Multi-layered self-driven microfluidic detection device and method for porcine disease and application

Abstract

The application provides a multi-pig disease hierarchical self-driven microfluidic detection device and method and application. The device comprises a self-driven microfluidic chip body, and sample pretreatment layers, multi-reaction layers and waste liquid absorption layers are sequentially layered and stacked along the fluid flow direction. The sample pretreatment layer is composed of a plasma separation membrane, an impurity filter membrane and a reagent release pad, realizing one-step plasma separation, filtration and marker release of whole blood samples; the multi-reaction layer is provided with at least one microfluid channel, and a binder pad, a hierarchical detection area and a quality control area are sequentially arranged in each microfluid channel, so that porcine blue ear disease, swine fever and porcine epidemic diarrhea can be simultaneously detected; the waste liquid absorption layer is overlapped with the end of the microfluid channel, and provides a capillary self-driven power, without the need of an external pump. The application also provides an application method of the device and application in preparation of a detection kit. The application does not need external equipment, is simple to operate, has high sensitivity and strong specificity, and is rapid in detection, and is suitable for on-site rapid detection scenes such as farms and primary veterinary stations.

Description

Technical Field

[0001] This invention relates to the field of animal disease detection technology, specifically to a multi-stage stratified self-driven microfluidic detection device, method, and application for swine diseases. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), classical swine fever (CSF), and porcine epidemic diarrhea (PED) are three highly contagious diseases threatening the pig industry. They often occur as mixed infections, making clinical diagnosis difficult and necessitating rapid, simultaneous detection of multiple pathogens. Among existing detection technologies, quantitative real-time PCR (qPCR) offers high sensitivity but requires specialized instruments and technicians, has a long testing cycle, and is difficult to apply on-site. Colloidal gold immunochromatography (CIGC) is simple to operate, but a single strip can only detect a single pathogen, and multiple strips are needed for multi-pathogen detection, resulting in high costs and susceptibility to matrix interference. Traditional microfluidic devices often rely on external pumps, are bulky and complex to operate, and suffer from cross-contamination and insufficient detection sensitivity. Current microfluidic devices for multi-pathogen detection in pigs primarily focus on nucleic acid amplification integration but lack efficient sample pretreatment modules, allowing blood cells and impurities to easily interfere with test results. Furthermore, their self-driving power design is imperfect, resulting in poor fluid flow stability and failing to meet the accuracy requirements of on-site testing. Therefore, developing a pig disease detection device that integrates efficient sample pretreatment, simultaneous multi-pathogen detection, and autonomous fluid drive is of great significance for improving the efficiency of disease prevention and control in the pig industry. Summary of the Invention

[0003] The purpose of this invention is to provide a multi-stage stratified self-driven microfluidic detection device, method, and application for swine diseases, in order to solve the problems of insufficient multi-stage detection capability, low sample pretreatment efficiency, reliance on external drive, and complex operation of existing swine disease detection devices.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0005] This invention provides a multi-layered self-driven microfluidic detection device for swine diseases, used to simultaneously detect at least one swine disease including porcine reproductive and respiratory syndrome (PRRS), classical swine fever (CSF), and porcine epidemic diarrhea (PED). The device is characterized by comprising a self-driven microfluidic chip body, wherein the chip body is provided with a sample pretreatment layer, a multi-layer reaction layer, and a waste liquid absorption layer stacked sequentially along the fluid flow direction.

[0006] The sample pretreatment layer includes a plasma separation membrane, an impurity filtration membrane, and a reagent release pad arranged sequentially along the fluid flow direction;

[0007] The multi-reaction layer is provided with at least one microchannel, and each microchannel is provided with a binding pad, a layered detection area and a quality control area in sequence along the fluid flow direction;

[0008] The waste liquid absorption layer overlaps with the end of the microchannel, providing capillary self-driving power to drive the sample fluid to flow sequentially through the sample pretreatment layer and the multi-reaction layer, and finally be absorbed by the waste liquid absorption layer.

[0009] Furthermore, the plasma separation membrane is made of glass fiber membrane or polyester membrane or hydrophilically treated porous membrane with a pore size of 0.22-0.45 μm and a thickness of 150-200 μm, with a plasma separation efficiency ≥95% and a blood cell retention rate ≥99%; the impurity filtration membrane is made of polyester nonwoven fabric or glass fiber with an areal density of 150-200 g / m² and a pore size of 5-10 μm; the reagent release pad is made of glass fiber membrane and pre-filled with one or more freeze-dried markers, wherein the markers are nanoparticles conjugated with specific antibodies against at least one of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSV), and porcine epidemic diarrhea virus (PEDV), or nanoparticles conjugated with specific nucleic acids.

[0010] Furthermore, the microchannel has a width of 1.5-2 mm and a depth of 0.8-1 mm, and its inner wall is treated with oxygen plasma, with a contact angle ≤60°; the conjugate pad is pre-loaded with three virus-specific antibodies or nucleic acids labeled with nanoparticles with a particle size of 100-200 nm, and the coupling concentration is 0.5-1 mg / mL; the layered detection area uses a nitrocellulose membrane, a cellulose carbonate membrane, or an AAO membrane, with a membrane pore size of 0.2-0.45 μm.

[0011] Furthermore, the layered detection area is immobilized with capture antigens, namely PRRSV N protein, CSFV E2 protein, and PEDV N protein, with a purity ≥95% and a spotting concentration of 0.6-0.8 mg / mL, respectively; the quality control area is immobilized with goat anti-rabbit IgG, with a spotting concentration of 0.7 mg / mL.

[0012] This invention also provides a detection method for the multi-connected stratified self-driven microfluidic detection device for swine diseases, comprising the following steps:

[0013] (1) Sample pretreatment: Take 50-200 μL of whole pig blood sample and add it to the plasma separation membrane. Filter naturally to obtain plasma. After the plasma flows through the impurity filter membrane, it enters the reagent release pad to dissolve and release the marker.

[0014] (2) Chip loading: The processed sample is dropped into the sample inlet of the multi-reaction layer. The fluid flows through the conjugate pad in the microchannel under the drive of capillary force, re-dissolves the label and forms a complex.

[0015] (3) Stratified detection: The complex flows into the stratified detection area and binds with the capture antibody to form a detection signal; the unbound labeled material flows into the quality control area and binds with goat anti-rabbit IgG to form a quality control signal;

[0016] (4) Result interpretation: The signal intensity of the detection area and the quality control area is read using a portable reader. When the signal intensity of the detection area is ≥ the mean of the negative control + 2 times the standard deviation and the color development of the quality control area is normal, it is judged as the corresponding virus positive. Quantitative detection is achieved by fitting the signal intensity with the standard curve.

[0017] Furthermore, the portable reader is a fluorescence reader with an excitation wavelength of 480-500nm, an emission wavelength of 520-540nm, and a detection resolution of ≤0.01 fluorescence intensity units.

[0018] Furthermore, the detection limits of the device for PRRSV, CSFV, and PEDV are 0.24 ng / mL, 0.38 ng / mL, and 0.56 ng / mL, respectively, with a detection time of ≤30 min, and it does not cross-react with porcine circovirus or porcine pseudorabies virus.

[0019] The present invention also discloses the application of the above method in the preparation of reagent kits or detection devices for the detection of porcine reproductive and respiratory syndrome (PRRS), classical swine fever, and / or porcine epidemic diarrhea (PED).

[0020] The present invention has the following beneficial effects:

[0021] This invention achieves one-step plasma separation, impurity filtration, and marker release from whole blood samples through a layered design of a sample pretreatment layer, a multi-reaction layer, and a waste liquid absorption layer, effectively reducing matrix interference. Multiple parallel microchannels combined with capillary self-drive enable autonomous fluid flow and simultaneous detection of multiple pathogens without any external pumps or centrifuges, making operation extremely simple and particularly suitable for on-site environments such as farms and grassroots veterinary stations. The use of nanoparticle labeling and a high-binding-strength detection zone significantly improves detection sensitivity and specificity, enabling simultaneous and accurate differentiation of porcine reproductive and respiratory syndrome (PRRS), classical swine fever (CSF), and porcine epidemic diarrhea (PED), without cross-reaction with other common swine pathogens. The device is compact, and the reagents can be stored at room temperature for extended periods, facilitating transportation and large-scale application. It provides an efficient and reliable technical means for the early rapid screening and precise control of highly contagious swine diseases. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0023] Figure 2 This is a schematic diagram of the layering of the present invention.

[0024] In the diagram: 1. Sample application well; 2. Waste liquid recovery area; 3. Porcine reproductive and respiratory syndrome virus (PRRSV) detection channel; 4. PRRSV conjugate pad; 5. PRRSV colorimetric detection area; 6. Classical swine fever virus (CSF) detection channel; 7. CSF conjugate pad; 8. STI virus colorimetric detection area; 9. STI virus conjugate pad; 10. STI virus colorimetric detection area. Detailed Implementation

[0025] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0026] refer to Figures 1-2 As shown, a multi-layered self-driven microfluidic detection device for swine diseases includes a self-driven microfluidic chip body. The self-driven microfluidic chip body has a sample pretreatment layer, a multi-layer reaction layer, and a waste liquid absorption layer stacked sequentially from top to bottom, with overall dimensions of 50mm × 30mm × 5mm. One end of the sample pretreatment layer has a sample dispensing port 1 for adding whole blood or plasma samples. The multi-layer reaction layer has three parallel detection channels: a porcine reproductive and respiratory syndrome virus (PRRSV) detection channel 3, a classical swine fever virus (CSF) detection channel 6, and a swine diarrhea virus (PDV) detection channel 9. Each detection channel has a corresponding conjugate pad and a colorimetric detection area arranged sequentially along the fluid flow direction; specifically, the PRRSV detection channel 3 has a PRRSV conjugate pad 4 and a PRRSV colorimetric detection area 5; the CSFV detection channel 6 has a CSFV conjugate pad 7 and a CSFV colorimetric detection area 8; and the PDVV detection channel 9 has a PDVV conjugate pad 10 and a PDVV colorimetric detection area 11. The ends of each detection channel are connected to the waste liquid recovery area 2, which overlaps with the waste liquid absorption layer to provide capillary self-driving power, so that the sample fluid flows autonomously through the pretreatment layer and each detection channel and enters the waste liquid recovery area 2, and the fluid flow and detection can be completed without external power.

[0027] Example 1: Preparation of the detection device

[0028] Sample pretreatment layer preparation: Plasma separation membrane: A glass fiber membrane (Whatman GF / D type) with a pore size of 0.45 μm and a thickness of 180 μm was selected. It was hydrophilically treated by immersing in a 10% (v / v) PEG-400 aqueous solution for 30 minutes, followed by drying in a vacuum drying oven at 40℃ for 2 hours, and then cut into 15 mm × 10 mm pieces for later use. Testing showed that this membrane achieved a plasma separation efficiency of ≥95% for porcine whole blood and a blood cell retention rate of ≥99%, effectively avoiding interference from blood cells in subsequent immune responses.

[0029] Impurity filtration membrane: A polyester nonwoven fabric (Shanghai Xingnuo Textile Materials Co., Ltd.) with a surface density of 180 g / m² and an average pore size of 8 μm is selected and cut to a size of 10 mm × 5 mm. It is stacked perpendicularly to the plasma separation membrane with partial overlap (overlap width 1-2 mm). This filtration membrane can efficiently retain particulate matter, fibrin clots, and cell debris in plasma, with a filtration efficiency ≥98%.

[0030] Reagent release pad: A glass fiber membrane (GF / D type, approximately 300 μm thick) was used. Three conjugates of porcine virus-specific antibodies and nanoparticles (anti-PRRSV, anti-CSFV, and anti-PEDV antibodies conjugated to 200 nm fluorescent microspheres, each at a concentration of 0.8 mg / mL) were applied using a spray-on method, with a loading volume of 2 μL / cm². After loading, the pads were dried at 37°C for 2 hours, vacuum-sealed, and stored at room temperature (25°C). Stability testing showed that after 6 months of storage at room temperature, the label release rate remained ≥90%, meeting practical application requirements.

[0031] Multi-reaction layer preparation: Substrate treatment: A transparent polycarbonate (PC) sheet (1.5 mm thick) was used. Three parallel microchannels were etched using a precision CNC milling machine. Each microchannel had dimensions of 20 mm in length, 1.8 mm in width, and 0.9 mm in depth, with a spacing of 3 mm between adjacent microchannels. After etching, the substrate was ultrasonically cleaned sequentially with deionized water and anhydrous ethanol for 10 minutes, and then dried with nitrogen. Subsequently, oxygen plasma surface treatment was performed (50 W power, 50 mL / min oxygen flow rate, 3 minutes treatment time). After treatment, the surface contact angle was measured with a contact angle tester, and the surface contact angle decreased from the initial 78° to 28°, indicating significantly enhanced hydrophilicity and sufficient capillary driving force.

[0032] Preparation of conjugate pads: Anti-PRRSV, anti-CSFV, and anti-PEDV monoclonal antibodies were conjugated to time-resolved fluorescent microspheres with a particle size of 200 nm (excitation wavelength 485 nm, emission wavelength 525 nm) using conventional EDC / NHS methods. The conjugation efficiency was determined to be ≥85% by UV spectrophotometry. The conjugates were diluted to 0.8 mg / mL with phosphate-buffered saline (PBS, containing 1% sucrose and 0.5% BSA) and evenly spread onto glass fiber membranes (GF / D type) at a sampling rate of 2 μL / cm². The membranes were dried at 37°C for 1 hour, then blocked with PBS containing 1% BSA for 30 minutes, and vacuum dried for later use.

[0033] Preparation of the layered detection zone: A 0.45 μm nitrocellulose membrane (NC membrane, Millipore HFC135 type) was used and fixed to the corresponding positions of the microchannels on the PC substrate. Three types of viral capture antigens were spotted separately using a spotting device.

[0034] PRRSV N protein (recombinant protein, purity ≥95%, spotting concentration 0.7 mg / mL);

[0035] CSFV E2 protein (recombinant protein, purity ≥95%, spotting concentration 0.6 mg / mL);

[0036] PEDV N protein (recombinant protein, purity ≥95%, spotting concentration 0.5 mg / mL). The spacing between each detection point was 2.5 mm, and the spotting volume was 1 μL / cm. After spotting, the sample was dried at 37℃ for 2 hours, then blocked with PBS containing 1% BSA for 1 hour, washed three times with deionized water, and dried for later use.

[0037] Preparation of the quality control zone: At a point 3 mm downstream of the final detection point, spot goat anti-rabbit IgG (concentration 0.5 mg / mL, spotting volume 1 μL / cm), dry at 37°C for 1 hour, and set aside. The quality control zone is used to verify the sample flow and the effectiveness of the reaction system.

[0038] Waste liquid absorption layer selection: Highly absorbent cellulose paper (Whatman 17 type, 0.8mm thick) was selected and cut to a size of 15mm × 15mm. It was overlapped with the end of the microchannel with an overlap width of 2mm to ensure continuous capillary siphon. This material has a water absorption ratio of ≥10 times its own weight and remains dry after completely absorbing all the reaction liquid (approximately 150μL), with no backflow.

[0039] Overall Assembly: Stack the prepared sample pretreatment layer, multi-response layer, and waste liquid absorption layer from top to bottom (sample pretreatment layer on top, waste liquid absorption layer on the bottom). Position and seal each layer with double-sided tape (ARcare® 88768), maintaining an overlap of 1-2 mm. The entire assembly is encapsulated in a transparent plastic shell, with a 3 mm diameter hole corresponding to the sample application port and a transparent observation window corresponding to the detection area. The final overall dimensions of the device are 50 mm (length) × 30 mm (width) × 5 mm (height). The assembled device should be stored at room temperature in a dry, light-protected environment for up to 6 months.

[0040] Example 2: Application and Performance Verification of Pig Sample Detection

[0041] Sample pretreatment: Collect whole blood (anticoagulated with EDTA) from live pigs suspected of being infected with porcine reproductive and respiratory syndrome (PRRS), classical swine fever, or porcine epidemic diarrhea (PED). Use a micropipette to add 80 μL of whole blood sample to the plasma separation membrane at the sample well of the device. At room temperature (25±2℃), the whole blood is naturally filtered for about 10 minutes by capillary action, and the plasma is separated and passes through the impurity filter membrane into the reagent release pad below. The lyophilized fluorescent microspheres labeled with antibodies in the reagent release pad are rapidly released upon reconstitution in the plasma and simultaneously undergo specific immune binding with the corresponding viral antigens that may be present in the plasma, forming an "antigen-antibody-fluorescent microsphere" complex.

[0042] Sample addition and reaction: The above mixture continues to flow into the three parallel microchannels of the multi-response layer (corresponding to PRRSV, CSFV, and PEDV detection, respectively) driven by capillary force. The conjugate pads within each microchannel further release and enrich the labeled antibody complexes. The fluid propels autonomously within the microchannels, with a total flow time of approximately 12 minutes. During flow, the complexes sequentially flow through the stratified detection zones. If the target viral antigen is present in the sample, it will bind to the corresponding capture antibody immobilized on the NC membrane, forming a "capture antibody-antigen-labeled antibody" sandwich immune complex, generating a fluorescent signal in the detection zone. Unbound labeled antibodies continue to flow to the control zone, where they bind to goat anti-rabbit IgG to form a control fluorescent band.

[0043] Result Interpretation: 15 minutes after the reaction begins, place the device into a portable fluorescence reader (excitation wavelength 485nm, emission wavelength 525nm, detection resolution 0.01 fluorescence intensity units) to read the fluorescence intensity of each detection area and control area. The interpretation criteria are as follows:

[0044] Effective detection: Fluorescence intensity in the quality control area ≥ background value + 3 times standard deviation;

[0045] Positive result determination: The fluorescence intensity of the detection area corresponding to a certain virus is ≥ (mean of negative control + 2 standard deviations), and the quality control area is valid;

[0046] Negative determination: The fluorescence intensity in the detection area is less than (mean of negative control + 2 times the standard deviation), and the quality control area is valid.

[0047] Meanwhile, the fluorescence intensity in the detection area is converted into the concentration of viral antigen by using a standard curve (a fluorescence intensity-concentration fitting equation is established in advance with a known concentration of recombinant protein, R²≥0.995), thus achieving quantitative detection.

[0048] Performance Validation and Data Comparison: This device was used to detect known concentrations of PRRSV N protein, CSFV E2 protein, and PEDVN protein standards. Negative controls (healthy pig plasma) and cross-reactivity controls (porcine circovirus type 2 (PCV2) and porcine pseudorabies virus (PRV) culture supernatant) were also included. Each experiment was repeated three times, and the intra- and inter-batch coefficients of variation (CV) were both ≤12.5%. The main performance indicators are summarized below:

[0049] Limit of detection (LOD, ng / mL) 0.24 0.38 0.56 Linear range (ng / mL) 0.24-50 0.38-50 0.56-50 Detection time (min) ≤30 ≤30 ≤30 Cross-reaction There is no overlap with PCV2, PRV, etc. There is no overlap with PCV2, PRV, etc. There is no overlap with PCV2, PRV, etc. Intra-batch coefficient of variation (%, n=3) 8.2-10.1 7.5-9.8 9.0-12.5 Inter-batch coefficient of variation (%, n=3) 9.5-11.2 8.9-11.5 10.2-12.4

[0050] Performance comparison with traditional detection methods: The detection performance of this device was compared with two commonly used methods: traditional colloidal gold immunochromatographic test strips (single strips, detecting three viruses separately) and enzyme-linked immunosorbent assay (ELISA) kits. The results are shown in the table below:

[0051] The device of the present invention 0.24-0.56 ≤30 min It is (a trilogy). Simple (one-step sample addition) No (Portable e-reader optional) Colloidal gold test strip (single sheet) Usually 5-10 15-20 min No (one of each) Simple no ELISA kit 0.5-1.0 2-3 h Multiple holes are possible. Complex (multi-step washing) ELISA reader

[0052] Data Summary and Analysis: Sensitivity: The detection limit of this invention (0.24-0.56 ng / mL) is significantly superior to that of traditional colloidal gold test strips (5-10 ng / mL), and comparable to or even better than that of high-quality ELISA kits (ELISA typically has a limit of 0.5-1.0 ng / mL). This is due to the high specific surface area of ​​the NC membrane detection zone, the signal amplification of the fluorescent microspheres, and optimized antibody pairing.

[0053] Detection speed: This invention takes no more than 30 minutes from sample addition to result interpretation, which is much faster than ELISA (2-3 hours) and similar to colloidal gold test strips. However, it achieves simultaneous detection of three pathogens, and the total time is only one-third of that of a single test strip.

[0054] Multi-mode capability: This invention enables the parallel detection of three viruses on the same chip, avoiding the independent operation of multiple test strips and the error in result integration, and significantly reducing detection costs and operation time.

[0055] Applicability: The device of this invention requires no external pumps or centrifuges, relying solely on capillary self-drive. Combined with a portable fluorescence reader, it enables on-site quantification, making it ideal for use in grassroots farms and veterinary stations. In contrast, ELISA relies on laboratory environments and professional personnel.

[0056] In summary, the multi-layered self-driven microfluidic detection device provided by this invention exhibits significant advantages in terms of sensitivity, detection throughput, ease of operation, and field adaptability, and is particularly suitable for rapid and simultaneous screening of porcine reproductive and respiratory syndrome (PRRS), classical swine fever, and porcine epidemic diarrhea (PED).

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-level stratified self-driven microfluidic detection device for swine diseases, used for the simultaneous detection of at least one swine disease, including porcine reproductive and respiratory syndrome (PRRS), classical swine fever (CSF), and porcine epidemic diarrhea (PED), characterized in that, It includes a self-driven microfluidic chip body, and the chip body is stacked in layers along the fluid flow direction, including a sample pretreatment layer, a multi-reaction layer, and a waste liquid absorption layer. The sample pretreatment layer includes a plasma separation membrane, an impurity filtration membrane, and a reagent release pad arranged sequentially along the fluid flow direction; The multi-reaction layer is provided with at least one microchannel, and each microchannel is provided with a binding pad, a layered detection area and a quality control area in sequence along the fluid flow direction; The waste liquid absorption layer overlaps with the end of the microchannel, providing capillary self-driving power to drive the sample fluid to flow sequentially through the sample pretreatment layer and the multi-reaction layer, and finally be absorbed by the waste liquid absorption layer.

2. The multi-unit stratified self-driven microfluidic detection device for swine diseases according to claim 1, characterized in that, The plasma separation membrane is made of glass fiber membrane or polyester membrane or hydrophilic porous membrane with a pore size of 0.22-0.45 μm and a thickness of 150-200 μm, with a plasma separation efficiency of ≥95% and a blood cell retention rate of ≥99%. The impurity filtration membrane is made of polyester nonwoven fabric or glass fiber with a surface density of 150-200 g / m² and a pore size of 5-10 μm. The reagent release pad is made of glass fiber membrane and pre-loaded with one or more freeze-dried markers. The markers are nanoparticles conjugated with specific antibodies against at least one of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSV), and porcine epidemic diarrhea virus (PEDV), or nanoparticles conjugated with specific nucleic acids.

3. The multi-unit stratified self-driven microfluidic detection device for swine diseases according to claim 1, characterized in that, The microchannel has a width of 1.5-2 mm and a depth of 0.8-1 mm, with its inner wall treated with oxygen plasma and a contact angle ≤60°. The conjugate pad is pre-loaded with three virus-specific antibodies or nucleic acids labeled with nanoparticles with a particle size of 100-200 nm, at a coupling concentration of 0.5-1 mg / mL. The layered detection zone uses a nitrocellulose membrane, a cellulose carbonate membrane, or an AAO membrane with a pore size of 0.2-0.45 μm.

4. The multi-unit stratified self-driven microfluidic detection device for swine diseases according to claim 1, characterized in that, The stratified detection zone is immobilized with capture antigens, namely PRRSV N protein, CSFV E2 protein, and PEDV N protein, with a purity ≥95% and a spotting concentration of 0.6-0.8 mg / mL, respectively; the quality control zone is immobilized with goat anti-rabbit IgG, with a spotting concentration of 0.7 mg / mL.

5. A detection method for the multi-stage stratified self-driven microfluidic detection device for swine diseases according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Sample pretreatment: Take 50-200 μL of whole blood sample from pigs and add it to the plasma separation membrane. Filter naturally to obtain plasma. After passing through the impurity filtration membrane, the plasma enters the reagent release pad, where it dissolves and releases the labeled substance. (2) Chip loading: The processed sample is dropped into the sample inlet of the multi-reaction layer. The fluid flows through the conjugate pad in the microchannel under the drive of capillary force, re-dissolves the label and forms a complex. (3) Stratified detection: The complex flows into the stratified detection area and binds with the capture antibody to form a detection signal; the unbound labeled material flows into the quality control area and binds with goat anti-rabbit IgG to form a quality control signal; (4) Result interpretation: The signal intensity of the detection area and the quality control area is read using a portable reader. When the signal intensity of the detection area is ≥ the mean of the negative control + 2 times the standard deviation and the color development of the quality control area is normal, it is judged as the corresponding virus positive. Quantitative detection is achieved by fitting the signal intensity with the standard curve.

6. The method according to claim 5, characterized in that, The portable reader is a fluorescence reader with an excitation wavelength of 480-500nm, an emission wavelength of 520-540nm, and a detection resolution of ≤0.01 fluorescence intensity units.

7. The method according to claim 5, characterized in that, The detection limits of the device for PRRSV, CSFV and PEDV are 0.24 ng / mL, 0.38 ng / mL and 0.56 ng / mL, respectively, with a detection time of ≤30 min, and it does not cross-react with porcine circovirus or porcine pseudorabies virus.

8. The use of the method according to any one of claims 5 to 7 in the preparation of a kit or detection device for detecting porcine reproductive and respiratory syndrome (PRRS), classical swine fever, and / or porcine epidemic diarrhea (PED).

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