CN118146913A - Kit, kit manufacturing method and amplification detection method - Google Patents

Abstract

The invention discloses a kit, a kit manufacturing method and an amplification detection method, which relate to the technical field of small-sized amplification equipment, in particular to a kit related to portable amplification equipment; the first sealing film can be attached to the substrate layer at first, so that the accommodating cavity and the connecting runner are opened on one side, at the moment, a specified liquid reagent or a specified solid reagent can be added into the specified accommodating cavity, and then the second sealing film is attached to the substrate layer, so that the various reagents in the multiple detection scene are pre-packaged easily in a low-cost manner, and the kit has great advantages in subsequent storage and transportation.

Description

Kit, kit manufacturing method and amplification detection method

Technical Field

The invention relates to the technical field of small-sized amplification equipment, in particular to a kit related to portable amplification equipment, and in particular relates to a kit, a kit manufacturing method and an amplification detection method.

Background

The molecular diagnosis is found from polymerase to be stably applied to perfection of a fluorescence detection scheme, the cost of various types of molecular diagnosis equipment which are developed at present is relatively high, the popularization and application difficulty in the medical weak areas is high, molecular POCT equipment has the advantage of small volume, most of molecular diagnosis operations performed in hospitals or inspection institutions are integrated on a single microfluidic chip, stronger professional skills are not required, and the molecular diagnosis equipment is the optimal choice for strengthening the medical capability in the medical weak areas.

The molecular POCT designed by most factories at present basically aims at simple operation of sample input and output, the whole machine design involves a plurality of different steps of sample processing, nucleic acid extraction and purification and amplification detection, in order to connect part of factories of each step in series, more chambers are designed and microfluidic valves are arranged between different chambers, in addition, the molecular POCT requires more amplification chambers in the development process to execute more multi-target detection, the patents with publication numbers US10793849B2 and US10919040B2 disclose an integrated detection kit, a user only needs to put a collecting cotton swab into a receiving position and then seal the collecting cotton swab, the matched equipment can fully automatically finish sample extraction and purification, and the purified samples are distributed into a plurality of different chambers to finish an independent amplification scheme of corresponding targets in the plurality of chambers, so that the whole operation is simple, but the transfer of fluid in the reagent kit in the different chambers depends on the driving of an external driving source, and a flow control valve is required to be arranged between the different chambers, so that the whole volume is finally larger, the cost of a single reagent kit is high, and the processing precision requirement of the reagent kit is particularly high; the kit disclosed in the publication number US11786906B2 is characterized in that different reagents are configured at different positions, and samples are transferred by driving of a related driving mechanism in the device, so that the flow path design is simpler, but in order to realize quantitative detection of digital PCR, an amplification detection cavity is configured into a honeycomb structure, the distribution resistance of the structure is larger, the amount of amplification detection targets is smaller, and 3-weight, 5-weight or even more detection expansion of the same sample is difficult to realize; in the scheme disclosed in the patent publication No. CN110283940B, a disc-shaped POCT kit is designed and 5 amplification detection chambers are configured to realize simultaneous detection of 10 targets listed by the kit, however, the technical scheme has more problems in actual mass production, such as small requirement on the size of a flow channel in a structure, high requirement on processing precision, large difficulty in packaging various reagents in the amplification chamber, no reliable sealing of the amplification chamber in the process of executing amplification, serious bubbles, evaporation and the like, and of course, similar centrifugal driving designs also have the schemes disclosed in application Nos. CN201710371949.6 and CN201711091035.0, the microfluidic valve and the unique return air flow channel configuration are adopted, but the problem of higher cost exists; in the scheme disclosed in the patent publication No. HK1235735A, an integrated valve structure capable of rotationally switching the communication relationship is designed, different chambers are communicated through rotation at different angles, and chambers which do not participate in the process are not communicated with each other at other angles, so that extraction, purification and amplification operations can be performed without interference, however, the processing precision of the most important integrated valve in the scheme directly determines the accuracy of communication control, especially in POCT equipment mainly comprising a micro-sized runner, so that the quality improvement of products under quantitative production is difficult; the technology disclosed in the patent with the publication number AU2023214374A1 is a constant temperature detection technology, the equipment design is simpler, but the primer probe design is more complex so that the single-person detection cost of the equipment is higher.

From the analysis, the development of the molecular POCT equipment has strong advantages for the improvement of the medical level in areas with weak basic level and medical capability, which is the consensus of various manufacturers, however, the single detection cost of the molecular POCT designed at present is not low, and most of the kits are difficult to execute multi-target multi-detection, the volume of the equipment capable of executing single multi-target detection on the market is very large, the processing cost is high, the processing precision requirement is high, and the design of a kit with lower cost, simple processing and low precision requirement and capable of executing multi-target detection is a technical problem to be solved.

Disclosure of Invention

The invention aims at: in view of the above problems, the present invention provides a kit, a method for manufacturing a kit, and an amplification detection method, in which a substrate layer of the kit is divided into two first sealing films and two second sealing films capable of being attached and sealed, and the substrate layer of the kit can be conveniently processed to form a required accommodating chamber and a connecting flow channel by using schemes such as mechanical cutting or integral injection molding, so that the processing is convenient and the cost is low; the first sealing film can be attached to the substrate layer at first, so that the accommodating cavity and the connecting runner are opened on one side, at the moment, a specified liquid reagent or a specified solid reagent can be added into the specified accommodating cavity, and then the second sealing film is attached to the substrate layer, so that the various reagents in the multiple detection scene are pre-packaged easily in a low-cost manner, and the kit has great advantages in subsequent storage and transportation.

The technical scheme adopted by the invention is as follows:

The kit comprises a multi-cavity body, wherein the multi-cavity body comprises a substrate layer, a first sealing film and a second sealing film, two sides of the thickness direction of the substrate layer are two opposite bonding surfaces, the first sealing film and the second sealing film are respectively correspondingly connected with the two bonding surfaces of the substrate layer, the substrate layer is provided with a containing cavity and a connecting runner, the containing cavity and the connecting runner are non-sealing structures with bonding openings formed on the bonding surfaces of the substrate layer, a plurality of containing cavities are mutually spaced and can be in fluid communication through the connecting runner, and the first sealing film and the second sealing film are mutually matched to seal the bonding openings of the containing cavity and the connecting runner; the accommodating chamber comprises a buffer chamber, a system chamber, an amplification chamber and a liquid separation driving chamber, wherein liquid reagents or solid reagents can be configured in the buffer chamber, the system chamber and the amplification chamber; the connecting flow channel comprises a first flow channel capable of communicating the amplification cavity with the liquid separation driving cavity, a second flow channel capable of communicating the system cavity with the amplification cavity, and a transfer flow channel communicating the buffer cavity with the system cavity.

The substrate layer comprises a convex processing part and a plane main body part, one side of the convex processing part is a flush side flush with the plane main body part, and the other side of the convex processing part is a protruding side protruding out of the plane main body part; the system cavity and the buffer cavity are accommodating cavities which are configured on the convex treatment part and are in groove shapes, and an opening is formed on the corresponding flush side of the system cavity and the buffer cavity and a corresponding protruding side is closed; the amplification cavity and the liquid separation driving cavity are accommodating cavities which are arranged on the plane main body part and have hollow structures.

The amplification cavity is internally provided with at least one group of primer probe reagents corresponding to the target, or the amplification cavity is internally provided with at least one group of primer probe reagents corresponding to the target and at least one group of primer probe reagents corresponding to the internal standard; the primer probe reagent is configured according to the concentration of a forward primer and the concentration of a reverse primer in amplification system liquid in an amplification cavity of 0.4 mu M-0.8 mu M and the concentration of a probe of 0.2 mu M-0.4 mu M; and an amplification reaction reagent is arranged in the system cavity.

The non-joint surface of the substrate layer is provided with a driving gas injection port and a sample injection port which can be communicated with the buffer cavity, and a vent port which can be communicated with the system cavity.

The liquid separation driving cavity, the first sealing membrane and the second sealing membrane are surrounded to form a liquid separation driving unit, and the first sealing membrane and/or the second sealing membrane can deform when being subjected to pressure and recover after losing the pressure; the first sealing film and the first sealing film are light-permeable.

The multi-cavity pressing part is matched with the multi-cavity body, the flow valve can be limited in the valve groove, and a control area is formed between the flow control valve and the groove bottom of the valve groove; the matching part of the transfer flow channel and the valve groove extends from the parallel side to the protruding side to form two parallel rotary flow channels, the two rotary flow channels are communicated with the control area to form a rotary structure, and the flow control valve can open or close the rotary structure under the action of the valve control.

The plurality of amplification cavities are arranged at intervals along the width direction of the substrate layer, the plurality of liquid separation driving cavities, the plurality of first flow channels and the plurality of second flow channels are in one-to-one correspondence with the plurality of amplification cavities, and each amplification cavity is connected with the corresponding liquid separation driving cavity through the corresponding first flow channel and connected with the distribution flow channel through the corresponding second flow channel; the distribution flow channel comprises a flow collecting and transferring section arranged along the vertical direction of the substrate layer, and a flow collecting and distributing section arranged along the horizontal direction, one end of the flow collecting and transferring section is connected with the system cavity, the other end of the flow collecting and transferring section is connected with the flow collecting and distributing section, the amplification cavity is arranged between the flow collecting and distributing section and the system cavity, one ends of the second flow channels are communicated with the flow collecting and distributing section, the other ends of the second flow channels extend to the bottoms of the corresponding amplification cavities along the vertical direction, the bottom contours of the amplification cavities are matched, the bottom contours of the amplification cavities are bent and extend to the middle parts of the corresponding amplification cavities, and finally extend to the middle parts of the corresponding amplification cavities along the horizontal direction and are communicated with the middle parts of the corresponding amplification cavities, and the two ends of the second flow channel are formed with height differences along the vertical direction; one end of each first flow channel is connected to the top of the corresponding amplification cavity, and the other end of each first flow channel is connected to the liquid separation driving cavity.

A method of manufacturing a kit comprising the steps of:

Preparing a matrix layer: the method comprises the steps that a set accommodating chamber and a connecting runner are processed on a substrate layer, two opposite joint surfaces are arranged on two sides of the thickness direction of the substrate layer, the accommodating chamber and the connecting runner are non-closed structures with joint openings formed on the joint surfaces of the substrate layer, and different accommodating chambers can be communicated through the set connecting runner;

a first sealing film attaching step: attaching a first sealing film to the bonding surface of one side of the substrate layer to seal the opening on the side of the substrate layer;

And a reagent loading step: adding a specified liquid reagent or a specified solid reagent into a specified accommodating chamber;

a second sealing film attaching step: and attaching the second sealing film to the bonding surface at the other side of the substrate layer, so that the bonding openings on the substrate layer are completely sealed.

The accommodating chamber comprises a buffer chamber, a system chamber and an amplification chamber, wherein in the reagent loading step, an amplification reaction reagent is added into the system chamber, a liquid primer probe reagent is added into the amplification chamber, and the primer probe reagent in the amplification chamber is dried to obtain a dried primer probe reagent.

An amplification detection method, which is performed using the kit described above, comprises the following steps:

sample adding: filling sample liquid into a buffer cavity which is encapsulated with a cracking reagent from a sample filling port, and cracking the sample liquid under the action of the cracking reagent to form a sample treatment liquid; or filling sample treatment liquid into the empty buffer cavity;

Amplification preparation step: closing a sample adding port, loading a kit on amplification equipment, pressing at least part of a first sealing film and/or a second sealing film attached to a liquid separation driving cavity by a clamping device in the amplification equipment, and deforming the first sealing film and/or the second sealing film, wherein an air source driving piece is communicated with a buffer cavity through a driving air injection port, a sample processing liquid in the buffer cavity is driven by the air source driving piece to be transferred to a system cavity, the sample processing liquid is driven by the air source driving piece to be mixed with an amplification reaction reagent packaged in the system cavity to form liquid to be distributed, and the liquid to be distributed is contained in the system cavity;

And (3) liquid separation: driving the clamping device to loosen the first sealing membrane and/or the second sealing membrane, recovering the deformation parts of the first sealing membrane and/or the second sealing membrane and forming a suction effect, so that liquid to be distributed in the liquid cavity to be amplified is driven to be filled into the amplification cavity, and the liquid to be distributed is mixed with primer probe reagents packaged in the amplification cavity to form amplification system liquid;

Amplification detection step: the heating module of the amplification equipment applies a constant temperature or variable temperature effect on the amplification cavity to enable the amplification system liquid in the amplification cavity to generate an amplification reaction, and the amplified amplification system liquid is subjected to fluorescent detection to obtain an amplification detection result.

In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:

1. the kit is divided into the substrate layer, the first sealing film and the second sealing film which can be attached and sealed, and the substrate layer of the kit can be conveniently processed to form a needed accommodating cavity and a connecting runner by using schemes such as mechanical cutting or integral injection molding, and the like, so that the kit is convenient to process and low in cost.

2. According to the invention, the first sealing film can be attached to the substrate layer, so that the accommodating cavity and the connecting runner are opened at one side, at the moment, a specified liquid reagent or a specified solid reagent can be added into the specified accommodating cavity, and then the second sealing film is attached to the substrate layer, so that the pre-packaging of various reagents in a multiple detection scene is realized with low cost and easy operation, and the kit has great advantages in subsequent storage and transportation.

3. The invention can be provided with a plurality of amplification chambers for receiving probe reagents of different target primers, thereby realizing the low-cost execution of multiplex amplification detection.

4. The first sealing membrane and the second sealing membrane can deform when being subjected to pressure and recover after losing the pressure, and can be matched with the liquid separation driving cavity and the clamping device to generate driving force for filling the amplification cavity, extra components are not needed to be additionally arranged, and the development cost of the kit is further reduced.

5. The first sealing film and the first sealing film can meet the requirements of film strength, light transmittance and heat conduction performance.

6. The primer probe reagent corresponding to the target in the amplification cavity is configured according to the concentration of the forward primer and the concentration of the reverse primer in the amplification system liquid in the amplification cavity of 0.4 mu M-0.8 mu M and the concentration of the probe of 0.2 mu M-0.4 mu M, compared with the existing amplification system liquid, the primer probe reagent has higher concentration, can be suitable for a rapid heating scene through a thin film partition wall, particularly in the cyclic amplification of temperature rise and fall, has higher temperature change speed and higher amplification efficiency.

7. According to the invention, the primer probe reagent is dried and then sealed, so that the primer probe reagent can be reliably stored to the maximum extent, and the liquid to be distributed containing the target can be fully contacted and mixed with the primer probe reagent in the process of being distributed to the amplification cavity, so that the accurate component amount of the final amplification system liquid is ensured, meanwhile, the storage and transportation cost and the requirements of the kit are lower, and the primer probe reagent in a dried state can be firmly attached to the packaging film by means of electrostatic adsorption force and the like, and cannot be excessively lost due to airflow.

8. The amplification cavity is arranged between the collecting and distributing section and the system cavity, one end of the second flow channel is communicated with the collecting and distributing section, the other end of the second flow channel extends to the bottom of the corresponding amplification cavity along the vertical direction, then the bottom profile of the amplification cavity is matched, the amplification cavity is bent and extends to the middle part of the corresponding amplification cavity, finally the amplification cavity extends to be communicated with the middle part of the corresponding amplification cavity along the horizontal direction, and the two ends of the second flow channel are provided with height differences along the vertical direction, so that liquid reagent can be effectively prevented from flowing into the amplification cavity by mistake when the liquid separation step is not executed.

Drawings

FIG. 1 is an exploded view of a substrate layer, a first closure film and a second closure film of the present invention;

FIG. 2 is a diagram showing various reagents added after the substrate layer is attached to the first sealing film;

FIG. 3 is a state diagram of a kit formed by attaching a first sealing film and a second sealing film to a substrate layer and pre-packaging various reagents;

FIG. 4 is an exploded view of the kit of the present invention;

FIG. 5 is a state diagram of the present invention in which a liquid reagent is placed in a buffer chamber and a system chamber;

FIG. 6 is a state diagram of the present invention in which solid reagents are placed in buffer chambers and system chambers;

FIG. 7 is a state diagram of the invention in which a sample solution is added to a buffer chamber and a lyophilized lysis reagent is dissolved;

FIG. 8 is a state diagram of transferring a sample processing liquid in a buffer chamber to a system chamber according to the present invention;

FIG. 9 is a state diagram of the system chamber of the present invention containing a liquid to be dispensed;

FIG. 10 is a schematic diagram of the structure of the extraction processing consumable of the present invention;

FIG. 11 is a state diagram of the invention for filling an amplification chamber;

FIG. 12 is a state diagram of the invention in which the filling of the amplification chamber is completed;

FIG. 13 is a test result of the present invention for performing an amplification test using one kit;

FIG. 14 is a test result of the present invention using another kit to perform an amplification test;

FIG. 15 is a side cross-sectional view of a fluidic valve in a cartridge actuated closed by a valve control in the device;

FIG. 16 is a side cross-sectional view of a fluidic valve in a cartridge actuated open by a valve control in the device;

FIG. 17 is a front view of the closure device in a closing operation;

Fig. 18 is a cross-sectional view taken in the direction A-A of the closure device of fig. 17.

The marks in the figure: 1-substrate layer, 2-first closing film, 3-second closing film, 4-extraction treatment consumable, 10-amplification chamber, 11-first runner, 12-second runner, 20-liquid separation driving part, 201-liquid separation driving unit, 211-closing auxiliary part, 30-system chamber, 31-distribution runner, 311-current collecting and transferring section, 312-current collecting and distributing section, 32-driving gas injection port, 321-buffer through hole, 40-buffer chamber, 41-flow control valve, 42-sample adding port, 421-box cover, 43-vent port, 431-vent through hole, 44-transfer runner, 340-air flow regulating part, 400-valve control part, 410-valve slot, 500-closing device, 510-multi-cavity pressing part, 512-valve pressing part, 520-multi-cavity body, 521-side clamping part, 522-top clamping unit, 523-valve control hole, 600-clamping device.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

Referring to fig. 1, which is an explosion state diagram of a substrate layer 1, a first sealing film 2 and a second sealing film 3 of the kit, the kit comprises a substrate layer 1, two opposite bonding surfaces are arranged on two sides of the substrate layer 1 in the thickness direction, the substrate layer 1 comprises a convex processing part at the upper part and a plane main body part at the lower part, in order to ensure that the substrate layer 1 is processed conveniently, the substrate layer 1 is obtained in an injection molding mode, and can be produced in batch by opening a mold, the overall cost is very low, and the manufacturing material of the substrate layer 1 can be one of acrylic (PMMA), acrylonitrile Butadiene Styrene (ABS), nylon polyamide PA), polycarbonate (PC), polyethylene (PE), polyoxymethylene (POM), polypropylene (PP), polystyrene (PS), thermoplastic elastomer (TPE) and Thermoplastic Polyurethane (TPU), so that a high-quality through/non-through accommodating chamber and a connecting runner with a set size can be obtained; of course, the substrate layer 1 can be obtained by molding and processing materials such as silicon base and resin base, and then the accommodating chamber and the connecting runner are processed by cutting, etching and other processes, and the material of the substrate layer 1 is optimally a nonmetallic material, so that the situation that serious heat dissipation does not exist in the accommodating chamber when heat treatment is required can be ensured, and the thermal performance of a key area can be ensured to the greatest extent, and the method is not limited. The system cavity 30 and the buffer cavity 40 are configured on the protruding side of the protruding processing part, the system cavity 30 and the buffer cavity 40 form a combined opening corresponding to the flush side, the corresponding protruding side is closed, the non-closed accommodating cavity is adopted in injection molding or other processing, on one hand, the material use can be reduced, the application of the external acting in the accommodating cavity is convenient, on the other hand, the processing quality in the accommodating cavity is higher, a valve hole for assembling a fluid control valve 41 is formed between the accommodating cavities, the valve hole is also configured to be of a non-through type, the opening side of the valve hole corresponds to the protruding side of the protruding processing part, the planar main body part is configured with a plurality of amplifying cavities 10 penetrating through the planar main body part, and a plurality of liquid separating driving cavities penetrating through the planar main body part, the number of the liquid separating driving cavities corresponds to the number of the amplifying cavities 10, and the liquid separating driving cavities are arranged at intervals with the amplifying cavities 10; in order to ensure that the amplification chamber 10 and the liquid-dividing driving chamber have preset reasonable volumes, the thickness range of the planar main body part is 1mm-3mm, the too small thickness indicates that the sectional area of the amplification chamber 10 must be increased, so that larger heating elements need to be matched, the strength and the reliability of the planar main body part are also affected, the too large thickness indicates that the sectional area of the amplification chamber 10 must be reduced, the heating power of the heating elements needs to be more concentrated, meanwhile, the too large thickness causes the heat transfer condition in the amplification chamber 10 to be deteriorated, and a rapid and uniform heating effect cannot be realized.

FIG. 2 is a state diagram of various reagents added after the substrate layer 1 of the kit is attached with the first sealing film 2; the first sealing film 2 is attached to the upper side of the bonding surface of the substrate layer 1 corresponding to the protruding side of the protruding processing portion, that is, the opposite side of the substrate layer 1 in the view of fig. 2, and the amplification chamber 10 and the side of the liquid-dividing driving chamber corresponding to the protruding side (that is, the opposite side as described above) are sealed, in which state, the amplification chamber 10 is in the shape of an open container, the amplification chamber 10 can receive the primer probe reagent therein, in which case, the amplification chamber 10 can receive the primer probe reagent in the liquid state, and then the drying process is performed for a predetermined time by the drying apparatus, and the primer probe reagent in the dried state is obtained, in addition, since the first sealing film 2 is attached only to the opposite side, both the system chamber 30 and the buffer chamber 40 are still in the open state, the method is characterized in that the amplification reaction reagent in the form of freeze-dried beads is only configured in the system cavity 30, then the second sealing film 3 attaching step can be executed, the second sealing film 3 is attached on the combination surface of the substrate layer 1 corresponding to the flush side of the convex treatment part, as shown in fig. 3, a state diagram of a kit formed by attaching the first sealing film 2 and the second sealing film 3 on the substrate layer 1 and pre-packaging various reagents is shown, the first sealing film 2 and the second sealing film 3 are respectively attached on the two opposite combination surfaces of the substrate layer 1, so that the buffer cavity 40, the system cavity 30, the amplification cavity 10, the liquid separation driving cavity and the combination openings formed on the substrate layer 1 in the communication flow passage are all sealed, thus the pre-packaging configuration of different reagents in different accommodating cavities can be simply and efficiently realized, compared with the existing POCT kit, the production efficiency is greatly improved, the cost is lower, the process complexity is smaller, the first sealing film 2 and the second sealing film 3 are attached to the two opposite bonding surfaces of the substrate layer 1 in the same way, the bonding can be glue connection or hot fusion bonding (including thermoplastic sealing and other ways), corresponding production equipment can be a glue beating machine or a plastic sealing machine, in addition, the materials of the first sealing film 2 and the second sealing film 3 are the same and are selected from at least one of materials such as propylene, polyethylene, polystyrene, cycloolefin copolymer (COC), polyester, polyacetate, leather, rubber and the like, in order to ensure that fluorescence detection has higher accuracy in the amplification process, here, the first sealing film 2 and the second sealing film 3 optimally have a light transmittance of 80% -95%, and in order to satisfy the requirements of film strength, light transmittance and heat conductive property, the thicknesses of the first sealing film 2 and the second sealing film 3 are optimally configured to be 0.02mm-0.12mm. In the closed kit, the amplification chamber 10 can directly contact with a heat source for heat transfer through the first sealing film 2 and/or the second sealing film 3, so that the first sealing film 2 and the second sealing film 3 with optimal thickness can ensure the highest heat transfer efficiency, the liquid separation driving chamber and the attached first sealing film 2 and second sealing film 3 surround to form a liquid separation driving unit 201, the clamping device can be pressed to at least part of the first sealing film 2 and/or the second sealing film 3 corresponding to the liquid separation driving unit 201, the first sealing film 2 and/or the second sealing film 3 at the pressing position of the clamping device are deformed, and after the clamping device is released from the liquid separation driving unit 201, the deformed first sealing film 2 and/or second sealing film 3 can form a suction effect in the deformation recovery process, so that the liquid to be distributed in the system cavity 30 is filled in the amplification cavity 10, thus the deformation recovery piece is not required to be independently configured to serve as driving force for filling the amplification cavity 10, the first sealing film 2 and the second sealing film 3 with proper thickness have enough strength and deformation recovery capability, the whole kit is simpler in design, the problem that the light transmittance is influenced by the excessively thick first sealing film 2 and the second sealing film 3, the attaching area of the first sealing film 2 is matched with the plane main body part, the attaching area of the second sealing film 3 is matched with the convex treatment part and the plane main body part, the attaching area of the first sealing film 2 is smaller than that of the second sealing film 3, wherein the attaching area refers to the area of the attaching area except for the hollow or notch on the substrate layer 1, the attaching area of the first sealing film 2 is preferably configured to be 0.6-0.9 times that of the second sealing film 3, an excessive area difference may cause poor attaching firmness of the first sealing film 2 and the second sealing film 3, and an excessive small area difference may cause small occupied space of the protruding treatment part, so that the buffer cavity 40 and the system cavity 30 do not have sufficient configuration space.

In the following, a kit in which the first sealing film 2 and the second sealing film 3 are not explicitly shown will be described in further detail, and fig. 4 is an exploded view of the kit; the kit is an integrated diagnostic kit, can be adapted to small molecular diagnostic POCT equipment, is hereinafter referred to as 'equipment', comprises different configuration areas, is vertically inserted into the equipment in order to smoothly drive liquid to accurately transfer between different accommodating chambers without leakage risk in actual operation, enables different parts in the equipment to act on the kit to enable the liquid to flow into different accommodating chambers according to a preset path, is provided with a sample adding port 42 in an upper area of the vertical direction of the kit, is detachably connected with a box cover 421 in order to ensure the requirements of reagent stability and sealing operation in the kit, the sample inlet 42 and the lid 421 can be detachably connected by fastening or screwing. In order to meet the requirements of convenience in production and processing and low cost, the kit can be split into a plurality of parts, including a multi-cavity body 520, a multi-cavity pressing member 510, a flow control valve 41 and a flow control fitting 340, wherein the multi-cavity body 520 is a combination of the substrate layer 1, the first sealing membrane 2 and the second sealing membrane 3 described above; the buffer cavity 40, the system cavity 30, the amplifying cavity 10, the liquid-dividing driving cavity and the connecting runner are all configured in the multi-cavity body 520, the connecting runner comprises a first runner 11 capable of communicating the amplifying cavity 10 with the liquid-dividing driving cavity, a distributing runner 31 capable of communicating the system cavity 30, a second runner 12 capable of communicating the amplifying cavity 10 with the distributing runner 31 and a transferring runner 44 capable of communicating the buffer cavity 40 with the system cavity 30, and a flow control valve 41 is configured at least at one position of the transferring runner 44 so as to realize the aim of changing the communicating relation between the buffer cavity 40 and the system cavity 30 by changing the opening of the flow control valve 41. The flow control valve 41 is arranged in the valve groove 410 on the multi-cavity body 520, the multi-cavity pressing member 510 which is matched and connected with the multi-cavity body 520 is also provided with a valve pressing part 512, when the multi-cavity pressing member 510 is matched with the multi-cavity body 520, the flow control valve 41 can be limited in the valve groove 410 through the valve pressing part 512, the middle part of the valve pressing part 512 is provided with a through valve control hole 523, the valve control 400 can act on the flow control valve 41 through the valve control hole 523, so that the flow control valve 41 can open or close the transfer flow passage 44, and in particular, a control area is formed between the flow control valve 41 and the groove bottom of the valve groove 410; the matching part of the transfer flow channel 44 and the valve groove 410 extends from the parallel side to the protruding side to form two parallel rotary flow channels, the two rotary flow channels are communicated with the control area to form a rotary structure, and the flow control valve 41 can open or close the rotary structure under the action of the valve control 400. The multi-cavity body 520 and the multi-cavity pressing member 510 are configured in a detachable fastening connection mode, in order to ensure the reliability of fastening connection, a side clamping portion 521 is arranged on the side wall of the multi-cavity body 520 corresponding to the convex processing portion, a top clamping unit 522 is arranged on the top, a side clamping unit capable of being clamped with the side clamping portion 521 and a top clamping portion capable of being clamped with the top clamping unit 522 are arranged at the multi-cavity pressing member 510, and of course, the reagent cartridge in actual use can also be configured in other detachable connection or directly configured in non-detachable connection such as gluing, fusing and the like. The air flow regulating member 340 is disposed in a regulating groove at the top of the multi-cavity body 520, the regulating groove comprises a buffer through hole 321 communicated with the buffer cavity 40, and further comprises a ventilation through hole 431 communicated with the system cavity 30, the top of the multi-cavity pressing member 510 is provided with a driving air injection port 32 and a ventilation port 43, and after the multi-cavity body 520 is cooperatively connected with the multi-cavity pressing member 510, the driving air injection port 32 is communicated with the buffer cavity 40 through the air flow regulating member 340, and the ventilation port 43 is communicated with the system cavity 30 through the air flow regulating member 340. In order to ensure that the buffer cavity 40 and the system cavity 30 have larger volumes and can basically transfer the liquid therein without residues, the buffer cavity 40 and the system cavity 30 are formed into basically the same structure, the buffer cavity 40 and the system cavity 30 are respectively provided with a column containing part with an upper part and a uniform section and a cone flow guiding part with a bottom section gradually reduced from top to bottom, one end of the transfer flow channel 44 is connected with the flow guiding part at the bottom of the buffer cavity 40, the other end is connected with the flow guiding part at the bottom of the system cavity 30, the bottom flow guiding part of the system cavity 30 is also connected with the distribution flow channel 31, and the distribution flow channel 31 and the transfer flow channel 44 are arranged at intervals.

The middle area of the kit is provided with at least two amplification cavities 10, the specific number of the amplification cavities 10 can be set according to the requirement, the amplification cavities 10 can be optimally configured into even numbers in order to ensure the balance of flow resistance, the amplification cavities 10 are optimally configured into shapes with variable cross-sectional areas, and the shapes comprise liquid drop types or liquid drop-like types, egg shapes, ellipsoids, cone-like bodies or other gyrorotors, so that the full development in the liquid filling process can be ensured, the probability of generating an air gap is smaller due to smaller turbulence degree, the filling degree in the final amplification cavity 10 is higher, the filling degree of 95% -100% can be realized, and the subsequent amplification is more reliable and the detection accuracy is higher. The liquid separation driving unit 20 is disposed at the lower part of the kit, and the liquid separation driving unit 20 may include liquid separation driving units 201 corresponding to the number of the amplification chambers 10 in order to ensure that the liquid separation driving unit 20 can apply uniform driving force to the liquid filling in the plurality of amplification chambers 10. In this embodiment, a connection port of the first flow channel 11 is configured at the top of the amplification chamber 10, and the other end of the first flow channel 11 is connected to the corresponding liquid separation driving unit 201, and optimally the first flow channel 11 needs to be connected to the top of the liquid separation driving unit 201, so that the top area of the amplification chamber 10 can be filled last, the possibility of gas residue is reduced, and the filling degree is ensured to be higher; in this embodiment, the area of the amplification chamber 10 near the top, specifically, the area above the center line of the amplification chamber 10, includes a configuration portion with a cross-sectional area gradually decreasing from bottom to top, and the configuration portion can exert a certain compression constraint on the liquid to be filled subsequently in the amplification chamber 10 during the liquid filling process, so as to have a rectifying effect, and finally, the filling degree of the amplification chamber 10 can be basically close to 100%, and basically no gas remains. The dispensing liquid inlet of the amplification chamber 10, that is, the connection port of the second flow channel 12, is disposed near the center line of the amplification chamber 10, so that the dual effects of gravity and abrupt cross-section change can be utilized, so that the liquid filled into the amplification chamber 10 can be basically adhered to the wall to reduce the probability of turbulence and air gap generation, and if the connection port of the second flow channel 12 is disposed at the bottom of the amplification chamber 10, a high-turbulence filling state similar to a fountain is more likely to be generated, which is unfavorable for high-filling and air gap-free filling. The distribution flow channel 31 comprises a flow collecting and transferring section 311 arranged along the vertical direction of the substrate layer, and a flow collecting and distributing section 312 arranged along the horizontal direction, wherein the flow collecting and transferring section 311 and the flow collecting and distributing section 312 form a T-shaped structure, one end of the flow collecting and transferring section 311 is connected with the system cavity 30, the other end is connected with the flow collecting and distributing section 312, the amplification cavity 10 is arranged between the flow collecting and distributing section 312 and the system cavity 30, one ends of a plurality of second flow channels 12 are communicated with the flow collecting and distributing section 312, the other ends extend to the bottom of the corresponding amplification cavity 10 along the vertical direction, the bottom outline of the matched amplification cavity 10 is bent and extended to the middle part of the corresponding amplification cavity 10, finally, the liquid reagent extends along the horizontal direction to be communicated with the middle part of the corresponding amplification cavity 10, and the two ends of the second flow channel 12 are formed with a height difference along the vertical direction, so that the liquid reagent can be effectively prevented from flowing into the amplification cavity 10 by mistake when the liquid separation step is not executed, the turbulence degree in the process of transferring the liquid in the system cavity 30 to the amplification cavity 10 is smaller due to the design that a plurality of second flow channels 12 are communicated with the collecting and distributing section 312, the liquid can be distributed to all amplification cavities 10 more uniformly, and the effect is similar to the flute-shaped tube effect in a fluid conveying scene. In order to ensure that the amplification chambers 10 can be amplified uniformly and reliably without interference in the present embodiment, the first flow channel 11 and the second flow channel 12 can be sealed by the sealing device 500 before amplification, and in order to reduce the material consumption of the sealing device 500 or reduce the energy consumption, and simultaneously ensure the sufficient release of the internal stress in the sealing process, a plurality of hollow closing auxiliary parts 211 are arranged between the first flow channel 11 and the second flow channel 12 at intervals, and because the sealing device 500 is close to the flush side of the convex treatment part, the closing auxiliary parts 211 are open grooves arranged on the substrate layer 1, the combined openings of the open grooves face the sealing device 500, and the open groove is closed by the second closing film 3, and the depth of the open groove is equal to or greater than the depths of the first flow passage 11 and the second flow passage 12.

As shown in fig. 5, in the state diagram of the liquid reagent in the buffer cavity and the system cavity, the liquid lysis reagent may be pre-packaged in the buffer cavity 40, the liquid amplification reaction reagent may be pre-packaged in the system cavity 30, the primer probe reagent in the dried state may be configured in the amplification cavity 10, the sample solution may be directly added into the buffer cavity 40, the sample solution is fully lysed after an external force is applied or no external force is applied for a certain time, the nucleic acid sequence fragment is released to obtain the sample processing solution, one side of the buffer cavity 40 corresponding to the second sealing membrane 3 is a membrane sealing side, and an external action may be applied to the membrane sealing side of the buffer cavity 40, for example, an ultrasonic vibration is applied to enable the sample solution in the buffer cavity 40 to be fully lysed by the pre-packaged lysis reagent. In another scenario, as shown in fig. 10, an extraction processing consumable 4 matched with a kit is configured, an extraction processing liquid can be configured in the extraction processing consumable 4, a collected sample is obtained through a swab, a blood collector can be used for obtaining a blood sample in some cases, the sample is fully shaken and uniformly mixed after being added into the extraction processing consumable 4 to obtain a sample processing liquid, the buffer cavity 40 can receive the sample processing liquid provided by the extraction processing consumable 4, the buffer cavity 40 in the kit can be not configured with any reagent and is only used for buffering and storage, the clamping device 600 applies clamping force to the liquid-separating driving part 20 at the bottom to deform the liquid-separating driving part, the air pressure in the amplification cavity 10 is increased, the liquid is prevented from entering the amplification cavity 10 when the liquid is transferred between the buffer cavity 40 and the system cavity 30, then the valve control 400 drives the flow control valve 41 to open the transfer flow channel 44, the air source driving part can drive the liquid in the buffer cavity 40 to enter the system cavity 30 and can also drive the liquid to transfer back and forth between the buffer cavity 40 and the system cavity 30 for a plurality of times, the liquid components to be fully mixed, the liquid to be distributed can be transferred, and the liquid to be transferred can be kept to the system cavity 30 is kept waiting for the step of the flow control valve control 41 to be closed, and the flow control 41 is closed when the flow control is performed.

Example 2

Example 2, which replaces the liquid lysis reagent and liquid amplification reaction reagent of example 1, is an alternative to example 1; further, the same components are not described herein, as shown in fig. 6, in order to provide a state diagram of placing solid reagents in the buffer chamber 40 and the system chamber 30, where the solid reagents may be freeze-dried beads, a freeze-dried lysis reagent is disposed in the buffer chamber 40, and a freeze-dried amplification reaction reagent is disposed in the system chamber 30, so that the reagents disposed in the chambers may not affect each other without disposing any other valve in the kit. When in use, as shown in fig. 7, a sample liquid is added into the buffer cavity 40, an external action can be applied to the membrane sealing side of the buffer cavity 40, for example, ultrasonic vibration is applied to enable the freeze-dried lysis reagent in the buffer cavity 40 to be fully dissolved into the sample liquid and enable the sample liquid to be fully lysed to form a sample processing liquid, then the flow control valve 41 is matched with the air source driving piece to drive the sample processing liquid into the system cavity 30, as shown in fig. 8, the freeze-dried lysis reagent in the system cavity 30 is dissolved into the sample processing liquid, at the moment, the liquid can be driven to reciprocate in the system cavity 30 and the buffer cavity 40, so that the sample processing liquid and the amplification reaction reagent in the system cavity 30 are fully mixed, finally, the liquid to be dispensed is obtained, as shown in fig. 9, the liquid to be dispensed is driven to be transferred into the system cavity 30, and the liquid separation step is waited for execution.

Example 3

Example 3 is a further modification of example 1, example 2; further, the same components are not described herein, and the primer probe reagent corresponding to the target in the amplification chamber 10 is configured according to the forward primer concentration and the reverse primer concentration in the amplification system liquid in the amplification chamber 10 being 0.4 μm to 0.8 μm and the probe concentration being 0.2 μm to 0.4 μm, so that compared with the existing amplification system liquid, the primer probe concentration is higher, the method can be suitable for a rapid heating scene through a thin film partition wall, particularly in the cycle amplification of a temperature rise and fall, the temperature change speed is faster, the amplification efficiency is higher, the primer probe reagent is dried and then sealed, the primer probe reagent can be stored reliably to the greatest extent, the liquid to be distributed containing the target can be fully contacted and mixed with the primer probe reagent in the process of being distributed to the amplification chamber 10, so as to ensure that the components of the final amplification system liquid are accurate, meanwhile, the cost and the requirement of the kit are lower, the primer probe reagent in a storage and transportation state can be firmly attached to the first film and the second film or the second film due to the fact that the air flow is lost (due to excessive liquid state and the air flow is not lost) by electrostatic adsorption force and the like. Two primer probe reagents for multiple detection of different objects are respectively pre-packaged in a plurality of amplification chambers 10, amplification reaction reagents containing reverse transcriptase, taq enzyme, mgCl2, glycerol, tris-HCl and dNTP are pre-packaged in a system chamber 30, so that after liquid to be distributed is driven into the amplification chambers 10, the liquid can be mixed with the primer probe reagents to form an amplification system liquid shown in the following table 1.

TABLE 1 multiplex detection of amplification System liquid Components for two different objects

Of course, the above table only shows the reagent contents of a part of the amplification system solution, and actually contains water, nucleic acid fragments, and the like. After the completion of the dispensing in the amplification chamber 10, the amplification chamber 10 is closed, and an amplification detection operation is performed, and in this embodiment, the rapid amplification of the amplification system liquid can be achieved using the amplification parameters as shown in table 2 below.

TABLE 2 amplification parameters

The amplification procedure can ensure that the whole detection process is not longer than 25min or shorter, and the reliability of the detection result of the kit is higher.

Fig. 13 and 14 show the detection results of performing amplification detection using two kits after the same target primer probe is disposed in each of the plurality of amplification chambers 10, wherein the detection targets are alphavirus nucleic acid fragments, the internal targets are mature commercial internal standard fragments, and the two detection results show that the target sequences disposed in each hole site show a more uniform amplification form as shown in a curve cluster a in the figure, and the internal standard sequences show a more uniform amplification form as shown in a curve cluster B in the figure, which also proves that the amplification system liquid can be uniformly and consistently distributed in the amplification chambers 10 of the kit according to the invention, and the heat transfer state in each amplification chamber 10 is also substantially uniform, so that the detection interference risk is lower.

Example 4

A method of manufacturing a kit comprising the steps of:

Preparing a substrate layer 1: the method comprises the steps that a set accommodating chamber and a connecting runner are processed on a substrate layer 1, two opposite joint surfaces are arranged on two sides of the substrate layer 1 in the thickness direction, the accommodating chamber and the connecting runner are non-closed structures with joint openings formed on the joint surfaces of the substrate layer 1, and different accommodating chambers can be communicated through the set connecting runner; the accommodating chamber comprises a buffer chamber 40, a system chamber 30, an amplifying chamber 10 and a liquid-separating driving chamber, the connecting flow passage comprises a first flow passage 11 capable of communicating the amplifying chamber 10 with the liquid-separating driving chamber, a second flow passage 12 capable of communicating the system chamber 30 with the amplifying chamber 10, and a transfer flow passage 44 communicating the buffer chamber 40 with the system chamber 30, a valve slot for loading a flow control valve 41 is processed at the position corresponding to the transfer flow passage 44, a blending groove is processed at the top of the substrate layer 1, a buffer through hole 321 for communicating the buffer chamber 40 with the blending groove is processed between the buffer chamber 40 and the blending groove, and a buffer through hole 431 for communicating the system chamber 30 with the blending groove is processed between the system chamber 30 and the blending groove;

The first closing film 2 attaching step: attaching a first sealing film 2 to the bonding surface of one side of the substrate layer 1 to seal the opening on the side of the substrate layer 1;

And a reagent loading step: adding a specified liquid reagent or a specified solid reagent into a specified accommodating chamber; wherein optionally a lysing agent is added to the buffer chamber 40; adding an amplification reaction reagent into the system chamber 30; adding a liquid primer probe reagent into the amplification cavity 10, and drying the primer probe reagent in the amplification cavity 10 to obtain a dried primer probe reagent;

The second sealing film 3 attaching step: the second sealing film 3 is attached to the bonding surface on the other side of the substrate layer 1, so that the bonding openings on the substrate layer 1 are all sealed to form the multi-cavity body 520.

Assembling: the air flow adjusting part 340 is assembled in the blending groove, the flow control valve 41 is assembled in the valve groove, the multi-cavity pressing part 510 is assembled in the multi-cavity body 520, the multi-cavity pressing part 510 is provided with a sample adding port 42 which can be communicated with the buffer part, and the box cover 421 is assembled in the sample adding port 42.

Example 5

An amplification detection method, performed using the kit provided in example 1, 2 or 3, as shown in FIGS. 15 to 18, comprises the following steps of:

Sample adding: filling sample liquid into the buffer cavity 40 which is packaged with the cracking reagent from the sample filling port 42, and cracking the sample liquid under the action of the cracking reagent to form sample treatment liquid; or filling the empty buffer chamber 40 with a sample processing liquid;

Amplification preparation step: closing the sample adding port 42, loading the kit into amplification equipment, pressing at least part of the first closing film 2 and/or the second closing film 3 attached to the liquid separation driving cavity by the clamping device 600 in the amplification equipment, and deforming the first closing film and/or the second closing film 3, wherein an air source driving piece is communicated with the buffer cavity 40 through driving the air injection port 32, the air source driving piece drives sample treatment liquid in the buffer cavity 40 to be transferred to the system cavity 30, the air source driving piece drives the sample treatment liquid to be mixed with amplification reaction reagent packaged in the system cavity 30 to form liquid to be distributed, and the liquid to be distributed is contained in the system cavity 30;

And (3) liquid separation: the clamping device 600 is driven to loosen the first sealing membrane 2 and/or the second sealing membrane 3, the deformation parts of the first sealing membrane 2 and/or the second sealing membrane 3 are restored and form a suction effect, so that liquid to be distributed in the liquid cavity to be amplified is driven to be filled into the amplification cavity 10, and the liquid to be distributed is mixed with primer probe reagents packaged in the amplification cavity 10 to form amplification system liquid;

And (3) a sealing step: after the amplification chamber 10 is filled, the sealing device 500 is driven to seal the first flow channel 11 and the second flow channel 12, so that the amplification chamber 10 in a filled state is isolated and sealed;

Amplification detection step: the heating module of the amplification equipment applies a constant temperature or a variable temperature action to the amplification cavity 10, so that amplification system liquid in the amplification cavity 10 generates an amplification reaction, and fluorescent detection is carried out on the amplified amplification system liquid to obtain an amplification detection result.

The principles and embodiments of the present invention have been described herein with reference to specific examples, which are intended to be merely illustrative of the methods of the present invention and their core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

In the description of the present invention, it should be noted that, directions or positional relationships indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., are directions or positional relationships based on those shown in the drawings, or are directions or positional relationships conventionally put in use of the inventive product, are merely for convenience of describing the present invention and simplifying the description, and are not indicative or implying that the apparatus or element to be referred to must have a specific direction, be constructed and operated in a specific direction, and thus should not be construed as limiting the present invention.

In the description of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.

Claims (10)

1. The kit is characterized by comprising a multi-cavity body, wherein the multi-cavity body comprises a substrate layer, a first sealing film and a second sealing film, two opposite bonding surfaces are arranged on two sides of the thickness direction of the substrate layer, the first sealing film and the second sealing film are respectively and correspondingly connected with the two bonding surfaces of the substrate layer, a containing cavity and a connecting runner are arranged on the substrate layer, the containing cavity and the connecting runner are non-sealing structures with bonding openings formed on the bonding surfaces of the substrate layer, a plurality of containing cavities are mutually spaced and can be in fluid communication through the connecting runner, and the first sealing film and the second sealing film are mutually matched to seal the bonding openings of the containing cavity and the connecting runner; the accommodating chamber comprises a buffer chamber, a system chamber, an amplification chamber and a liquid separation driving chamber, wherein liquid reagents or solid reagents can be configured in the buffer chamber, the system chamber and the amplification chamber; the connecting flow channel comprises a first flow channel capable of communicating the amplification cavity with the liquid separation driving cavity, a second flow channel capable of communicating the system cavity with the amplification cavity, and a transfer flow channel communicating the buffer cavity with the system cavity.

2. The kit of claim 1, wherein the base layer comprises a raised treatment portion and a planar body portion, the raised treatment portion having one side that is flush with the planar body portion and the other side that is a protruding side that protrudes from the planar body portion; the system cavity and the buffer cavity are accommodating cavities which are configured on the convex treatment part and are in groove shapes, and an opening is formed on the corresponding flush side of the system cavity and the buffer cavity and a corresponding protruding side is closed; the amplification cavity and the liquid separation driving cavity are accommodating cavities which are arranged on the plane main body part and have hollow structures.

3. The kit of claim 1, wherein the amplification chamber is configured with no less than one set of primer probe reagents corresponding to the target, or the amplification chamber is configured with no less than one set of primer probe reagents corresponding to the target, and no less than one set of primer probe reagents corresponding to the internal standard; the primer probe reagent is configured according to the concentration of a forward primer and the concentration of a reverse primer in amplification system liquid in an amplification cavity of 0.4 mu M-0.8 mu M and the concentration of a probe of 0.2 mu M-0.4 mu M; and an amplification reaction reagent is arranged in the system cavity.

4. The kit according to claim 1, wherein the non-bonding surface of the substrate layer is provided with a driving gas injection port and a sample injection port which can be communicated with the buffer cavity, and a vent port which can be communicated with the system cavity.

5. The kit of claim 1, wherein the liquid separation driving chamber and the first and second sealing films are surrounded to form a liquid separation driving unit, and the first sealing film and/or the second sealing film can deform when being subjected to pressure and recover after losing pressure; the first sealing film and the first sealing film are light-permeable.

6. The kit according to claim 2, further comprising a multi-chamber pressing member and a flow control valve for controlling the on-off of the transfer flow passage, wherein a valve groove is provided at the boss processing portion, an assembly opening of the valve groove corresponds to the protruding side, the multi-chamber pressing member can restrict the flow valve in the valve groove when being matched with the multi-chamber body, and a control area is formed between the flow control valve and a bottom of the valve groove; the matching part of the transfer flow channel and the valve groove extends from the parallel side to the protruding side to form two parallel rotary flow channels, the two rotary flow channels are communicated with the control area to form a rotary structure, and the flow control valve can open or close the rotary structure under the action of the valve control.

7. The kit of any one of claims 1-6, wherein a plurality of amplification chambers are arranged at intervals along the width direction of the substrate layer, a plurality of liquid separation driving chambers, a plurality of first flow channels and a plurality of second flow channels are in one-to-one correspondence with the plurality of amplification chambers, and each amplification chamber is connected with the corresponding liquid separation driving chamber through the corresponding first flow channel and is connected with the distribution flow channel through the corresponding second flow channel; the distribution flow channel comprises a flow collecting and transferring section arranged along the vertical direction of the substrate layer, and a flow collecting and distributing section arranged along the horizontal direction, one end of the flow collecting and transferring section is connected with the system cavity, the other end of the flow collecting and transferring section is connected with the flow collecting and distributing section, the amplification cavity is arranged between the flow collecting and distributing section and the system cavity, one ends of the second flow channels are communicated with the flow collecting and distributing section, the other ends of the second flow channels extend to the bottoms of the corresponding amplification cavities along the vertical direction, the bottom contours of the amplification cavities are matched, the bottom contours of the amplification cavities are bent and extend to the middle parts of the corresponding amplification cavities, and finally extend to the middle parts of the corresponding amplification cavities along the horizontal direction and are communicated with the middle parts of the corresponding amplification cavities, and the two ends of the second flow channel are formed with height differences along the vertical direction; one end of each first flow channel is connected to the top of the corresponding amplification cavity, and the other end of each first flow channel is connected to the liquid separation driving cavity.

8. A method of manufacturing a kit, comprising the steps of:

Preparing a matrix layer: the method comprises the steps that a set accommodating chamber and a connecting runner are processed on a substrate layer, two opposite joint surfaces are arranged on two sides of the thickness direction of the substrate layer, the accommodating chamber and the connecting runner are non-closed structures with joint openings formed on the joint surfaces of the substrate layer, and different accommodating chambers can be communicated through the set connecting runner;

a first sealing film attaching step: attaching a first sealing film to the bonding surface of one side of the substrate layer to seal the opening on the side of the substrate layer;

And a reagent loading step: adding a specified liquid reagent or a specified solid reagent into a specified accommodating chamber;

a second sealing film attaching step: and attaching the second sealing film to the bonding surface at the other side of the substrate layer, so that the bonding openings on the substrate layer are completely sealed.

9. The method of manufacturing a kit according to claim 8, wherein the accommodating chamber comprises a buffer chamber, a system chamber and an amplification chamber, and in the step of loading the reagent, an amplification reaction reagent is added into the system chamber, a liquid primer probe reagent is added into the amplification chamber, and the primer probe reagent in the amplification chamber is dried to obtain the primer probe reagent in a dried state.

10. An amplification detection method performed using the kit according to any one of claims 1 to 7, comprising the steps of:

sample adding: filling sample liquid into a buffer cavity which is encapsulated with a cracking reagent from a sample filling port, and cracking the sample liquid under the action of the cracking reagent to form a sample treatment liquid; or filling sample treatment liquid into the empty buffer cavity;

Amplification preparation step: closing a sample adding port, loading a kit on amplification equipment, pressing at least part of a first sealing film and/or a second sealing film attached to a liquid separation driving cavity by a clamping device in the amplification equipment, and deforming the first sealing film and/or the second sealing film, wherein an air source driving piece is communicated with a buffer cavity through a driving air injection port, a sample processing liquid in the buffer cavity is driven by the air source driving piece to be transferred to a system cavity, the sample processing liquid is driven by the air source driving piece to be mixed with an amplification reaction reagent packaged in the system cavity to form liquid to be distributed, and the liquid to be distributed is contained in the system cavity;

And (3) liquid separation: driving the clamping device to loosen the first sealing membrane and/or the second sealing membrane, recovering the deformation parts of the first sealing membrane and/or the second sealing membrane and forming a suction effect, so that liquid to be distributed in the liquid cavity to be amplified is driven to be filled into the amplification cavity, and the liquid to be distributed is mixed with primer probe reagents packaged in the amplification cavity to form amplification system liquid;

Amplification detection step: the heating module of the amplification equipment applies a constant temperature or variable temperature effect on the amplification cavity to enable the amplification system liquid in the amplification cavity to generate an amplification reaction, and the amplified amplification system liquid is subjected to fluorescent detection to obtain an amplification detection result.