 The schematic diagram of the second surface of the nucleic acid extraction chip of the reagent cartridge according to an embodiment of the present application;

[0073] Figure 7 The schematic diagram of the lower cover of the reagent cartridge according to an embodiment of the present application;

[0074] Figure 8 The schematic diagram of the lower bottom plate of the reagent cartridge according to an embodiment of the present application;

[0075] Figure 9 The schematic diagram of the first surface of the lower bottom plate of the reagent cartridge according to an embodiment of the present application;

[0076] Figure 10 The schematic diagram of the second surface of the lower bottom plate of the reagent cartridge according to an embodiment of the present application;

[0077] Figure 11 The schematic diagram of the rotating assembly of the reagent cartridge according to an embodiment of the present application;

[0078] Figure 12 The schematic diagram of the second surface of the valve of the rotating assembly of the reagent cartridge according to an embodiment of the present application;

[0079] Figure 13 The exploded view of the amplification chip of the reagent cartridge according to an embodiment of the present application;

[0080] Figure 14 The schematic diagram of the chip main body of the amplification chip of the reagent cartridge according to an embodiment of the present application;

[0081] Figure 15 A schematic diagram of a chip main body of an amplification chip of a reagent cartridge according to an embodiment of the present application;

[0082] Figure 16 A schematic diagram of a reagent cartridge according to an embodiment of the present application after assembly.

[0083] Explanation of reference numerals

[0084] 10. Reagent cartridge

[0085] 1. Upper cover body; 101, first single-sided film; 102, upper cover plate; 102a, first surface of cover plate; 102b, second surface of cover plate; 1021, 1022, 1023, 1024, 1025, 1026, 1027, air passage; 1028, air outlet hole; 10211, air hole; 10212, sample addition hole; 10213, first communication passage;

[0086] 2. Nucleic acid extraction chip; 201a, first surface of chip; 201b, second surface of chip; 2011, central pool; 2012, 2013, 2014, 2015, reagent cavity; 2016, 2017, 2018, storage pool; 20110, gas buffer pool; 20111, aerosol adsorption column storage cavity; 20112, 20113, 20114, 20115, 20116, 20117, 20118, first liquid passage; 20119, second liquid passage; 202, aerosol adsorption column;

[0087] 3. Lower cover body; 301, lower base plate; 301a, first surface of base plate; 301b, second surface of base plate; 3011, valve placement slot; 3013, communication hole; 3014, flow passage through hole; 3015, film placement slot; 3017, third liquid passage; 3018, chip insertion slot; 3019, 30110, 30111, 30112, 30113, 30114, 30115, rotation alignment mark slot; 302, gasket; 3021, first clearance hole; 30211, central hole; 30212, 30213, 30214, 30215, 30216, 30217, 30218, second clearance hole; 3023, third clearance hole; 3024, 3025, 3026, 3027, fourth clearance hole; 3028, 3029, 30210, fifth clearance hole; 303, second single-sided film; 304, silica gel film; 305, rotation assembly; 305a, first surface of valve; 305b, second surface of valve; 3051, rotation passage; 3052, interface piece; 306, gasket;

[0088] 4, amplification chip; 401, chip main body; 4011, chip through hole; 4012, matching clamping piece; 4013, clamping piece through hole; 4014, amplification detection cavity; 4015, annular protruding part; 4016, chip flow channel; 402, lower film; 403, upper film; 404, waterproof and breathable film. DETAILED DESCRIPTION

[0089] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a variety of ways beyond the specific embodiments described herein, and skilled persons in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0090] In the description of the present application, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0091] In the present application, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection", "fixing" and the like should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrated; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0092] In the present application, unless otherwise explicitly specified and limited, the first feature is "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "over" and "on" the second feature can be that the first feature is directly above or obliquely above the second feature, or only means that the first feature is higher in horizontal height than the second feature. The first feature "below", "under" and "under" the second feature can be that the first feature is directly below or obliquely below the second feature, or only means that the first feature is lower in horizontal height than the second feature. In the description of the present application, if the meaning of several is more than one, the meaning of multiple is more than two, greater than, less than, more than, etc. Understand as not including the number, above, below, within, etc. Understand as including the number.

[0093] 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 the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0094] The embodiment of the present application provides a reagent cartridge 10 to solve the problem that the mainstream nucleic acid diagnostic equipment on the market is to separate and complete each step of molecular diagnosis independently, each step needs to be completed by independent equipment, a plurality of equipment is needed in a nucleic acid detection process, the equipment occupies a large space; the sample needs to be moved to the subsequent equipment after the previous step is completed, the operation is cumbersome, time-consuming is long, the environment and personnel requirements are high; meanwhile, the non-integrated reagent product is prone to be contaminated by the external environment or contaminate the detection environment during the switching from the previous step to the subsequent step. The reagent cartridge 10 will be described below in combination with the drawings.

[0095] The reagent cartridge 10 provided by the embodiment of the present application is exemplarily shown in Figure 1 and Figure 16 The reagent cartridge 10 provided by the embodiment of the present application is exemplarily shown in Figure 1 The structural schematic diagram of the reagent cartridge 10 provided by the embodiment of the present application. The reagent cartridge 10 of the present application can be used for all detection processes such as nucleic acid extraction, purification, amplification and detection.

[0096] In order to more clearly illustrate the structure of the reagent cartridge 10, the reagent cartridge 10 will be introduced below in combination with the drawings.

[0097] The reagent cartridge 10 provided by the embodiment of the present application is exemplarily shown in Figure 1 and Figure 1 The structural schematic diagram of the reagent cartridge 10 provided by the embodiment of the present application.

[0098] At least one embodiment of the present application provides a reagent cartridge 10.

[0099] A reagent cartridge 10, the reagent cartridge 10 is provided with a sample adding hole 10212, a ventilation hole 10211, an air outlet hole 1028 and a plurality of ventilation channels; the reagent cartridge 10 is also provided with a center pool 2011, a gas buffer pool 20110, a plurality of reagent cavities, a plurality of liquid storage pools and a plurality of first liquid channels, each reagent cavity and each liquid storage pool is respectively communicated with one end of a first liquid channel, each reagent cavity and each liquid storage pool is respectively communicated with one end of a ventilation channel, the other end of the ventilation channel is communicated with the air outlet hole 1028, the ventilation hole 10211 is communicated with the center pool 2011; the reagent cartridge 10 is also provided with a valve placing groove 3011, a membrane placing groove 3015 and a chip inserting groove 3018, the valve placing groove 3011 is provided with a rotating assembly 305, the rotating assembly 305 has a rotating channel 3051, the rotating channel 3051 can be communicated with the other end of the different first liquid channels with the rotating assembly 305 rotating, the membrane placing groove 3015 is provided with a nucleic acid adsorption silica gel membrane 304, the membrane placing groove 3015 is communicated with the center pool 2011, the membrane placing groove 3015 is also communicated with the valve placing groove 3011, the chip inserting groove 3018 is used for inserting the amplification chip 4.

[0100] In some embodiments, the rotating assembly 305 can be a rotating valve structure.

[0101] In some embodiments, the reagent cartridge 10 further comprises an amplification chip 4, the amplification chip 4 can be detachably inserted into the chip inserting groove 3018 and can be communicated with the rotating channel 3051 for amplifying nucleic acid.

[0102] In some embodiments, the reagent cartridge 10 is also provided with an aerosol adsorption column storage cavity 20111, the aerosol adsorption column storage cavity 20111 is used for placing the aerosol adsorption column 202, the ventilation hole 10211 is communicated with the center pool 2011 through the aerosol adsorption column storage cavity 20111; the reagent cartridge 10 is also provided with a gas buffer pool 20110, the other end of the ventilation channel is communicated with the gas buffer pool 20110, the gas buffer pool 20110 is communicated with the air outlet hole 1028.

[0103] In one specific example, the reagent cartridge 10 comprises an upper cover body 1, a nucleic acid extraction chip 2, a lower cover body 3 and an amplification chip 4. The upper cover body 1, the nucleic acid extraction chip 2 and the lower cover body 3 are connected in sequence from top to bottom.

[0104] At this time, the upper cover body 1 is provided with a sample adding hole 10212, a ventilation hole 10211, an air outlet hole 1028 and a plurality of ventilation channels (1021, 1022, 1023, 1024, 1025, 1026, 1027).

[0105] The nucleic acid extraction chip 2 is provided with a center pool 2011, a gas buffer pool 20110, an aerosol adsorption column storage cavity 20111, a plurality of reagent cavities (2012, 2013, 2014, 2015), a plurality of liquid storage pools (2016, 2017, 2018), and a plurality of first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118). Each reagent cavity (2012, 2013, 2014, 2015) and each liquid storage pool (2016, 2017, 2018) is respectively communicated with one end of a first liquid channel (20112, 20113, 20114, 20115, 20116, 20117, 20118). Specifically, the reagent cavity 2012 is communicated with one end of the first liquid channel 20112, the reagent cavity 2013 is communicated with one end of the first liquid channel 20115, the reagent cavity 2014 is communicated with one end of the first liquid channel 20114, the reagent cavity 2015 is communicated with one end of the first liquid channel 20113, the liquid storage pool 2016 is communicated with one end of the first liquid channel 20116, the liquid storage pool 2017 is communicated with one end of the first liquid channel 20117, and the liquid storage pool 2018 is communicated with one end of the first liquid channel 20118. The aerosol adsorption column storage cavity 20111 is provided with an aerosol adsorption column 202. The aerosol adsorption column 202 can effectively prevent external aerosol from entering the cartridge and introducing false positive results, and also avoid the diffusion of nucleic acid aerosol in the nucleic acid extraction chip 2 to the outside of the nucleic acid extraction chip 2 to cause environmental pollution. Each reagent cavity (2012, 2013, 2014, 2015) and each liquid storage pool (2016, 2017, 2018) is respectively communicated with one end of a ventilation channel (1021, 1022, 1023, 1024, 1025, 1026, 1027). Specifically, as shown in FIGS. 10A and 10B, the reagent cavity 2012 is communicated with the ventilation channel 1021, the reagent cavity 2013 is communicated with the ventilation channel 1022, the reagent cavity 2014 is communicated with the ventilation channel 1027, the reagent cavity 2015 is communicated with the ventilation channel 1026, the liquid storage pool 2016 is communicated with the ventilation channel 1023, the liquid storage pool 2017 is communicated with the ventilation channel 1024, and the liquid storage pool 2018 is communicated with the ventilation channel 1025. The other end of the ventilation channel (1021, 1022, 1023, 1024, 1025, 1026, 1027) is communicated with the gas buffer pool 20110. The gas buffer pool 20110 is internally placed with a water-absorbing resin to prevent liquid from splashing out and contaminating other reagents. The gas buffer pool 20110 is communicated with an air outlet hole 1028, the ventilation hole 10211 is communicated with the aerosol adsorption column storage cavity 20111, and the aerosol adsorption column storage cavity 20111 is communicated with the center pool 2011. Figure 3 With Figure 5 the ventilation channel 1027, the reagent cavity 2015 is communicated with the ventilation channel 1026, the liquid storage pool 2016 is communicated with the ventilation channel 1023, the liquid storage pool 2017 is communicated with the ventilation channel 1024, and the liquid storage pool 2018 is communicated with the ventilation channel 1025. The other end of the ventilation channel (1021, 1022, 1023, 1024, 1025, 1026, 1027) is communicated with the gas buffer pool 20110. The gas buffer pool 20110 is internally placed with a water-absorbing resin to prevent liquid from splashing out and contaminating other reagents. The gas buffer pool 20110 is communicated with an air outlet hole 1028, the ventilation hole 10211 is communicated with the aerosol adsorption column storage cavity 20111, and the aerosol adsorption column storage cavity 20111 is communicated with the center pool 2011.

[0106] The lower cover body 3 is provided with a valve placement groove 3011, a membrane placement groove 3015, and a chip insertion groove 3018. The valve placement groove 3011 is provided with a rotating assembly 305, and the rotating assembly 305 has a rotating channel 3051. The rotating channel 3051 can be in communication with the other end of different first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) as the rotating assembly 305 rotates. The membrane placement groove 3015 is provided with a nucleic acid adsorption silica gel membrane 304. The membrane placement groove 3015 is in communication with the center pool 2011 and the valve placement groove 3011. The chip insertion groove 3018 is in communication with one of the liquid storage pools 2017.

[0107] The amplification chip 4 is inserted into the chip insertion groove 3018 and can be in communication with the rotating channel 3051 for amplifying nucleic acid.

[0108] In some embodiments, the silica gel membrane 304 is made of a mixed solution of silica and other components. The diameter of the silica gel membrane 304 is 60mm-80mm, and the thickness is 2mm-4mm. The silica gel membrane 304 is used to adsorb nucleic acid under high salt conditions and elute nucleic acid under low salt conditions. The size and thickness of the silica gel membrane 304 used in the present application can ensure a large adsorption capacity and a fast liquid passing speed during nucleic acid adsorption, and by adjusting the external air pressure, the quality of nucleic acid extraction and purification is ensured while the time of nucleic acid extraction and purification is reduced. By controlling the size of the external air pressure through the vent hole 10211, the silica gel membrane 304 can be fully contacted with each reaction reagent, achieving better adsorption, cleaning, and elution effects.

[0109] In some embodiments, please refer to Figure 2 As shown in the figure, the upper cover body 1 includes an upper cover plate 102 and a first single-sided film 301. The upper cover plate 102 has a cover plate first surface 102a connected with the first single-sided film 301 and a cover plate second surface 102b connected with the nucleic acid extraction chip 2. The cover plate first surface 102a is provided with a plurality of vent grooves, and the groove bottoms at both ends of the plurality of vent grooves are respectively penetrated through the upper cover plate 102. The first single-sided film 301 covers the cover plate first surface 102a and seals each vent groove to form a plurality of vent channels (1021, 1022, 1023, 1024, 1025, 1026, 1027). The sample addition hole 10212 and the vent hole 10211 are both penetrated through the upper cover plate 102 and the first single-sided film 301.

[0110] In some embodiments, please refer to Figure 3As shown, the first surface 102a of the cover plate is further provided with a first communication groove, and the first single-sided film 301 covers the first surface 102a of the cover plate and seals the first communication groove to form a first communication channel 10213, one end of the first communication channel 10213 extends to communicate with the aerosol adsorption column storage cavity 20111, and the other end of the first communication channel 10213 extends to communicate with the central pool 2011.

[0111] In some embodiments, please refer to Figure 4 As shown, the number of aerosol adsorption column storage cavities 20111 is multiple, multiple aerosol adsorption column storage cavities 20111 are connected in series, and multiple aerosol adsorption column storage cavities 20111 are connected in series in a winding manner, wherein the first aerosol adsorption column storage cavity 20111 communicates with the air hole 10211, and the last aerosol adsorption column storage cavity 20111 communicates with the first communication channel 10213.

[0112] In some embodiments, please refer to Figure 5 , Figure 6As shown, the nucleic acid extraction chip 2 has a chip first surface 201a matched with the upper cover body 1 and a chip second surface 201b matched with the lower cover body 3, the chip second surface 201b is provided with a plurality of first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118), and the chip first surface 201a is provided with a central pool 2011, a gas buffer pool 20110, an aerosol adsorption column storage cavity 20111, a plurality of reagent cavities (2012, 2013, 2014, 2015), and a plurality of liquid storage pools (2016, 2017, 2018). The bottom surface of the aerosol adsorption column storage cavity 20111, the bottom surface of the plurality of reagent cavities (2012, 2013, 2014, 2015), and the bottom surface of the plurality of liquid storage pools (2016, 2017, 2018) all penetrate to the chip second surface 201b. The bottom surface of the plurality of reagent cavities (2012, 2013, 2014, 2015) and the bottom surface of the plurality of liquid storage pools (2012, 2013, 2014, 2015) penetrate to the chip second surface and are respectively communicated with one end of one of the first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118), and the other end of the first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) all extend to a first preset position and are communicated with the valve placement groove 3011, and the first preset position corresponds to the valve placement groove 3011. Specifically, the reagent cavity 2012 corresponds to the first liquid channel 20112, the reagent cavity 2013 corresponds to the first liquid channel 20115, the reagent cavity 2014 corresponds to the first liquid channel 20114, the reagent cavity 2015 corresponds to the first liquid channel 20113, the liquid storage pool 2016 corresponds to the first liquid channel 20116, the liquid storage pool 2017 corresponds to the first liquid channel 20117, and the liquid storage pool 2018 corresponds to the first liquid channel 20118.

[0113] In some embodiments, please refer to Figure 6 As shown, the chip second surface 201b is also provided with a second liquid channel 20119, one end of the second liquid channel 20119 extends to a first preset position and is communicated with the valve placement groove 3014, and the other end of the second liquid channel 20119 is communicated with the membrane placement groove 3015.

[0114] In some embodiments, the gas buffer pool 20110, the aerosol adsorption column storage cavity 20111, the reagent cavities (2012, 2013, 2014, 2015), and the liquid storage pools (2016, 2017, 2018) all have a funnel structure.

[0115] In some embodiments, when the number of the aerosol adsorption column storage cavities 20111 is two, a second communication passage is arranged on the second surface 201b of the chip between the two aerosol adsorption column storage cavities 20111, and the second communication passage is used for communicating the two aerosol adsorption column storage cavities 20111.

[0116] In some embodiments, referring to Figure 7 As shown, the lower cover body 3 has a lower bottom plate 301 and a second single-sided film 303, the lower bottom plate 301 has a bottom plate first surface 301a matched with the nucleic acid extraction chip 2 and a bottom plate second surface 301b sealed with the second single-sided film 303, the lower bottom plate 301 has a valve placement groove 3011 and a film placement groove 3015 penetrating the bottom plate first surface 301a and the bottom plate second surface 301b, the bottom plate second surface 301b has a chip insertion groove 3018, and the bottom plate first surface 301a is provided with a communication hole 3013 communicating with the chip insertion groove 3018, and the chip insertion groove 3018 communicates with one of the liquid storage pools 2017 through the communication hole 3013.

[0117] In some embodiments, referring to Figure 9 As shown, the bottom plate second surface 301b is further provided with a third liquid passage 3017. One end of the third liquid passage 3017 communicates with the film placement groove 3015 on the bottom plate second surface 301b, and the other end of the third liquid passage 3017 extends to the bottom plate first surface 301a and communicates with the valve placement groove 3011. Specifically, the other end of the third liquid passage 3017 extends to the bottom plate first surface 301a and communicates with the valve placement groove 3011 through the second liquid passage 20119 on the second surface 201b of the chip.

[0118] In some embodiments, referring to Figure 9 As shown, the bottom plate first surface 301a is further provided with a flow channel through hole 3014 for communicating with the third liquid passage 3017 on the bottom plate second surface 301b. The flow channel through hole 3014 further communicates with the second liquid passage 20119 on the second surface 201b of the chip 2.

[0119] In some embodiments, referring to Figure 7 As shown, the lower cover body 3 further has a sealing gasket 302 connected to the bottom plate first surface 301a, and the sealing gasket 302 is used for sealing the bottom plate first surface 301a and the nucleic acid extraction chip 2. Referring to Figure 10As shown, the sealing gasket 302 is provided with a central hole 30211 communicating with the third liquid channel 3017, a first accommodating hole 3021 communicating with the membrane accommodating groove 3015, and second accommodating holes (30212, 30213, 30214, 30215, 30216, 30217, 30218) communicating with the valve accommodating groove 3011. The central pool 2011 communicates with the membrane accommodating groove 3015 through the first accommodating hole 3021. The valve accommodating groove 3011 can communicate with the end of the plurality of first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) through the second accommodating holes (30212, 30213, 30214, 30215, 30216, 30217, 30218), specifically, the second accommodating hole 30212 corresponds to the end of the first liquid channel 20114, the second accommodating hole 30213 corresponds to the end of the first liquid channel 20115, the second accommodating hole 30214 corresponds to the end of the first liquid channel 20112, the second accommodating hole 30215 corresponds to the end of the first liquid channel 20116, the second accommodating hole 30216 corresponds to the end of the first liquid channel 20117, the second accommodating hole 30217 corresponds to the end of the first liquid channel 20118, the second accommodating hole 30214 corresponds to the end of the first liquid channel 20119, and the second accommodating hole 30219 corresponds to the end of the first liquid channel 20113. The lower cover body 3 is laser-bonded with the sealing gasket 302, which can ensure flatness and bonding strength. The sealing gasket 302 is elastic, and the elastic sealing gasket 302 can tightly seal the rotating assembly 305, avoiding liquid leakage caused by gaps under the rotating assembly 305.

[0120] In some embodiments, see Figure 10As shown, the sealing gasket 302 is further provided with a third displacement hole 3023, a plurality of fourth displacement holes (3024, 3025, 3026, 3027), and a plurality of fifth displacement holes (3028, 3029, 30210). The third displacement hole 3023 is in communication with the aerosol adsorption column storage cavity 20111. The plurality of fourth displacement holes (3024, 3025, 3026, 3027) are respectively in communication with the ends of the plurality of first liquid channels (20112, 20113, 20114, 20115). Specifically, the fourth displacement hole 3024 is in communication with the other end of the first liquid channel 20112, the fourth displacement hole 3025 is in communication with the other end of the first liquid channel 20115, the fourth displacement hole 3026 is in communication with the other end of the first liquid channel 20114, and the fourth displacement hole 3027 is in communication with the other end of the first liquid channel 20113. The plurality of fifth displacement holes (3028, 3029, 30210) are respectively in communication with the plurality of liquid storage pools (2016, 2017, 2018). Specifically, the fifth displacement hole 3028 is in communication with the liquid storage pool 2016 and the first liquid channel 20116, the fifth displacement hole 3029 is in communication with the liquid storage pool 2017 and the first liquid channel 20117, and the fifth displacement hole 30210 is in communication with the liquid storage pool 2018 and the first liquid channel 20118.

[0121] In some embodiments, refer to Figure 11 、 Figure 12 As shown, the rotating assembly 305 has a valve first surface 305a facing the nucleic acid extraction chip 2 and a valve second surface 305b facing the valve placement groove 3011. The rotating assembly 305 has a rotating channel 3051 penetrating the valve first surface 305a and the valve second surface 305b.

[0122] In some embodiments, at least one of the following technical features is further included:

[0123] The valve second surface 305b is provided with an alignment station. Correspondingly, the inner wall of the valve placement groove 3011 has a plurality of rotating alignment mark grooves matched with the alignment station. The number of the rotating alignment mark grooves is equal to the sum of the number of the first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) and the number of the second liquid channels 20119.

[0124] Refer to Figure 12 As shown, the valve second surface 305b is further provided with an interface 3052 for cooperating with an external driving motor.

[0125] In some embodiments, a sealing ring 306 is further connected to the rotating assembly 305, and is located on the second surface 305b of the valve. The sealing ring 306 is used for sealing the second surface 305b of the valve with the bottom surface of the valve placement groove 3011. The sealing ring 306 can be made of an elastic material such as silicone, latex, rubber, etc.

[0126] In some embodiments, the rotating assembly 305 of the nucleic acid extraction step is unidirectional rotation. The unidirectional rotation can avoid the back-and-forth rotation of the rotating assembly 305, so that the waste liquid of the previous step does not enter the reagent of the subsequent step, thereby reducing the contamination of the reagent and improving the extraction efficiency and quality of the nucleic acid.

[0127] In some embodiments, as shown in Figure 13 The amplification chip 4 includes a chip body 401, an upper cover film 403, a lower cover film 402, and a waterproof and breathable film. The chip body 401 is provided with a chip flow channel 4016 and an amplification detection cavity 4014. One end of the chip flow channel 4016 is in communication with the rotating channel 3051, and the other end is in communication with the amplification detection cavity 4014. The upper cover film 403 is sealingly connected to the upper surface of the chip body 401, and the lower cover film 402 is sealingly connected to the lower surface of the chip body 401. The waterproof and breathable film covers the amplification detection cavity 4014. The waterproof and breathable film on the amplification chip 4 can ensure that the amplification reagent fills the amplification chamber when pressure is applied without diffusing to the outside of the chip.

[0128] In some embodiments, the amplification chip 4 further includes a matching clamping member 4012 connected to the upper surface of the chip body 401. The matching clamping member 4012 can be embedded and matched in the chip insertion groove 3018. The matching clamping member 4012 is provided with a clamping member through hole 4013 in communication with the rotating channel 3051, and the clamping member through hole 4013 is also in communication with the chip flow channel 4016.

[0129] In some embodiments, as shown in Figure 14 The number of amplification detection cavities 4014 is multiple, and the multiple amplification detection cavities 4014 are connected in parallel to the chip flow channel 4016. For example, in one specific embodiment, the number of amplification detection cavities 4014 is four. The four reaction amplification cavities encapsulate different reaction reagents to realize the multiplex detection of nucleic acid.

[0130] In some embodiments, as shown in Figure 14 , Figure 15 The chip through hole 4011 is further provided on the pipeline between the chip flow channel 4016 and the amplification detection cavity 4014.

[0131] In some embodiments, as shown in Figure 14As shown, the amplification chip 4 further comprises a ring-shaped protruding part 4015 connected to the upper surface of the chip main body 401, the plurality of amplification detection cavities 4014 are located within the ring-shaped protruding part 4015, and the waterproof and breathable film is sealingly connected to the ring-shaped protruding part 4015.

[0132] In some embodiments, for example, the number of central pools 2011 is one, the number of gas buffer pools 20110 is one, the number of aerosol adsorption column storage cavities 20111 is two, the number of reagent cavities (2012, 2013, 2014, 2015) is four, the number of liquid storage pools (2016, 2017, 2018) is three, the number of first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) is seven, and the number of second liquid channels 20119 is one. One first liquid channel (20112, 20113, 20114, 20115, 20116, 20117, 20118) is respectively connected to one of the four reagent cavities (2012, 2013, 2014, 2015) and one of the three liquid storage pools (2016, 2017, 2018). Correspondingly, the number of third accommodation holes 203 on the sealing gasket 302 is two, the two third accommodation holes 3023 correspond to the two aerosol adsorption column storage cavities 20111 respectively; the number of second accommodation holes (30212, 30213, 30214, 30215, 30216, 30217, 30218) is eight, the eight second accommodation holes (30212, 30213, 30214, 30215, 30216, 30217, 30218) correspond to one end of one of the seven first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) and the second liquid channel 20119 respectively; the number of fourth accommodation holes (3024, 3025, 3026, 3027) is four, the four fourth accommodation holes (3024, 3025, 3026, 3027) correspond to the openings of the four reagent cavities (2012, 2013, 2014, 2015) on the second surface 201b of the chip respectively; the number of fifth accommodation holes (3028, 3029, 30210) is three, the three fifth accommodation holes (3028, 3029, 30210) correspond to the openings of the three liquid storage pools (2016, 2017, 2018) on the second surface 201b of the chip respectively. Correspondingly, the number of rotation alignment mark grooves is eight.

[0133] Further, the other ends of the first liquid channels (20112, 20113, 20114, 20115, 20116, 20117, 20118) and the other end of the second liquid channel 20119 extend to the first preset position, and then are evenly distributed on the same circumference according to eight points, that is, the eight position rotation alignment mark grooves are evenly distributed on the same circumference, and the included angle between adjacent rotation alignment mark grooves is 45°. When the rotation assembly 305 is rotated, the rotation assembly 305 can be switched between adjacent rotation alignment mark grooves by rotating 45°.

[0134] The reagent cartridge 10 can quickly, sensitively and accurately detect pathogenic microorganisms on site, and is crucial for preventing infectious diseases and controlling disease outbreaks. The reagent cartridge 10 can integrate various steps required for detection and analysis on a chip to complete sample pretreatment, manual sample addition, reagent mixing, optical detection and other complex operations, and can provide a closed environment required for diagnosis, can be made into a disposable chip, has low cost, and reduces false positive results and infection risks caused by repeated use. Compared with the traditional detection method, the on-site detection of the present application has the following advantages: faster detection speed, higher sensitivity and specificity, lower cost, higher efficiency, rapid detection at any location, and non-professionals can complete the operation in a short time.

[0135] The nucleic acid extraction, purification and amplification integrated method provided by another embodiment of the present application also provides a nucleic acid extraction, purification and amplification integrated method.

[0136] A nucleic acid extraction, purification and amplification integrated method comprises the following steps:

[0137] The lysing solution, the washing solution and the elution solution are respectively packaged in different reagent chambers and different liquid storage pools of the reagent cartridge 10; the rotation assembly 305 is rotated to be connected to the reagent chamber in which the lysing solution is packaged, negative pressure is applied through the air hole 10211, the lysing solution is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015, and the silica gel membrane 304 can adsorb nucleic acids in the lysing solution; positive pressure is applied through the air hole 10211 to make the liquid in the center pool 2011 backflow;

[0138] The positive pressure is stopped, and the rotation assembly 305 is rotated to be connected to the reagent chamber in which the washing solution is packaged, negative pressure is applied through the air hole 10211, the washing solution is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to wash the silica gel membrane 304; and positive pressure is applied through the air hole 10211 to make the liquid in the center pool 2011 backflow;

[0139] controlling the positive pressure to stop and rotating the rotating assembly 305 to be communicated with the reservoir containing the eluent, applying negative pressure through the air hole 10211 to adsorb the eluent to the silica gel membrane 304 in the membrane placement groove 3015 into the center pool 2011 to elute the silica gel membrane 304;

[0140] rotating the rotating assembly 305 to be communicated with the amplification chip 4, applying positive pressure through the air hole 10211 to transfer the eluent in the center pool 2011 to the amplification chip 4; and

[0141] controlling the amplification chip 4 to complete nucleic acid amplification and detection.

[0142] The operation principle of the reagent cartridge 10 of the application is as follows: controlling the gas source to apply positive pressure through the air hole to realize that the liquid in the reagent cavity or the reservoir sequentially passes through the corresponding first liquid channel, the rotating assembly, the membrane placement groove and enters the center pool, and controlling the gas source to apply negative pressure through the air hole to realize that the liquid in the center pool sequentially passes through the membrane placement groove, the rotating assembly and the first liquid channel and flows back to the reagent cavity or the reservoir. Different from the traditional centrifugal method of adding liquid from the top in each step and then centrifuging once, the various reagents of the application can flow forward and at least once in reverse, for example, contact the silica gel membrane twice, can operate the silica gel membrane twice in a single step, realize more sufficient reaction, and at the same time, by controlling the pressure of the external gas pressure, the reaction reagent can have more contact time with the silica gel membrane, avoid the situation that the nucleic acid is not completely adsorbed but is transferred to the waste liquid bin in the traditional technology, and improve the nucleic acid extraction quality.

[0143] Another embodiment of the application further provides a nucleic acid extraction, purification and amplification integrated method.

[0144] A nucleic acid extraction, purification and amplification integrated method, comprising the following steps: encapsulating a lysis solution, a washing solution I, a washing solution II, a washing solution III and an eluent in the reagent cavities 2012, 2013, 2013, 2015 and the reservoir 2016 on the nucleic acid extraction chip 2 respectively; wherein in some embodiments, 300-800 μL of the lysis solution, 300-900 μL of the washing solution I, 300-900 μL of the washing solution II, 300-900 μL of the washing solution III and 50-300 μL of the eluent are respectively encapsulated in the first reagent cavity, the second reagent cavity, the third reagent cavity, the fourth reagent cavity and the first reservoir on the nucleic acid extraction chip.

[0145] different reaction reagents are respectively encapsulated in the plurality of amplification and detection cavities 4014 of the amplification chip 4 to realize multiple detection of nucleic acid;

[0146] adding the sample to be tested to the reagent cavity containing the lysis solution through the sample adding hole 10212 to perform lysis to form a lysis solution;

[0147] Rotate the rotating assembly 305 to be connected to the first liquid channel 20112 connected to the reagent cavity 2012, control the external air pressure to apply negative pressure through the air hole 10211, and adsorb the lysis solution to the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015, and the silica gel membrane 304 can adsorb nucleic acid in the lysis solution; control the external air pressure to apply positive pressure through the air hole 10211, and transfer the liquid in the center pool 2011 to the reagent cavity 2012;

[0148] Control the positive pressure to stop and rotate the rotating assembly 305 to be connected to the first liquid channel 20113 connected to the reagent cavity 2013, control the external air pressure to apply negative pressure through the air hole 10211, and adsorb the washing solution I to the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to wash the silica gel membrane 304; control the external air pressure to apply positive pressure through the air hole 10211, and transfer the liquid in the center pool 2011 to the reagent cavity 2013;

[0149] Control the positive pressure to stop and rotate the rotating assembly 305 to be connected to the first liquid channel 20114 connected to the reagent cavity 2014, control the external air pressure to apply negative pressure through the air hole 10211, and adsorb the washing solution II to the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to wash the silica gel membrane 304; control the external air pressure to apply positive pressure through the air hole 10211, and transfer the liquid in the center pool 2011 to the reagent cavity 2014;

[0150] Control the positive pressure to stop and rotate the rotating assembly 305 to be connected to the first liquid channel 20113 connected to the reagent cavity 2015, control the external air pressure to apply negative pressure through the air hole 10211, and adsorb the washing solution III to the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to wash the silica gel membrane 304; control the external air pressure to apply positive pressure through the air hole 10211, and transfer the liquid in the center pool 2011 to the reagent cavity 2015;

[0151] Control the positive pressure to stop and rotate the rotating assembly 305 to be connected to the first liquid channel 20116 connected to the liquid storage pool 2018, control the external air pressure to apply positive pressure through the air hole 10211 to dry the silica gel membrane 304;

[0152] Control the positive pressure to stop and rotate the rotating assembly 305 to be connected to the first liquid channel 20116 connected to the liquid storage pool 2016, control the external air pressure to apply negative pressure through the air hole 10211, and adsorb the elution solution to the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to elute the silica gel membrane 304, stop the negative pressure and keep still, so that the silica gel membrane 304 is in full contact with the elution solution, and the nucleic acid is fully eluted for 5-10 min;

[0153] rotating the rotating assembly 305 to be connected to the amplification chip 4, controlling the external air pressure to apply positive pressure through the air hole 10211, and transferring the eluent in the center pool 2011 to the amplification chip 4 through the first liquid channel 20117; and

[0154] controlling the amplification chip 4 to complete nucleic acid amplification and detection under the cooperation and action of the external instrument heating module and the fluorescence detection module.

[0155] In some embodiments, the positive pressure is 0-20 kPa, and the negative pressure is 0-10 kPa.

[0156] Embodiment 1

[0157] The embodiment also provides a nucleic acid extraction, purification and amplification integrated method.

[0158] The controlled positive pressure is 0-20 kPa, and the controlled negative pressure is 0-10 kPa.

[0159] A nucleic acid extraction, purification and amplification integrated method comprises the following steps: 600 μL of a lysis solution, 500 μL of a cleaning solution I, 700 μL of a cleaning solution II, 700 μL of a cleaning solution III and 200 μL of an eluent are respectively packaged in the reagent cavities 2012, 2013, 2013, 2015 and the liquid storage pool 2016 on the nucleic acid extraction chip 2.

[0160] The reagent cartridge 10 is placed in the matched automatic instrument, the rotating rod in the automatic instrument is connected with the connector 3052 on the rotating assembly 305, the center point of the rotating assembly 305 is always connected with the center hole 30211 on the sealing gasket 302, the second liquid channel 20119 on the nucleic acid extraction chip 2, the third liquid channel 3017 and the center pool 2011 on the nucleic acid extraction chip 2, at this time, the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves (30114) on the lower cover body 3, and at this time, the center pool 2011 is not connected with any reagent cavity (2012, 2013, 2014, 2015).

[0161] Different reaction reagents are respectively packaged in the four amplification and detection cavities 4014 of the amplification chip 4 to realize multiple detection of nucleic acid. The sample to be detected is added to the reagent cavity 2012 packaged with the lysis solution through the sample adding hole 10212 to form a lysis solution.

[0162] counterclockwise rotation of the rotating assembly 305 by 45°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves 30113 on the lower cover body 3, at this time the center pool 2011 is connected with one of the second accommodation holes 30213 on the sealing gasket 302, the first liquid channel 20112 on the nucleic acid extraction chip 2, and the reagent cavity 2012 through the rotating channel 3051 on the rotating assembly 305, the external air pressure is controlled to apply negative pressure through the air hole 10211, the lysis solution is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015, at this time the mixed solution can adsorb the nucleic acid of the sample lysed through the silica gel membrane 304, and then the external air pressure is controlled to apply positive pressure through the air hole 10211 to transfer the lysis solution back to the reagent cavity 2012, and the mixed solution passes through the silica gel membrane 304 again, at this time the silica gel membrane 304 can adsorb the nucleic acid again, realizing one liquid transfer and twice nucleic acid adsorption, and the nucleic acid adsorption is more sufficient.

[0163] After the liquid transfer is completed, the positive pressure is controlled to stop, and the rotating assembly 305 is counterclockwise rotated by 45°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves (30112) on the lower cover body 3, at this time the center pool 2011 is connected with the second accommodation hole 30212 on the sealing gasket 302, the first liquid channel 20115 on the nucleic acid extraction chip 2, and the reagent cavity 2013 through the rotating channel 3051 on the rotating assembly 305, the external air pressure is controlled to apply negative pressure through the air hole 10211, the washing solution I is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to perform the first washing of the silica gel membrane 304, the external air pressure is controlled to apply positive pressure through the air hole 10211 to transfer the liquid in the center pool 2011 to the reagent cavity 2012, and the washing solution I passes through the silica gel membrane 304 to perform the second washing of the silica gel membrane 304.

[0164] After the liquid transfer is completed, the positive pressure is controlled to stop, and the rotating assembly 305 is counterclockwise rotated by 45°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves (30112) on the lower cover body 3, at this time the center pool 2011 is connected with the second accommodation hole 30212 on the sealing gasket 302, the first liquid channel 20115 on the nucleic acid extraction chip 2, and the reagent cavity 2013 through the rotating channel 3051 on the rotating assembly 305, the external air pressure is controlled to apply negative pressure through the air hole 10211, the washing solution I is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to perform the first washing of the silica gel membrane 304, the external air pressure is controlled to apply positive pressure through the air hole 10211 to transfer the liquid in the center pool 2011 to the reagent cavity 2012, and the washing solution I passes through the silica gel membrane 304 to perform the second washing of the silica gel membrane 304.

[0165] After the transfer of the cleaning solution II is completed, the positive pressure is stopped, the rotating assembly 305 is counterclockwise rotated by 45°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves 30110 on the lower cover body 3, at this time, the center pool 2011 is communicated with the second displacement hole 30218 on the sealing gasket 302, the first liquid channel 20114 on the nucleic acid extraction chip 2 and the reagent cavity 2014 through the rotating channel 3051 on the rotating assembly 305, the external air pressure is controlled to apply negative pressure through the air hole 10211, the cleaning solution III is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to clean the silica gel membrane 304, the external air pressure is controlled to apply positive pressure through the air hole 10211, the liquid in the center pool 2011 is transferred into the reagent cavity 2014, and the cleaning solution III is used to clean the silica gel membrane 304 for the second time.

[0166] After the transfer of the cleaning solution III is completed, the positive pressure is stopped, the rotating assembly 305 is counterclockwise rotated by 45°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves 3019 on the lower cover body 3, at this time, the center pool 2011 is communicated with the second displacement hole 30217 on the sealing gasket 302, the first liquid channel 20118 on the nucleic acid extraction chip 2 and the liquid storage pool 2018 through the rotating channel 3051 on the rotating assembly 305, and the silica gel membrane 304 is blown dry by controlling the external air pressure to apply positive pressure through the air hole 10211.

[0167] The positive pressure is stopped, the rotating assembly 305 is counterclockwise rotated by 90°, so that the alignment station on the rotating assembly 305 is aligned with one of the rotating alignment mark grooves 30115 on the lower cover body 3, at this time, the center pool 2011 is communicated with the second displacement hole 30214 on the sealing gasket 302, the first liquid channel 20116 on the nucleic acid extraction chip 2 and the liquid storage pool 2016 through the rotating channel 3051 on the rotating assembly 305, the eluent is adsorbed into the center pool 2011 through the silica gel membrane 304 in the membrane placement groove 3015 to elute the silica gel membrane 304 by controlling the external air pressure to apply negative pressure, the negative pressure is stopped, and the silica gel membrane 304 is fully contacted with the eluent to elute the nucleic acid for 5-10 min.

[0168] Clockwise rotation of the rotating assembly 305 45°, so that the rotating assembly 305 on the alignment station with one of the lower cover body 3 on the rotating alignment mark slot 3018 alignment, at this time the center pool 2011 through the rotating channel 3051 on the rotating assembly 305 and sealing pad 302 on the second let hole 30216, nucleic acid extraction chip 2 on the first liquid channel 20117, clamping piece through hole 4013 communication, so that the rotating assembly 305 is rotated to be connected to the amplification chip 4, the external air pressure is applied by the air hole 10211 to apply positive pressure, the eluent in the center pool 2011 is transferred to the amplification chip 4 through the first liquid channel 20117, the external air pressure is transferred to the four amplification detection cavities 4014 through the chip flow channel 4016, when the four amplification chambers are filled with eluent, immediately rotate the rotating assembly 305 counterclockwise 90°, align the mark on the rotating assembly 305 with the rotating alignment mark slot 30113 on the lower cover body 3, so that the rotating assembly 305 returns to the initial state, and the amplification chip 4 is closed. And

[0169] The eluent is mixed with different lyophilized reagents in the amplification detection cavity 4014, and the amplification chip 4 is controlled to complete nucleic acid amplification and detection under the cooperation and action of the external instrument heating module and the fluorescence detection module.

[0170] In summary, compared with the prior art, the reagent cartridge 10 of the application has the following beneficial effects:

[0171] 1. The column membrane method nucleic acid extraction is integrated into a cartridge type microfluidic chip, the cartridge design is compact, small in size, and few in operation steps, so that automatic nucleic acid extraction and purification can be completed in a short time.

[0172] 2. The nucleic acid detection reagents are all integrated into a cartridge type microfluidic chip, which reduces the errors and pollution caused by manual operation, and reduces the problems of false positives and inaccurate detection results.

[0173] 3. The application realizes the whole detection process of nucleic acid extraction, purification, amplification and detection in a closed system, avoiding the aerosol pollution caused by nucleic acid amplification and the possible false positive results caused by external aerosol;

[0174] 4. The application can realize multiplex nucleic acid detection in a chip system, improve the nucleic acid detection throughput, and reduce the sample consumption.

[0175] 5. The application realizes the automatic nucleic acid detection with the external control instrument, reduces the personnel time and uncertainty, improves the accuracy and stability of nucleic acid detection, and avoids the reagent pollution and environmental pollution in the nucleic acid detection process.

[0176] 6. This invention transfers the same liquid first to the central chamber 2011 containing the silica membrane 304, and then transfers the liquid away to complete each step of nucleic acid adsorption, washing, or elution. Unlike traditional centrifugation methods where liquid is added from above and centrifuged again for each step, in this invention, the reagent contacts the silica membrane 304 twice, allowing for two operations on the silica membrane 304 in a single step, resulting in a more complete reaction in each step. Simultaneously, by controlling the external air pressure, the reaction reagents can have more contact time with the silica membrane 304, reducing the likelihood of nucleic acids being transferred to the waste liquid chamber before complete adsorption.

[0177] In the above embodiments, the descriptions of each embodiment have their own emphasis. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0178] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A reagent cartridge, characterized in that, The reagent cartridge is provided with a central pool, multiple reagent chambers, multiple liquid storage pools, multiple ventilation channels, and multiple first liquid channels. Each of the reagent chambers and each of the liquid storage pools is connected to one end of a first liquid channel, and each of the reagent chambers and each of the liquid storage pools is connected to one end of a ventilation channel. The reagent cartridge is also equipped with a rotating assembly and an amplification chip. The rotating assembly has a rotating channel that connects to the other end of different first liquid channels as the assembly rotates. A nucleic acid adsorption silica membrane is installed in the passage between the central pool and the rotating assembly. The rotating channel can also communicate with the amplification chip. The central pool can be externally connected to a negative pressure to allow the liquid in the reagent chamber and the liquid in the storage pool to pass through the nucleic acid adsorption silica membrane and enter the central pool, and to allow the liquid entering the central pool to reflux. The nucleic acid adsorption silica membrane is used to adsorb and purify nucleic acids in the lysis buffer. The reagent cartridge is equipped with a chip insertion slot, and the amplification chip can be detachably inserted into the chip insertion slot and communicate with the rotating channel for nucleic acid amplification. The rotating assembly rotates unidirectionally during the nucleic acid extraction step.

2. The reagent cartridge according to claim 1, characterized in that, The reagent cartridge is equipped with a sample application hole, a vent hole, and a vent hole; The reagent cartridge is also provided with a gas buffer pool, the other end of the ventilation channel is connected to the vent, and the vent is connected to the central pool; The reagent cartridge is also provided with a valve placement slot and a membrane placement slot. The rotating component is provided in the valve placement slot, and the nucleic acid adsorption silica membrane is provided in the membrane placement slot. The membrane placement slot is connected to the central pool and is also connected to the valve placement slot.

3. The reagent cartridge according to claim 2, characterized in that, The reagent cartridge is also provided with an aerosol adsorption column storage cavity, which is used to place the aerosol adsorption column. The vent is connected to the central pool through the aerosol adsorption column storage cavity. The other end of the ventilation channel is connected to the gas buffer pool, and the gas buffer pool is connected to the air outlet.

4. The reagent cartridge according to claim 3, characterized in that, The reagent cartridge includes an upper cover, a nucleic acid extraction chip, a lower cover, and an amplification chip, wherein the upper cover, the nucleic acid extraction chip, and the lower cover are connected in an overlapping manner from top to bottom; The upper cover is provided with the sample addition hole, the vent hole, the air outlet hole, and multiple ventilation channels; The nucleic acid extraction chip is provided with the central pool, the gas buffer pool, the aerosol adsorption column storage chamber, multiple reagent chambers, multiple liquid storage pools, and multiple first liquid channels; The lower cover is provided with the valve placement groove, the membrane placement groove, and the chip insertion groove.

5. The reagent cartridge according to claim 4, characterized in that, The upper cover includes an upper cover plate and a first single-sided membrane. The upper cover plate has a first surface that is connected to the first single-sided membrane and a second surface that is connected to the nucleic acid extraction chip. A plurality of ventilation grooves are provided on the first surface of the cover plate. The bottom surfaces of the grooves at both ends of the plurality of ventilation grooves penetrate the upper cover plate. The first single-sided membrane covers the first surface of the cover plate and closes each of the ventilation grooves to form a plurality of ventilation channels. The sample application hole and the ventilation hole both penetrate the upper cover plate and the first single-sided membrane.

6. The reagent cartridge according to claim 5, characterized in that, The first surface of the upper cover plate is also provided with a first connecting groove. The first single-sided film covers the first surface of the cover plate and closes the first connecting groove to form a first connecting channel. One end of the first connecting channel extends to communicate with the storage cavity of the aerosol adsorption column, and the other end extends to communicate with the central pool.

7. The reagent cartridge according to claim 6, characterized in that, The aerosol adsorption column storage cavity is multiple, and the multiple aerosol adsorption column storage cavities are connected in series and the multiple aerosol adsorption column storage cavities are connected in a meandering manner. The first aerosol adsorption column storage cavity is connected to the vent hole, and the last aerosol adsorption column storage cavity is connected to the first connecting channel.

8. The reagent cartridge according to any one of claims 4-7, characterized in that, The nucleic acid extraction chip has a first chip surface that mates with the upper cover and a second chip surface that mates with the lower cover. The second chip surface is provided with a plurality of first liquid channels. The first chip surface is provided with a central pool, a gas buffer pool, an aerosol adsorption column storage cavity, a plurality of reagent chambers, and a plurality of liquid storage pools. The bottom surfaces of the aerosol adsorption column storage cavity, the plurality of reagent chambers, and the plurality of liquid storage pools all extend to the second chip surface. The bottom surfaces of the plurality of reagent chambers and the plurality of liquid storage pools all extend to the second chip surface and are respectively connected to one end of a first liquid channel. The other end of each first liquid channel extends to a first preset position and communicates with the valve placement slot. The first preset position corresponds to the valve placement slot.

9. The reagent cartridge according to claim 8, characterized in that, A second liquid channel is also provided on the second surface of the chip. One end of the second liquid channel extends to the first preset position and communicates with the valve placement groove, and the other end of the second liquid channel communicates with the membrane placement groove.

10. The reagent card holder according to any one of claims 3-7 and 9, characterized in that, The gas buffer pool, the aerosol adsorption column storage chamber, the reagent chamber, and the liquid storage pool are all funnel-shaped structures.

11. The reagent cartridge according to any one of claims 4-7 and 9, characterized in that, The lower cover has a lower base plate and a second single-sided membrane. The lower base plate has a first base plate surface that is connected to the nucleic acid extraction chip and a second base plate surface that is sealed to the second single-sided membrane. The lower base plate has a valve placement groove and a membrane placement groove that penetrate the first base plate surface and the second base plate surface. The second base plate surface has a chip insertion groove. The first base plate surface has a connecting hole that communicates with the chip insertion groove. The chip insertion groove communicates with one of the liquid storage tanks through the connecting hole.

12. The reagent cartridge according to claim 11, characterized in that, A third liquid channel is also provided on the second surface of the base plate. One end of the third liquid channel is connected to the membrane placement groove on the second surface of the base plate, and the other end of the third liquid channel extends to the first surface of the base plate and is connected to the valve placement groove.

13. The reagent cartridge according to claim 12, characterized in that, The lower cover also has a sealing gasket, which is connected to the first surface of the base plate. The sealing gasket is used to seal the first surface of the base plate and the nucleic acid extraction chip. The sealing gasket is provided with a central hole communicating with the third liquid channel, a first clearance hole communicating with the membrane placement groove, and a second clearance hole communicating with the valve placement groove. The central pool communicates with the membrane placement groove through the first clearance hole, and the valve placement groove communicates with the ends of the plurality of first liquid channels through the second clearance hole.

14. The reagent cartridge according to claim 13, characterized in that, The sealing gasket is also provided with a third clearance hole, a plurality of fourth clearance holes and a plurality of fifth clearance holes. The third clearance hole is connected to the storage cavity of the aerosol adsorption column. The plurality of fourth clearance holes are connected to the ends of the plurality of first liquid channels one by one. The plurality of fifth clearance holes are connected to the plurality of liquid storage tanks one by one.

15. The reagent cartridge according to any one of claims 4-7, 9, and 12-14, characterized in that, The rotating assembly has a first valve surface facing the nucleic acid extraction chip and a second valve surface facing the valve placement slot, and the rotating assembly has a rotating channel penetrating the first valve surface and the second valve surface.

16. The reagent cartridge according to claim 15, characterized in that, The valve's second surface is provided with an alignment station, and correspondingly, the inner wall of the valve placement groove has multiple rotating alignment mark grooves that cooperate with the alignment station.

17. The reagent cartridge according to claim 15, characterized in that, The valve's second surface is also provided with a connecting member, which is used to cooperate with an external drive motor.

18. The reagent cartridge according to claim 15, characterized in that, The rotating assembly is also connected to a sealing ring, which is located on the second surface of the valve and is used to seal the second surface of the valve with the bottom surface of the valve placement groove.

19. The reagent card holder according to any one of claims 4-7, 9, 12-14, and 17-18, characterized in that, The amplification chip includes a chip body, an upper cover film, a lower cover film, and a waterproof and breathable membrane. The chip body is provided with a chip flow channel and an amplification detection chamber. One end of the chip flow channel is connected to the rotating channel and the other end is connected to the amplification detection chamber. The upper cover film is sealed to the upper surface of the chip body, the lower cover film is sealed to the lower surface of the chip body, and the waterproof and breathable membrane covers the amplification detection chamber.

20. The reagent cartridge according to claim 19, characterized in that, The amplification chip also includes a mating connector, which is connected to the upper surface of the chip body. The mating connector can be embedded in the chip insertion slot. The mating connector has a connector through hole that communicates with the rotation channel and is also connected to the chip flow channel.

21. The reagent cartridge according to claim 19, characterized in that, The number of amplification detection cavities is multiple, and the multiple amplification detection cavities are connected in parallel to the chip channel.

22. The reagent cartridge according to claim 19, characterized in that, The amplification chip also includes an annular protrusion component connected to the upper surface of the chip body, the amplification detection chamber located inside the annular protrusion component, and the waterproof and breathable membrane sealed to the annular protrusion component.

23. The reagent card holder according to any one of claims 4-7, 9, 12-14, 17-18, and 20-22, characterized in that, The reagent cartridge also includes an aerosol adsorption column, and the aerosol adsorption column is disposed in the storage cavity of the aerosol adsorption column.

24. An integrated method for nucleic acid extraction, purification, and amplification, characterized in that, Using the reagent cartridge according to any one of claims 1 to 23, the following steps are included: The lysis buffer, cleaning buffer, and elution buffer are respectively encapsulated in different reagent chambers and different storage pools of the reagent cartridge; The rotating assembly is rotated to connect to the reagent chamber containing the lysis buffer. Negative pressure is applied to the central chamber through the vent, causing the lysis buffer to be adsorbed into the central chamber through the silica membrane in the membrane placement tank. The silica membrane can adsorb nucleic acids in the lysis buffer. Positive pressure is applied through the vent, causing the liquid in the central chamber to flow back. The positive pressure is controlled to stop and the rotating component is rotated to connect to the reagent chamber containing the cleaning solution. Negative pressure is applied through the vent hole to draw the cleaning solution through the silica membrane in the membrane placement tank into the central pool to clean the silica membrane. Positive pressure is applied through the vent hole to cause the liquid in the central pool to flow back. The positive pressure is controlled to stop and the rotating component is rotated to a storage tank containing the eluent. Negative pressure is applied through the vent to draw the eluent through the silica membrane in the membrane placement tank into the central tank to elute the silica membrane. The rotating assembly is rotated to connect with the amplification chip, and positive pressure is applied through the vent to transfer the elution solution in the central cell into the amplification chip; as well as The amplification chip is controlled to complete nucleic acid amplification and detection.

25. An integrated method for nucleic acid extraction, purification, and amplification, characterized in that, Using the reagent cartridge according to any one of claims 1 to 23 includes the following steps: encapsulating lysis buffer, washing buffer I, washing buffer II, washing buffer III, and elution buffer in the first reagent chamber, second reagent chamber, third reagent chamber, fourth reagent chamber, and first storage tank on the nucleic acid extraction chip, respectively. Different reaction reagents are encapsulated in multiple amplification and detection chambers of the amplification chip to achieve multiplex detection of nucleic acids; The sample to be tested is added through the sample addition hole into the reagent chamber containing the lysis solution for lysis to form the lysis solution; The rotating assembly is rotated to connect to the first liquid channel connected to the first reagent chamber. External air pressure is controlled to apply negative pressure through the vent, causing the lysis buffer to be adsorbed into the central pool through the silica membrane in the membrane placement tank. The silica membrane can adsorb nucleic acids in the lysis buffer. External air pressure is controlled to apply positive pressure through the vent, transferring the liquid in the central pool into the first reagent chamber. The positive pressure is controlled to stop and the rotating assembly is rotated to connect to the first liquid channel connected to the second reagent chamber. The external air pressure is controlled to apply negative pressure through the vent hole, so that the cleaning liquid I is adsorbed into the central pool through the silica membrane in the membrane placement tank to clean the silica membrane. The external air pressure is controlled to apply positive pressure through the vent hole, so that the liquid in the central pool is transferred to the second reagent chamber. Control the positive pressure to stop and rotate the rotating assembly to connect to the first liquid channel connected to the third reagent chamber; control the external air pressure to apply negative pressure through the vent hole, so that the cleaning solution II is adsorbed into the central pool through the silica membrane in the membrane placement tank to clean the silica membrane; control the external air pressure to apply positive pressure through the vent hole, so that the liquid in the central pool is transferred to the third reagent chamber; Control the positive pressure to stop and rotate the rotating assembly to connect to the first liquid channel connected to the fourth reagent chamber; control the external air pressure to apply negative pressure through the vent hole, so that the cleaning solution III is adsorbed into the central pool through the silica membrane in the membrane placement tank to clean the silica membrane; control the external air pressure to apply positive pressure through the vent hole, so that the liquid in the central pool is transferred to the fourth reagent chamber; Control the positive pressure to stop and rotate the rotating component to connect to the first liquid channel connected to the second liquid storage tank; control the external air pressure to apply positive pressure through the vent hole to dry the silicone membrane. Stop the positive pressure and rotate the rotating assembly to connect to the first liquid channel connected to the first storage tank. Control the external air pressure to apply negative pressure through the vent hole. Adsorb the eluent through the silica membrane in the membrane placement tank into the central tank to elute the silica membrane. Stop the negative pressure and keep it still so that the silica membrane and the eluent are in full contact. The nucleic acid is fully eluted for 5-10 minutes. The rotating component is rotated to connect with the amplification chip, and external air pressure is controlled to apply positive pressure through the vent hole to transfer the elution solution in the central cell into the amplification chip. as well as The amplification chip is controlled to complete nucleic acid amplification and detection in conjunction with the heating module and fluorescence detection module of the external instrument.

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