Animal epidemic disease integrated rapid detection card based on microfluidic technology and preparation method thereof

Abstract

The application belongs to the technical field of animal epidemic disease detection and microfluidic chip, and provides an animal epidemic disease integrated rapid detection card based on microfluidic technology and a preparation method thereof, wherein the animal epidemic disease integrated rapid detection card based on microfluidic technology comprises a card shell, a gradient purification sample processing unit, a self-constant-temperature microfluidic amplification unit and a double-labeled specific immune detection unit are coaxially and sealingly connected in the card shell in sequence along a fluid transmission direction, and a fluid transmission path is formed in a full-closed and breakpoint-free mode; the application solves the pain points of the existing epidemic disease detection technology, such as dispersed process, easy cross contamination, dependence on laboratory equipment and high operation threshold, and the whole process is full-closed and non-opening, non-professional personnel can complete the whole process detection by simple pressing operation, the application is suitable for laboratory-free scenes such as primary breeding farms and field ports, the modular structure is easy to be industrialized and batch-prepared, the reagent storage stability is excellent, and multiple target diseases can be synchronously and accurately screened.

Description

Technical Field

[0001] This invention belongs to the fields of animal disease detection and microfluidic chip technology, and particularly relates to an integrated rapid detection card for animal diseases based on microfluidic technology and its preparation method. Background Technology

[0002] With the large-scale development of livestock and poultry farming, rapid on-site screening of animal diseases has become a core link in disease prevention and control and public health security. Existing animal disease detection technologies mostly rely on large-scale laboratory equipment, with cumbersome operating procedures, long testing cycles, and the need for professional personnel. They cannot meet the on-site testing needs of grassroots farms, port quarantine, and field sampling where there are no laboratory facilities.

[0003] Microfluidic technology, due to its high integration, low reagent consumption, and fast detection speed, has become a core research and development direction for rapid on-site detection. However, existing related technologies still have many unresolved problems:

[0004] First, the integration level is insufficient. Sample pretreatment, nucleic acid amplification, and immunoassay are mostly segmented operations, requiring multiple openings and transfers of the sample, which can easily cause contamination and prevent true sample input and result output.

[0005] Secondly, it has poor adaptability to complex samples. Impurities and amplification inhibitors in samples such as whole blood, feces, and tissue homogenates cannot be effectively removed, which can easily lead to flow channel blockage and detection failure.

[0006] Third, the nucleic acid amplification process relies on external temperature control equipment. Stable amplification cannot be completed without the necessary equipment on-site, thus limiting its applicability.

[0007] Fourth, simultaneous detection of multiple targets is prone to cross-contamination and lacks specificity.

[0008] Fifth, the valve control structure is complex and the operation steps are cumbersome, making it difficult for non-professionals to complete the operation. In addition, the preparation process is complex and large-scale production is difficult.

[0009] Therefore, an integrated rapid detection card for animal diseases based on microfluidic technology and its preparation method are needed to solve the above problems. Summary of the Invention

[0010] The purpose of this invention is to provide an integrated rapid detection card for animal diseases based on microfluidic technology and its preparation method, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: an integrated rapid detection card for animal diseases based on microfluidic technology, comprising a card shell, wherein a gradient purification sample processing unit, a self-temperature-controlled microfluidic amplification unit, and a dual-label specific immunoassay unit are sequentially and coaxially connected and sealed along the fluid transport direction inside the card shell, forming a fully enclosed and uninterrupted fluid transport pathway; the upper shell of the card shell has a sample application port and a visualization observation window, the sample application port being directly opposite and sealed to the sample inlet of the gradient purification sample processing unit, and the visualization observation window being directly opposite the position of the dual-label specific immunoassay unit.

[0012] The gradient purification sample processing unit is composed of a gradient pore size three-dimensional sample filter membrane, a silicon-modified lysis-binding membrane, and a nucleic acid-specific purification membrane stacked sequentially along the fluid transport direction. The outlet of the nucleic acid-specific purification membrane is connected to the inlet of the self-temperature-controlled microfluidic amplification unit through a first guide channel. The gradient pore size three-dimensional sample filter membrane is arranged with a gradient increase in pore size along the fluid transport direction, which can filter large particulate impurities and cell debris in the sample in stages to avoid flow channel blockage. The silicon-modified lysis-binding membrane can simultaneously complete the lysis of sample cells and the binding of nucleic acids. The nucleic acid-specific purification membrane can retain and purify nucleic acids and remove amplification inhibitors from the sample.

[0013] The self-isothermal microfluidic amplification unit includes a cyclic olefin copolymer chip substrate. A directional flow-guiding microfluidic channel is formed on the surface of the cyclic olefin copolymer chip substrate. This directional flow-guiding microfluidic channel includes a liquid inlet channel, a gradient expansion-contraction chaotic mixing channel, and an isothermal amplification reaction chamber connected sequentially. The inner wall of the gradient expansion-contraction chaotic mixing channel is symmetrically arranged with periodically arrayed arc-shaped flow-guiding protrusions. Adjacent arc-shaped flow-guiding protrusions form expansion-contraction channel units. The inner diameter of the channel increases in a segmented gradient along the fluid transport direction, which reduces capillary resistance and simultaneously creates a chaotic mixing effect, improving the uniformity of sample and reagent mixing. The cyclic olefin copolymer chip substrate also integrates a fully pre-packaged reagent pre-storage chamber assembly. This reagent pre-storage chamber assembly is connected to the gradient expansion-contraction chaotic mixing channel through independent branch channels, enabling full-process pre-packaging of the detection reagents without the need for on-site reagent addition.

[0014] The casing is equipped with a shaped phase change energy storage temperature control layer that is precisely coupled to the position of the isothermal amplification reaction chamber. The shaped phase change energy storage temperature control layer is equipped with a heat insulation structure on the outside, which can maintain the temperature stability of the isothermal amplification reaction chamber under normal ambient temperature, meet the temperature requirements of nucleic acid isothermal amplification, and eliminate the need for external temperature control equipment.

[0015] The dual-label specific immunoassay unit includes a polyvinyl chloride (PVC) base plate. A sample pad, a label binding pad, a solid-phase carrier membrane, and an absorbent pad are sequentially overlapped on the surface of the PVC base plate along the fluid transport direction. The solid-phase carrier membrane has parallel and spaced multi-target detection lines and control lines. Each detection line is coated with a specific capture substance paired with a hapten label of the corresponding pathogen amplification product. The control lines are coated with a specific secondary antibody, enabling simultaneous specific detection of multiple pathogens and avoiding cross-contamination.

[0016] The casing also houses a two-stage press-type sequential valve control assembly, which includes a single press-type valve rod. The valve rod has elastic sealing plugs and reagent chamber puncture structures corresponding to the flow channels. The press-type valve rod achieves two-stage locking control via a limiting groove within the casing. When pressed to the first position, the lysis reagent chamber and washing reagent chamber open, while the first flow guide remains normally closed, completing the sample lysis and impurity washing process. When pressed to the second position, the elution reagent chamber and isothermal amplification reagent chamber open, and the first and second flow guides open sequentially, completing the entire process of nucleic acid elution, isothermal amplification, and immunoassay. This achieves programmed sequential on / off switching of the fully enclosed flow channel, making operation simple.

[0017] In a further technical solution, the gradient pore size three-dimensional sample filter membrane is made of glass fiber, the silicon-modified lysis binding membrane is a glass fiber membrane modified with silanol, the membrane surface is pre-coated with cell lysis reagent and nucleic acid specific binding reagent, and the nucleic acid specific purification membrane group is composed of multiple silicon-modified polyvinylidene fluoride membranes stacked together, the pore size of the multiple membranes gradually decreases, which can improve the nucleic acid purification effect.

[0018] In a further technical solution, the arc-shaped guiding protrusions on the inner wall of the gradient expansion-contraction chaotic mixing channel are arranged symmetrically and periodically. The protrusion structure is integrally formed with the inner wall of the channel. The expansion-contraction channel unit can disrupt the laminar flow state of the fluid, achieve dead volume-free mixing, and improve the utilization rate of nucleic acid templates.

[0019] A further technical solution is that the fully pre-packaged reagent pre-storage chamber group includes a lysis reagent chamber, a washing reagent chamber, an elution reagent chamber, and an isothermal amplification reagent chamber. Each reagent chamber is sealed in the groove of the cyclic olefin copolymer chip substrate by heat-pressing with a sealing film. Each reagent chamber corresponds to an independent branch channel. The opening and closing of the branch channel is controlled by a two-stage press-type timing valve control component to match the standard procedure timing of nucleic acid detection.

[0020] A further technical solution is that the shaped phase change energy storage temperature control layer is made of shaped composite phase change material and encapsulated in a sealed protective structure. It has a high coverage area with the isothermal amplification reaction chamber. The heat insulation structure is made of heat insulation cotton material, which can reduce heat exchange and maintain the temperature stability of the amplification chamber.

[0021] A further technical solution is that the detection line group of the dual-label specific immunoassay unit includes multiple independent detection lines, with the detection lines and control lines arranged in parallel and spaced apart. The specific capture substance coated on each detection line is paired one-to-one with the hapten labeled at the end of the corresponding pathogen amplification primer. The label binding pad is pre-coated with multiple specific label conjugates, which specifically bind to the amplification products of different target pathogens, thereby achieving simultaneous detection of multiple targets without cross-interference.

[0022] In a further technical solution, the push-type valve stem of the two-stage push-type sequential valve control assembly is made of food-grade polymer material, the elastic sealing plug is made of silicone material, and it is press-fitted with the flow channel to achieve a normally closed seal. The reagent chamber puncture structure is made of metal material, with the needle tip facing the sealing membrane of the reagent pre-storage chamber group. An anti-rebound buckle is set in the limiting groove, which automatically locks after being pressed to the corresponding position to avoid operational errors.

[0023] A further technical solution is that the casing includes an upper shell and a lower shell that interlock with each other, both of which are precision injection molded from polymer material. A sealing ring is provided on the interlocking surface of the upper shell and the lower shell, and a fully sealed enclosure is achieved by ultrasonic welding. The surface of the lower shell is provided with positioning slots for fixing each functional unit. The coaxiality deviation of the flow channels of each functional unit is small, ensuring smooth fluid transmission.

[0024] The preparation method of the integrated rapid detection card for animal diseases based on microfluidic technology, applied to any of the above-mentioned integrated rapid detection cards for animal diseases based on microfluidic technology, includes the following steps:

[0025] S1 housing and temperature control component preparation: The upper and lower housings are prepared by precision injection molding. The sample feeding hole, visualization window, positioning slot, and two-stage press-type sequential valve control component mounting position are processed by machining. The transparent sheet is heat-sealed at the visualization window position, and the shaped phase change energy storage temperature control layer and heat insulation structure are fixed at the corresponding position of the lower housing to complete the temperature control component assembly.

[0026] Preparation of S2 gradient purification sample processing unit: Cut gradient pore size three-dimensional sample filter membrane, silicon-modified lysis-binding membrane, and nucleic acid specific purification membrane according to design size. Quantitatively spray cell lysis reagent and nucleic acid binding reagent onto the surface of silicon-modified lysis-binding membrane and vacuum dry it. Assemble each membrane material sequentially by automated assembly equipment and fix it in the corresponding positioning slot of the lower shell.

[0027] S3 self-temperature microfluidic amplification unit preparation: A cyclic olefin copolymer chip substrate with directional flow-guiding microfluidic channels is prepared using a precision hot-pressing process. A reagent pre-storage chamber is prepared by hot-pressing and sealing the corresponding groove of the chip substrate with a sealing film. The corresponding reagent is quantitatively injected and sealed. The cyclic olefin copolymer chip substrate is fixed in the positioning slot of the lower shell to complete the coaxial docking and sealing connection of the flow channels.

[0028] Preparation of S4 dual-label specific immunoassay unit: The detection lines and control lines are sequentially sprayed onto the surface of the solid carrier membrane using a three-dimensional spraying platform. After vacuum drying, the sample pad, the pre-coated labeled conjugate pad, the sprayed solid carrier membrane, and the absorbent pad are sequentially overlapped on the surface of the polyvinyl chloride base plate using automated equipment. The resulting strips are then cut into standard sizes and fixed in the corresponding positioning slots of the lower shell.

[0029] S5 Assembly and Finished Product Preparation: Assemble the two-stage press-type sequential valve control assembly into the corresponding installation position, ensuring that the elastic sealing plug is completely sealed with the corresponding flow channel, and that the reagent chamber puncture structure is aligned with the reagent pre-storage chamber group; place sealing rings on the interlocking surfaces of the upper and lower shells, and complete the fully enclosed interlocking seal through ultrasonic welding; use ethylene oxide sterilization process for processing, and aseptically vacuum seal to obtain the finished product.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] This invention integrates a gradient purification sample processing unit, a self-temperature-controlled microfluidic amplification unit, and a dual-label specific immunoassay unit into a single casing through a fully enclosed, coaxially connected, and sealed integrated architecture. Combined with a fully pre-packaged reagent storage chamber, it achieves a closed-loop integration of the entire process from sample pretreatment to nucleic acid amplification and result detection. This eliminates the need for manual sample transfer, completely avoids aerosol contamination and cross-contamination during the detection process, and ensures the accuracy of the test results.

[0032] This invention employs a three-dimensional sample filter membrane with progressively increasing pore size, a silicon-modified lysis-binding membrane, and a nucleic acid-specific purification membrane to form a three-stage progressive purification structure. It can be directly adapted to loading complex samples such as whole blood, feces, and tissue homogenates without the need for additional pretreatment operations such as centrifugation and dilution. It effectively removes impurities and amplification inhibitors from the sample, ensuring the efficiency and stability of subsequent nucleic acid amplification. At the same time, the progressively increasing pore size design can avoid flow channel blockage and improve the service life and reliability of the test card.

[0033] This invention, through the precise coupling design of the shaped phase change energy storage temperature control layer and the isothermal amplification reaction chamber, eliminates the need for any external temperature control equipment. Relying on the energy storage characteristics of the phase change material and the protective effect of the thermal insulation structure, it can maintain the temperature stability of the isothermal amplification reaction chamber, meet the temperature requirements of nucleic acid isothermal amplification, completely eliminate the dependence on laboratory temperature control equipment, and can be directly applied to grassroots sites without laboratory conditions, greatly expanding the applicable scenarios of the detection technology.

[0034] This invention employs a single-stem, two-stage press-type sequential valve control assembly. Through two-stage locking, it achieves programmed sequential on / off of the reagent chamber and flow channel. Operators only need to complete sample addition and two press operations to complete the entire detection process. The operation steps are extremely simple, requiring no professional training. Non-professionals can quickly learn to use it, solving the problems of cumbersome operation and high barriers to entry in existing technologies.

[0035] This invention employs a dual-label detection scheme combining hapten labeling and paired specific capture. Multiple target detection lines are independently arranged and specifically paired, enabling simultaneous detection of various animal disease pathogens. There is no cross-interference between targets, resulting in high detection specificity. Furthermore, each functional unit adopts a modular design, coupled with high-precision positioning slots, enabling fully automated assembly and production. This simplifies the preparation process, ensures good batch consistency, and has value for large-scale industrialization and promotion.

[0036] This invention employs a fully enclosed flow channel design and aseptic packaging process, resulting in excellent overall sealing of the test card. Reagents are pre-stored within the sealed cavity, preventing them from becoming damp or inactivated, and ensuring excellent storage stability. Test results can be directly read by the naked eye through a visual observation window, eliminating the need for additional reading equipment and significantly improving the convenience of on-site testing.

[0037] To more clearly illustrate the structural features and effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0038] Figure 1 This is a block diagram of the overall architecture of the present invention;

[0039] Figure 2 This is a block diagram of the gradient purification sample processing unit submodule of the present invention;

[0040] Figure 3 This is a block diagram of the self-temperature-controlled microfluidic amplification unit submodule of the present invention;

[0041] Figure 4 This is a block diagram of the dual-label specific immunoassay unit submodule of the present invention;

[0042] Figure 5 This is a block diagram of a submodule of the two-stage press-type sequential valve control component of the present invention;

[0043] Figure 6 This is a schematic diagram of the overall process of the present invention.

[0044] In the diagram: 1. Chassis; 2. Gradient purification sample processing unit; 21. Gradient pore size three-dimensional sample filter membrane; 22. Silicon-modified lysis binding membrane; 23. Nucleic acid-specific purification membrane assembly; 24. First flow channel; 3. Self-heating microfluidic amplification unit; 31. Cyclic olefin copolymer chip substrate; 32. Liquid inlet channel; 33. Gradient contraction-expansion chaotic mixing channel; 34. Isothermal amplification reaction chamber; 35. Second flow channel; 36. Reagent pre-storage chamber Group; 4. Dual-label specific immunoassay unit; 41. Polyvinyl chloride base plate; 42. Sample pad; 43. Label binding pad; 44. Solid phase carrier membrane; 441. Detection line; 442. Quality control line; 45. Absorbent pad; 5. Shaped phase change energy storage temperature control layer; 6. Two-stage press-type sequential valve control assembly; 61. Press-type valve stem; 62. Elastic sealing plug; 63. Reagent chamber puncture structure; 64. Limiting groove; 65. Anti-rebound buckle. Detailed Implementation

[0045] The present invention will be further described below with reference to embodiments.

[0046] The following embodiments are used to illustrate the present invention, but should not be used to limit the scope of protection of the present invention. The conditions in the embodiments can be further adjusted according to specific conditions, and simple improvements to the method of the present invention under the premise of the concept of the present invention are all within the scope of protection claimed by the present invention.

[0047] Example 1

[0048] like Figure 1-6 As shown, this embodiment of the invention provides an integrated rapid detection card for single-target animal diseases, including a card case 1. Inside the card case 1, a gradient purification sample processing unit 2, a self-temperature microfluidic amplification unit 3, and a dual-label specific immunoassay unit 4 are sequentially and coaxially connected and sealed along the fluid transport direction.

[0049] The upper shell 13 of the casing 1 has a sample loading port and a visualization window. The sample loading port is sealed and connected to the sample inlet of the gradient purification sample processing unit 2. The visualization window is directly opposite the position of the dual-label specific immunoassay unit 4. The gradient purification sample processing unit 2 is sequentially stacked with a gradient pore size three-dimensional sample filter membrane 21, a silicon-modified lysis binding membrane 22, and a nucleic acid specific purification membrane group 23. The liquid outlet is connected to the self-temperature-controlled microfluidic amplification unit 3 through the first guide channel 24.

[0050] The surface of the cyclic olefin copolymer chip substrate 31 of the isothermal microfluidic amplification unit 3 has a liquid inlet channel 32, a gradient expansion chaotic mixing channel 33, and an isothermal amplification reaction chamber 34. The inner wall of the gradient expansion chaotic mixing channel 33 is symmetrically provided with arc-shaped guiding protrusions. The cyclic olefin copolymer chip substrate 31 integrates a reagent pre-storage chamber group 36, including a lysis reagent chamber, a washing reagent chamber, an elution reagent chamber, and an isothermal amplification reagent chamber. Each reagent chamber is sealed by heat-pressing with a sealing film.

[0051] The casing 1 has a fixed phase change energy storage temperature control layer 5 coupled to the isothermal amplification reaction chamber 34 inside, and is equipped with a heat insulation structure on the outside; the surface of the polyvinyl chloride base plate 41 of the dual-label specific immunoassay unit 4 is sequentially connected to the sample pad 42, the label binding pad 43, the solid phase carrier membrane 44, and the water-absorbing pad 45, and the solid phase carrier membrane 44 is provided with a single detection line 441 and a quality control line 442.

[0052] The housing 1 is equipped with a two-stage press-type sequential valve control assembly 6, including a press-type valve stem 61, an elastic sealing plug 62, a reagent chamber puncture structure 63, a limit groove 64, and an anti-rebound buckle 65, to realize two-stage gear sequential control.

[0053] In this embodiment, the detection card is designed for a single animal disease pathogen, with a simplified structure, and is suitable for on-site screening of a single disease. The gradient purification sample processing unit 2 can efficiently purify the nucleic acid of a single pathogen, the self-isothermal microfluidic amplification unit 3 completes specific isothermal amplification, and the dual-label specific immunoassay unit 4 enables accurate result interpretation. The entire detection process can be completed by a two-stage press operation.

[0054] Example 2

[0055] The difference between this embodiment and Embodiment 1 is that: the surface of the solid-phase carrier membrane 44 of the dual-label specific immunodetection unit 4 is provided with multiple independent detection lines 441, which are arranged in parallel and at equal intervals. Each detection line 441 is coated with different specific capture substances, corresponding to the amplification products of different animal disease pathogens; the isothermal amplification reagent chamber in the reagent pre-storage chamber group 36 is pre-encapsulated with isothermal amplification reagents for multiple pathogens, which can simultaneously complete the amplification of nucleic acids of multiple pathogens.

[0056] In this embodiment, the detection card is a multi-target universal detection card that can simultaneously detect multiple common animal disease pathogens. Each detection line 441 is independently paired and there is no cross-interference. Multiple diseases can be screened in one test, which greatly improves the efficiency of on-site detection. It is suitable for scenarios such as farms and quarantine stations where multiple diseases are screened simultaneously.

[0057] Example 3

[0058] The difference between this embodiment and embodiment 2 is that: the nucleic acid-specific purification membrane 23 of the gradient purification sample processing unit 2 adopts a multi-layer composite membrane structure, and the membrane surface is hydrophilic to improve the nucleic acid adsorption and elution efficiency; the shaped phase change energy storage temperature control layer 5 adopts a high latent heat shaped composite phase change material, and the thickness of the heat insulation structure is increased to improve the temperature stability effect; the elastic sealing plug 62 of the two-stage press-type sequential valve control component 6 adopts a high elastic silicone material, which improves the sealing performance and service life.

[0059] In this embodiment, the detection card is a high-performance enhanced version, adapted for use in extreme field environments. The nucleic acid purification efficiency, temperature stability, and sealing reliability have been further optimized, and it can maintain stable detection performance in complex field environments. It is suitable for disease detection in harsh environments such as field sampling and remote areas.

[0060] Working principle and usage process of this invention:

[0061] This invention is based on the innovative coupling of microfluidic technology, nucleic acid isothermal amplification technology, immunolabeling detection technology, and phase change energy storage temperature control technology. Through a fully enclosed integrated architecture and time-sequential valve control design, it enables rapid on-site detection of animal disease pathogens. The specific workflow is as follows:

[0062] Sample loading and gradient purification stage: Whole blood, feces, tissue homogenate, and other samples awaiting testing are added to the test card through the loading wells. The samples first flow through a gradient-pore three-dimensional sample filter membrane 21. The gradient-increasing pore size structure filters out large particulate impurities, cell debris, fibrous tissue, etc., in the sample, preventing impurities from entering subsequent channels and causing blockage. The filtered samples continue to flow through a silicon-modified lysis-binding membrane 22. The lysis reagent on the membrane surface rapidly lyses the sample cells, releasing nucleic acid. The nucleic acid specifically binds to the silicon-modified sites on the membrane surface, completing the solid-phase capture of nucleic acid. The nucleic acid-bound membrane continues to pass through a nucleic acid-specific purification membrane 23. The membrane progressively retains nucleic acid and removes amplification inhibitors such as lipids, proteases, and salts from the sample, completing the gradient purification process of the sample.

[0063] First-position press-lysis-wash stage: Press the press-type valve stem 61 of the two-stage press-type sequential valve control component 6 to the first position. The anti-rebound buckle 65 automatically locks the position. At this time, the reagent chamber piercing structure 63 pierces the sealing membrane of the lysis reagent chamber and the washing reagent chamber. The lysis reagent and the washing reagent enter the gradient purification sample processing unit 2 through independent branch channels. The lysis reagent further enhances the cell lysis effect and ensures that the nucleic acid is fully released. The washing reagent rinses the membrane structure to remove unbound impurities and residual inhibitors. The nucleic acid is still stably retained on the nucleic acid specific purification membrane 23. During this stage, the first guide channel 24 remains normally closed to prevent the reagent and sample from entering the amplification unit in advance.

[0064] Second-position press-elution amplification stage: Press the press-type valve rod 61 to the second position, the anti-rebound buckle 65 locks again, the reagent chamber piercing structure 63 pierces the sealing membrane of the elution reagent chamber and the isothermal amplification reagent chamber, and the first guide groove 24 opens automatically at the same time; the elution reagent flows through the nucleic acid specific purification membrane group 23, eluting the retained nucleic acid. The eluted nucleic acid and the isothermal amplification reagent are mixed in the gradient shrinking and expanding chaotic mixing channel 33. The arc-shaped guide protrusion and the shrinking and expanding channel unit make the fluid produce a chaotic mixing effect. After the sample and reagent are fully mixed, they enter the isothermal amplification reaction chamber 34; the phase change energy storage temperature control layer 5 continuously releases or absorbs heat, and with the heat insulation structure, reduces the exchange of environmental heat, maintains the temperature stability of the isothermal amplification reaction chamber 34, and meets the reaction conditions for nucleic acid isothermal amplification. The nucleic acid template completes specific amplification under the action of the amplification reagent, generating amplification products with hapten labels.

[0065] Immunoassay and Result Interpretation Stage: After amplification, the second flow channel 35 automatically opens, and the amplified product enters the dual-labeled specific immunoassay unit 4 under capillary driving force, flowing sequentially through the sample pad 42 and the label conjugate pad 43. The specific label conjugate on the label conjugate pad 43 specifically binds to the amplified product, forming a composite detectable. The composite detectable continues to move along the solid-phase carrier membrane 44 by chromatography and is captured by the specific capture substance on the corresponding detection line 441, enriching to form a visually visible colored band. The unbound free label continues to chromatography to the control line 442 and is captured by the secondary antibody on the control line 442. The color development of the control line 442 completes the detection quality verification. Finally, the color development of the bands is directly observed through the visual observation window to complete the result interpretation. Color development of the control line indicates that the detection is valid, color development of the corresponding detection line indicates that the pathogen is positive, and no color development indicates that it is negative.

[0066] This invention adopts a fully enclosed, seamless flow channel design, eliminating the need for opening the lid throughout the entire process and completely preventing aerosol contamination and sample cross-contamination. The two-stage press-type operation is extremely simple, requiring no external equipment or professional skills, making it suitable for use by non-professionals at the grassroots level. The integrated design enables fully automated detection of the entire process from sample entry to result exit, providing a reliable technical solution for rapid on-site screening of animal diseases.

[0067] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An integrated rapid detection card for animal diseases based on microfluidic technology, characterized in that: The device includes a casing (1), inside which a gradient purification sample processing unit (2), a self-temperature microfluidic amplification unit (3), and a dual-label specific immunoassay unit (4) are sequentially and coaxially connected and sealed along the fluid transport direction, forming a fully enclosed and uninterrupted fluid transport pathway; the upper shell of the casing (1) has a sample loading hole and a visualization observation window, the sample loading hole is directly opposite and sealed to the sample inlet of the gradient purification sample processing unit (2), and the visualization observation window is directly opposite to the position of the dual-label specific immunoassay unit (4); The gradient purification sample processing unit (2) is provided with a gradient pore size three-dimensional sample filter membrane (21), a silicon-modified lysis binding membrane (22) and a nucleic acid specific purification membrane group (23) stacked sequentially along the fluid transport direction. The liquid outlet of the nucleic acid specific purification membrane group (23) is connected to the liquid inlet of the self-temperature microfluidic amplification unit (3) through the first guide groove (24). The self-temperature microfluidic amplification unit (3) includes a cyclic olefin copolymer chip substrate (31). The surface of the cyclic olefin copolymer chip substrate (31) is opened with a directional flow-guiding microfluidic channel. The directional flow-guiding microfluidic channel includes a liquid inlet channel (32), a gradient expansion-contraction chaotic mixing channel (33), and an isothermal amplification reaction chamber (34) connected in sequence. The inner wall of the gradient expansion-contraction chaotic mixing channel (33) is symmetrically provided with periodically arranged arc-shaped flow-guiding protrusions. An expansion-contraction channel unit is formed between adjacent arc-shaped flow-guiding protrusions. The inner diameter of the channel increases in a segmented gradient along the fluid transport direction. The cyclic olefin copolymer chip substrate (31) is also integrated with a fully pre-packaged reagent pre-storage chamber group (36). The reagent pre-storage chamber group (36) is connected to the gradient expansion-contraction chaotic mixing channel (33) through an independent branch channel. The casing (1) is provided with a shaped phase change energy storage temperature control layer (5) that is precisely coupled to the position of the isothermal amplification reaction chamber (34), and the shaped phase change energy storage temperature control layer (5) is provided with a heat insulation structure on the outside. The dual-label specific immunoassay unit (4) includes a polyvinyl chloride base plate (41). A sample pad (42), a label binding pad (43), a solid-phase carrier membrane (44), and an absorbent pad (45) are sequentially overlapped on the surface of the polyvinyl chloride base plate (41) along the fluid transport direction. The solid-phase carrier membrane (44) is provided with a group of multi-target detection lines and a quality control line (442) arranged in parallel and spaced intervals. Each detection line (441) is coated with a specific capture substance that is paired with the hapten label of the corresponding pathogen amplification product. The quality control line (442) is coated with a specific secondary antibody. The casing (1) is also equipped with a two-stage press-type sequential valve control assembly (6). The two-stage press-type sequential valve control assembly (6) includes a single press-type valve rod (61). The press-type valve rod (61) is equipped with an elastic sealing plug (62) corresponding to the flow channel and a reagent chamber puncture structure (63). The press-type valve rod (61) achieves two-stage locking control through the limiting groove (64) in the casing (1). When pressed to the first position, the lysis reagent chamber and the washing reagent chamber are opened, and the first guide groove (24) remains normally closed. When pressed to the second position, the elution reagent chamber and the isothermal amplification reagent chamber are opened, and the first guide groove (24) and the second guide groove (35) are opened in sequence.

2. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The gradient pore size three-dimensional sample filter membrane (21) is made of glass fiber, and the pore size is set in a gradient increasing manner along the fluid transmission direction; the silicon-modified lysis binding membrane (22) is a glass fiber membrane modified with silanol, and the surface of the membrane is pre-coated with cell lysis reagent and nucleic acid specific binding reagent; the nucleic acid specific purification membrane group (23) is composed of multiple silicon-modified polyvinylidene fluoride membranes stacked together.

3. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The arc-shaped guide protrusions on the inner wall of the gradient expansion chaotic mixing channel (33) are arranged symmetrically and periodically. The protrusion structure is integrally formed with the inner wall of the channel. The expansion channel unit can destroy the laminar flow state of the fluid to achieve mixing without dead volume.

4. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The fully pre-packaged reagent pre-storage chamber group (36) includes a lysis reagent chamber, a washing reagent chamber, an elution reagent chamber, and an isothermal amplification reagent chamber. Each reagent chamber is sealed in the groove of the cyclic olefin copolymer chip substrate (31) by hot pressing with a sealing film. Each reagent chamber corresponds to an independent branch channel.

5. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The shaped phase change energy storage temperature control layer (5) is made of shaped composite phase change material and is encapsulated in a sealed protective structure, with a high coverage area of ​​the contact area with the isothermal amplification reaction chamber (34); the heat insulation structure is made of heat insulation cotton.

6. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The detection line group of the dual-label specific immunoassay unit (4) includes multiple independent detection lines (441), and the detection lines (441) and the control lines (442) are arranged in parallel and spaced apart; the specific capture substance coated on each detection line (441) is paired one-to-one with the hapten labeled at the end of the corresponding pathogen amplification primer, and the label conjugate pad (43) is pre-coated with multiple specific label conjugates.

7. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The push-type valve stem (61) of the two-stage push-type sequential valve control assembly (6) is made of food-grade polymer material, the elastic sealing plug (62) is made of silicone material, and it is press-fitted with the flow channel to achieve normal closed sealing; the reagent chamber puncture structure (63) is made of metal material, and the limit groove (64) is equipped with anti-rebound buckle (65).

8. The integrated rapid detection card for animal diseases based on microfluidic technology according to claim 1, characterized in that: The casing (1) includes an upper casing and a lower casing that interlock with each other, both of which are precision injection molded from polymer materials; the interlocking surfaces of the upper casing and the lower casing are provided with sealing rings, and a fully sealed enclosure is achieved by ultrasonic welding; the surface of the lower casing is provided with positioning slots for fixing each functional unit.

9. A method for preparing an integrated rapid detection card for animal diseases based on microfluidic technology, applicable to the integrated rapid detection card for animal diseases based on microfluidic technology as described in any one of claims 1-8, characterized in that, Includes the following steps: S1 housing (1) and temperature control component preparation: The upper and lower housings are prepared by precision injection molding process. The sample feeding hole, visualization window, positioning slot, and two-stage press-type sequential valve control component (6) installation position are processed by machining. The transparent sheet is heat-sealed at the visualization window position, and the shaped phase change energy storage temperature control layer (5) and heat insulation structure are fixed at the corresponding position of the lower housing to complete the temperature control component assembly. Preparation of S2 gradient purification sample processing unit (2): Cut gradient pore size three-dimensional sample filter membrane (21), silicon-modified lysis binding membrane (22) and nucleic acid specific purification membrane group (23) according to the design size. Quantitatively spray cell lysis reagent and nucleic acid binding reagent on the surface of silicon-modified lysis binding membrane (22) and vacuum dry it. Assemble each membrane material sequentially by automated assembly equipment and fix it in the corresponding positioning slot of the lower shell. S3 self-temperature microfluidic amplification unit (3) preparation: a cyclic olefin copolymer chip substrate (31) with a directional flow microfluidic channel is prepared by precision hot pressing process. A reagent pre-storage cavity group (36) is prepared by hot pressing and sealing the corresponding groove of the chip substrate with a sealing film. The corresponding reagent is quantitatively injected and the sealing process is completed. The cyclic olefin copolymer chip substrate (31) is fixed in the positioning slot of the lower shell to complete the coaxial docking and sealing connection of the flow channel. Preparation of S4 dual-label specific immunoassay unit (4): The detection lines (441) and quality control lines (442) are sequentially sprayed on the surface of the solid carrier membrane (44) using a three-dimensional spraying platform. After vacuum drying, the sample pad (42), the pre-coated label conjugate pad (43), the sprayed solid carrier membrane (44), and the absorbent pad (45) are sequentially overlapped on the surface of the polyvinyl chloride base plate (41) using automated equipment. The strips are then cut into standard sizes and fixed in the corresponding positioning slots of the lower shell. S5 Assembly and Finished Product Preparation: Assemble the two-stage press-type sequential valve control assembly (6) to the corresponding installation position, ensuring that the elastic sealing plug (62) is completely sealed with the corresponding flow channel, and that the reagent chamber puncture structure (63) is aligned with the reagent pre-storage chamber group (36); place sealing rings on the interlocking surfaces of the upper and lower shells, and complete the fully enclosed interlocking seal by ultrasonic welding; use ethylene oxide sterilization process, and sterile vacuum seal to obtain the finished product.

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