A porcine epidemic diarrhea virus kit

Abstract

This application relates to the field of virus detection technology, specifically disclosing a porcine epidemic diarrhea virus (PEDV) kit. The kit includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell. The preparation method involves sequentially attaching the sample pad, conjugate pad, chromatography membrane, and absorbent pad to the PVC carrier, with adjacent components overlapping by 2-3 mm to obtain a test strip. The test strip is then cut into 3-5 mm wide pieces using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with sample wells from the sample pad and observation windows from the nitrocellulose membrane, thus obtaining the desired PEDV kit. The PEDV kit prepared in this application can rapidly and easily detect PEDV with good accuracy and high sensitivity.

Description

Technical Field

[0001] This application relates to the field of virus detection technology, and more specifically, to a reagent kit for porcine epidemic diarrhea virus. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is an RNA virus belonging to the genus Coronavirus in the family Coronaviridae. It can infect pigs of all ages and breeds, causing porcine epidemic diarrhea (PDV). PDV is a highly contagious intestinal infectious disease characterized by diarrhea, vomiting, and dehydration. It is particularly harmful to suckling piglets, with a mortality rate of up to 100% in infected piglets. Early screening for PEDV is the most critical and effective way to control its spread.

[0003] Common methods for detecting PEDV include conventional reverse transcription PCR (RT-PCR), quantitative real-time PCR, loop-mediated isothermal amplification PCR (RT-LAMP), indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). These common methods require a high level of expertise from the personnel conducting the tests and require expensive equipment, making them unsuitable for rapid on-site inspections in pig farms.

[0004] PEDV test strips do not require professional personnel and can be used for rapid detection at pig farms. However, currently available PEDV test strips suffer from insufficient sensitivity, resulting in frequent false negatives and false positives. Therefore, there is a need for a highly sensitive, accurate, easy-to-use, and rapid detection kit for porcine epidemic diarrhea virus. Based on the above, this application provides a porcine epidemic diarrhea virus kit. Summary of the Invention

[0005] To address the problems in the background art, this application provides a porcine epidemic diarrhea virus kit.

[0006] Firstly, this application provides a porcine epidemic diarrhea virus kit, which adopts the following technical solution: A porcine epidemic diarrhea virus (PEDV) kit includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell. The conjugate pad is coated with colloidal gold-labeled mouse anti-PEDV monoclonal antibody 1. The chromatography membrane has a detection line composed of mouse anti-PEDV monoclonal antibody 2 and a control line composed of goat anti-mouse IgG polyclonal antibody.

[0007] Preferably, the conjugate pad is prepared by the following method: A1. Mix mouse anti-PEDV monoclonal antibody 1 and stabilizer to obtain a mixture. Vortex the colloidal gold solution and the mixture, and let it stand at room temperature for 1 hour. Then add 1-2% PBS-BSA solution, vortex, and let it stand at room temperature for 1 hour. Centrifuge at low temperature for 15-25 minutes, discard the supernatant, and resuspend the precipitate in 2-3% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. A2. Use a pipette to draw 8-12 µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 30-40℃ for 1-2 hours to obtain the conjugate pad.

[0008] Preferably, in step A1, the stabilizer is a mixture of alginate and pullulan in a mass ratio of 0.4-1:3-7; the mass ratio of mouse anti-PEDV monoclonal antibody 1 to the stabilizer is 1-2:3-5; and the mass ratio of the mixture, colloidal gold solution, 1-2% PBS-BSA solution, and 2-3% PBS-BSA solution is 2-3:10-12:4-6:1.5-2.5.

[0009] Preferably, the colloidal gold solution in step A1 is prepared as follows: B1. Heat double-distilled water to boiling, add 1-2% tetrachloroauric acid solution, stir and mix, then add 1-2% trisodium citrate solution, stir and heat to boiling for 10-20 minutes, cool to room temperature to obtain mixture 1. B2. Add polyethylene glycol dithiol to mixture 1 and stir to react for 0.5-1.5 hours to obtain mixture 2; B3. Stir and mix 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and dicarboxylated polyethylene glycol for 10-20 min to obtain activated dicarboxylated polyethylene glycol; mix the activated dicarboxylated polyethylene glycol with mixture 2 and react for 2-4 h to obtain colloidal gold solution.

[0010] Preferably, in step B1, the mass ratio of double-distilled water, tetrachloroauric acid solution, and trisodium citrate solution is 220-260:2-3:6-8.

[0011] Preferably, in step B2, the mass ratio of polyethylene glycol dithiol to mixture 1 is 1-3:8000-10000; the molecular weight of polyethylene glycol dithiol is 900-1100.

[0012] Preferably, in step B3, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol is 2-3:0.4-1:8-12; the molecular weight of dicarboxylated polyethylene glycol is 1800-2200; the mass ratio of activated dicarboxylated polyethylene glycol to mixture 2 is 2-4:8000-10000; and the particle size of colloidal gold particles in the colloidal gold solution is 10-20 nm.

[0013] Preferably, the method for preparing the chromatography membrane is as follows: The mouse anti-PEDV monoclonal antibody 2 was streaked onto the nitrocellulose membrane at a rate of 1-2 µL / cm using a three-dimensional planar streaking apparatus to obtain the test line; the goat anti-mouse IgG polyclonal antibody was streaked onto the nitrocellulose membrane at a rate of 1-2 µL / cm to obtain the control line; the distance between the test line and the control line was 4-6 mm. The streaked nitrocellulose membrane was then dried at 30-40℃ for 16-20 h to obtain the desired chromatographic membrane.

[0014] Preferably, the concentration of the mouse anti-PEDV monoclonal antibody 2 is 1-2 mg / mL; the concentration of the goat anti-mouse IgG polyclonal antibody is 1-2 mg / mL.

[0015] Secondly, this application provides a method for preparing a porcine epidemic diarrhea virus kit, which adopts the following technical solution: A method for preparing a porcine epidemic diarrhea virus kit includes the following steps: The sample pad, conjugate pad, chromatography membrane, and absorbent pad are sequentially attached to the PVC carrier, with adjacent components overlapping by 2-3 mm to obtain the test strip. The test strip is then cut into 3-5 mm wide strips using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with the sample well from the sample pad and the observation window from the nitrocellulose membrane on the polypropylene shell, thus obtaining the required porcine epidemic diarrhea virus kit.

[0016] In summary, this application has the following beneficial effects: This application describes the preparation of a stabilizer using alginate and pullulan in the preparation of gold-labeled antibody solutions. Alginate, an anionic polysaccharide, forms a negatively charged layer on the surface of colloidal gold particles, preventing aggregation between particles through electrostatic repulsion and thus maintaining the stability of the colloidal gold solution. It also helps protect mouse anti-PEDV monoclonal antibody 1 from environmental factors, ensuring the antibody maintains its antigen recognition function stably throughout the detection process. The high viscosity of pullulan helps regulate the flowability of the gold-labeled antibody solution, ensuring uniform distribution of colloidal gold particles during detection. Simultaneously, pullulan provides a stable microenvironment for the antibody, contributing to improved sensitivity and specificity of the detection system. The combined effect of alginate and pullulan forms two protective barriers: preventing particle aggregation through charge shielding and reducing non-specific binding through steric hindrance, significantly improving the stability of the gold-labeled antibody solution. Their synergistic effect also better protects the antibody and colloidal gold particles, preventing them from losing activity or aggregating due to prolonged storage, thereby extending the shelf life of the test strip. Pullulan's film-forming and water-soluble properties, combined with the steric hindrance effect of alginate, can effectively reduce background noise and improve the sensitivity and specificity of the detection system.

[0017] This application involves adding polyethylene glycol dithiol and activated dicarboxylated polyethylene glycol to the preparation of colloidal gold solutions. The polyethylene glycol dithiol forms stable and strong Au-S bonds with gold atoms in the colloidal gold solution through its thiol (-SH) groups, effectively preventing the aggregation of colloidal gold particles and thus improving the long-term stability of the colloidal gold solution, thereby enhancing detection sensitivity. The activation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide makes the carboxyl groups of the dicarboxylated polyethylene glycol more reactive, readily reacting with gold atoms or other functional groups on the surface of colloidal gold particles. The carboxyl groups can form stable chemical bonds with the gold nanoparticle surface, further enhancing the stability of the colloidal gold. Furthermore, the carboxyl groups can react with other functional groups on the gold surface, forming additional stabilizing mechanisms. Stable colloidal gold particles help maintain the consistency and reliability of the test strips. Detailed Implementation

[0018] The present application will be further described in detail below with reference to the embodiments.

[0019] The raw materials used in the embodiments and comparative examples of this application are as follows: Mouse anti-PEDV monoclonal antibody 1 was purchased from Shanghai Yuduo Biotechnology Co., Ltd., named mouse anti-porcine epidemic diarrhea (PEDV) N protein monoclonal antibody, catalog number YDX374; Mouse anti-PEDV monoclonal antibody 2 was purchased from Shanghai Yuduo Biotechnology Co., Ltd., named porcine viral diarrhea virus PEDV membrane protein S monoclonal antibody, catalog number YDD-009-1; Goat anti-mouse IgG polyclonal antibody was purchased from Beijing APPLYGEN Gene Technology Co., Ltd., catalog number C1710; Polyethylene glycol dithiol was purchased from Merck Millipore, product number 717142; Dicarboxylated polyethylene glycol was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., product number PS2-CM-2K; Alginate was purchased from Merck Millipore, product number 180947; Pullulan was purchased from Merck Millipore, product number P4516; PBS-BSA solution was purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml28777-1; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was purchased from Merck Millipore, catalog number 39391; N-hydroxysuccinimide was purchased from Merck Millipore, catalog number 130672.

[0020] Examples 1-3 provide a porcine epidemic diarrhea virus kit.

[0021] Example 1 A porcine epidemic diarrhea virus kit includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell.

[0022] The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 1% tetrachloroauric acid solution and stir at 80 rpm. Then add a 1% trisodium citrate solution, stir at 80 rpm and heat to boiling for 10 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 220:2:6. A2. Polyethylene glycol dithiol was added to mixture 1 and stirred at 80 rpm for 0.5 h to obtain mixture 2; wherein the mass ratio of polyethylene glycol dithiol to mixture 1 was 1:8000; and the molecular weight of polyethylene glycol dithiol was 1000. A3. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and dicarboxylated polyethylene glycol were stirred and mixed at 80 rpm for 10 min to obtain activated dicarboxylated polyethylene glycol; wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol was 2:0.4:8; the molecular weight of dicarboxylated polyethylene glycol was 2000; A4. The activated dicarboxylated polyethylene glycol is mixed with mixture 2 and reacted for 2 hours to obtain a colloidal gold solution; wherein the mass ratio of activated dicarboxylated polyethylene glycol to mixture 2 is 2:8000; the particle size of the colloidal gold particles in the colloidal gold solution is 10 nm.

[0023] The preparation method of the binding pad is as follows: B1. A mixture of mouse anti-PEDV monoclonal antibody 1 and stabilizer was prepared. The colloidal gold solution and the mixture were vortexed and incubated at room temperature for 1 hour. Then, 1% PBS-BSA solution was added, vortexed, and incubated at room temperature for 1 hour. The mixture was centrifuged at 8000 rpm for 15 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. The stabilizer was prepared by combining alginate and pullulan polysaccharide in a mass ratio of 0.4:3, and the mass ratio of mouse anti-PEDV monoclonal antibody 1 to stabilizer was 1:3. The mass ratio of the mixture, colloidal gold solution, 1% PBS-BSA solution, and 2% PBS-BSA solution was 2:10:4:1.5. B2. Use a pipette to draw 8µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 30℃ for 1 hour to obtain the conjugate pad.

[0024] The preparation method of the chromatography membrane is as follows: Using a three-dimensional planar streaking apparatus, a detection line was obtained by streaking a 1 mg / mL mouse anti-PEDV monoclonal antibody 2 onto a nitrocellulose membrane at a spray volume of 1 µL / cm; a control line was obtained by streaking a 1 mg / mL goat anti-mouse IgG polyclonal antibody onto a nitrocellulose membrane at a spray volume of 1 µL / cm; the distance between the detection line and the control line was 4 mm. The streaked nitrocellulose membrane was then dried at 30 °C for 16 h to obtain the desired chromatographic membrane.

[0025] A method for preparing a porcine epidemic diarrhea virus kit includes the following steps: The sample pad, conjugate pad, chromatography membrane, and absorbent pad are sequentially attached to the PVC carrier, with adjacent components overlapping by 2 mm to obtain the test strip. The test strip is then cut into 3 mm wide pieces using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with the sample well from the sample pad and the observation window from the nitrocellulose membrane on the polypropylene shell, thus obtaining the required porcine epidemic diarrhea virus kit.

[0026] Example 2 A porcine epidemic diarrhea virus kit includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell.

[0027] The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 1.5% tetrachloroauric acid solution and stir at 90 rpm. Then add a 1.5% trisodium citrate solution, stir at 90 rpm and heat to boiling for 15 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 240:2.5:7. A2. Polyethylene glycol dithiol was added to mixture 1 and stirred at 90 rpm for 1 hour to obtain mixture 2; wherein the mass ratio of polyethylene glycol dithiol to mixture 1 was 2:9000; and the molecular weight of polyethylene glycol dithiol was 1000. A3. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and dicarboxylated polyethylene glycol were stirred and mixed at 90 rpm for 15 min to obtain activated dicarboxylated polyethylene glycol; wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol was 2.5:0.7:10; the molecular weight of dicarboxylated polyethylene glycol was 2000; A4. The activated dicarboxylated polyethylene glycol was mixed with mixture 2 and reacted for 3 hours to obtain a colloidal gold solution; wherein the mass ratio of the activated dicarboxylated polyethylene glycol to mixture 2 was 3:9000; the particle size of the colloidal gold particles in the colloidal gold solution was 15 nm.

[0028] The preparation method of the binding pad is as follows: B1. A mixture of mouse anti-PEDV monoclonal antibody 1 and stabilizer was prepared. The colloidal gold solution and the mixture were vortexed and incubated at room temperature for 1 hour. Then, 1.5% PBS-BSA solution was added, vortexed, and incubated at room temperature for 1 hour. The mixture was then centrifuged at 8000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2.5% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. The stabilizer was prepared by combining alginate and pullulan polysaccharide in a mass ratio of 0.7:5, and the mass ratio of mouse anti-PEDV monoclonal antibody 1 to stabilizer was 1.5:4. The mass ratio of the mixture, colloidal gold solution, 1.5% PBS-BSA solution, and 2.5% PBS-BSA solution was 2.5:11:5:2. B2. Use a pipette to draw 10µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 35℃ for 1.5h to obtain the conjugate pad.

[0029] The preparation method of the chromatography membrane is as follows: Using a three-dimensional planar streaking apparatus, a detection line was obtained by streaking a 1.5 mg / mL mouse anti-PEDV monoclonal antibody 2 onto a nitrocellulose membrane at a spray volume of 1.5 µL / cm; a control line was obtained by streaking a 1.5 mg / mL goat anti-mouse IgG polyclonal antibody onto a nitrocellulose membrane at a spray volume of 1.5 µL / cm; the distance between the detection line and the control line was 5 mm. The streaked nitrocellulose membrane was then dried at 35 °C for 18 h to obtain the desired chromatography membrane.

[0030] A method for preparing a porcine epidemic diarrhea virus kit includes the following steps: The sample pad, conjugation pad, chromatography membrane, and absorbent pad are sequentially attached to the PVC carrier, with adjacent components overlapping by 2.5 mm to obtain the test strip. The test strip is then cut into 3.5 mm wide pieces using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with the sample well from the sample pad and the observation window from the nitrocellulose membrane on the polypropylene shell, thus obtaining the required porcine epidemic diarrhea virus kit.

[0031] Example 3 A porcine epidemic diarrhea virus kit includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell.

[0032] The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 2% tetrachloroauric acid solution and stir at 100 rpm. Then add a 2% trisodium citrate solution, stir at 100 rpm and heat to boiling for 20 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 260:3:8. A2. Polyethylene glycol dithiol was added to mixture 1 and stirred at 100 rpm for 1.5 h to obtain mixture 2; wherein the mass ratio of polyethylene glycol dithiol to mixture 1 was 3:10000; and the molecular weight of polyethylene glycol dithiol was 1000. A3. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and dicarboxylated polyethylene glycol were stirred and mixed at 100 rpm for 20 min to obtain activated dicarboxylated polyethylene glycol; wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol was 3:1:12; the molecular weight of dicarboxylated polyethylene glycol was 2000; A4. The activated dicarboxylated polyethylene glycol was mixed with mixture 2 and reacted for 4 hours to obtain a colloidal gold solution; wherein the mass ratio of activated dicarboxylated polyethylene glycol to mixture 2 was 4:10000; the particle size of the colloidal gold particles in the colloidal gold solution was 20 nm.

[0033] The preparation method of the binding pad is as follows: B1. A mixture of mouse anti-PEDV monoclonal antibody 1 and stabilizer was prepared. The colloidal gold solution and the mixture were vortexed and incubated at room temperature for 1 hour. Then, 2% PBS-BSA solution was added, vortexed, and incubated at room temperature for 1 hour. The mixture was centrifuged at 8000 rpm for 25 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 3% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. The stabilizer was prepared by combining alginate and pullulan polysaccharide in a mass ratio of 1:7, and the mass ratio of mouse anti-PEDV monoclonal antibody 1 to stabilizer was 2:5. The mass ratio of the mixture, colloidal gold solution, 2% PBS-BSA solution, and 3% PBS-BSA solution was 3:12:6:2.5. B2. Use a pipette to draw 12µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 40℃ for 2 hours to obtain the conjugation pad.

[0034] The preparation method of the chromatography membrane is as follows: Using a three-dimensional planar streaking apparatus, a detection line was obtained by streaking a 2 mg / mL mouse anti-PEDV monoclonal antibody 2 onto a nitrocellulose membrane at a spray volume of 2 µL / cm; a control line was obtained by streaking a 2 mg / mL goat anti-mouse IgG polyclonal antibody onto a nitrocellulose membrane at a spray volume of 2 µL / cm; the distance between the detection line and the control line was 6 mm. The streaked nitrocellulose membrane was then dried at 40 °C for 20 h to obtain the desired chromatography membrane.

[0035] A method for preparing a porcine epidemic diarrhea virus kit includes the following steps: The sample pad, conjugate pad, chromatography membrane, and absorbent pad are sequentially attached to a PVC carrier, with adjacent components overlapping by 3 mm to obtain a test strip. The test strip is then cut into 5 mm wide strips using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with sample wells from the sample pad and observation windows from the nitrocellulose membrane on the polypropylene shell, thus obtaining the desired porcine epidemic diarrhea virus kit.

[0036] To verify the overall performance of the porcine epidemic diarrhea virus kits prepared in Examples 1-3 of this application, comparative examples 1-5 are provided, as follows: Comparative Example 1 Comparative Example 1 is the same as Example 1, except that the stabilizer used in the preparation of the conjugate pad is only alginate, as detailed below: The preparation method of the binding pad is as follows: B1. A mixture of mouse anti-PEDV monoclonal antibody 1 and stabilizer was prepared. The mixture was then vortexed with colloidal gold solution and allowed to stand at room temperature for 1 hour. 1.5% PBS-BSA solution was added, and the mixture was vortexed and allowed to stand at room temperature for 1 hour. The mixture was then centrifuged at 8000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2.5% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. The stabilizer was alginate, and the mass ratio of mouse anti-PEDV monoclonal antibody 1 to stabilizer was 1.5:4. The mass ratio of the mixture, colloidal gold solution, 1.5% PBS-BSA solution, and 2.5% PBS-BSA solution was 2.5:11:5:2. B2. Use a pipette to draw 10µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 35℃ for 1.5h to obtain the conjugate pad.

[0037] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that the stabilizer used in the preparation of the binding pad is pullulan, as detailed below: The preparation method of the binding pad is as follows: B1. Mouse anti-PEDV monoclonal antibody 1 and stabilizer were mixed to obtain a mixture. Colloidal gold solution and the mixture were vortexed and incubated at room temperature for 1 hour. Then, 1.5% PBS-BSA solution was added, vortexed, and incubated at room temperature for 1 hour. The mixture was then centrifuged at 8000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in 2.5% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. The stabilizer was pullulan, and the mass ratio of mouse anti-PEDV monoclonal antibody 1 to stabilizer was 1.5:4. The mass ratio of the mixture, colloidal gold solution, 1.5% PBS-BSA solution, and 2.5% PBS-BSA solution was 2.5:11:5:2. B2. Use a pipette to draw 10µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 35℃ for 1.5h to obtain the conjugate pad.

[0038] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that polyethylene glycol dithiol is not added in the preparation of the colloidal gold solution, as detailed below: The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 1.5% tetrachloroauric acid solution and stir at 90 rpm. Then add a 1.5% trisodium citrate solution, stir at 90 rpm and heat to boiling for 15 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 240:2.5:7. A2. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and dicarboxylated polyethylene glycol were stirred and mixed at 90 rpm for 15 min to obtain activated dicarboxylated polyethylene glycol; wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol was 2.5:0.7:10; the molecular weight of dicarboxylated polyethylene glycol was 2000; A3. The activated dicarboxylated polyethylene glycol was mixed with mixture 1 and reacted for 3 hours to obtain a colloidal gold solution; wherein the mass ratio of the activated dicarboxylated polyethylene glycol to mixture 1 was 3:9000; the particle size of the colloidal gold particles in the colloidal gold solution was 15 nm.

[0039] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that activated dicarboxylated polyethylene glycol is not added in the preparation of the colloidal gold solution, as detailed below: The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 1.5% tetrachloroauric acid solution and stir at 90 rpm. Then add a 1.5% trisodium citrate solution, stir at 90 rpm and heat to boiling for 15 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 240:2.5:7. A2. Polyethylene glycol dithiol was added to mixture 1 and stirred at 90 rpm for 1 hour to obtain a colloidal gold solution; the particle size of the colloidal gold particles in the colloidal gold solution was 15 nm.

[0040] Comparative Example 5 Comparative Example 5 is the same as Example 1, except that the activated dicarboxylated polyethylene glycol is replaced with dicarboxylated polyethylene glycol in the preparation of the colloidal gold solution, as detailed below: The preparation of colloidal gold solution is as follows: A1. After heating double-distilled water to boiling, add a 1.5% tetrachloroauric acid solution and stir at 90 rpm. Then add a 1.5% trisodium citrate solution, stir at 90 rpm and heat to boiling for 15 minutes. Cool to room temperature to obtain mixture 1. The mass ratio of double-distilled water, tetrachloroauric acid solution and trisodium citrate solution is 240:2.5:7. A2. Polyethylene glycol dithiol was added to mixture 1 and stirred at 90 rpm for 1 hour to obtain mixture 2; wherein the mass ratio of polyethylene glycol dithiol to mixture 1 was 2:9000; and the molecular weight of polyethylene glycol dithiol was 1000. A3. Mix dicarboxylated polyethylene glycol with mixture 2 and react for 3 hours to obtain a colloidal gold solution; wherein the mass ratio of dicarboxylated polyethylene glycol to mixture 2 is 3:9000; the particle size of the colloidal gold particles in the colloidal gold solution is 15 nm.

[0041] Performance testing The overall performance of the porcine epidemic diarrhea virus kits prepared in Examples 1-3 and Comparative Examples 1-5 of this application was tested respectively.

[0042] 1. Accuracy: 100 negative samples and 100 positive samples were tested using the porcine epidemic diarrhea virus kits prepared in Examples 1-3 and Comparative Examples 1-5, respectively. The negative concordance rate (%) and positive concordance rate (%) were statistically analyzed after the test. 2. Sensitivity: The porcine epidemic diarrhea virus antigen was serially diluted to 4000, 2000, 1000, 500, and 250 pg / mL, respectively, and the sensitivity of the porcine epidemic diarrhea virus kits prepared in Examples 1-3 and Comparative Examples 1-5 was tested. "+" represents positive, "±" represents weak positive, and "-" represents negative.

[0043] The results are shown in the table below: As shown in the table above, the porcine epidemic diarrhea virus kits prepared in Examples 1-3 have significantly better overall performance than those in Comparative Examples 1-5, exhibiting higher accuracy and sensitivity.

[0044] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A porcine epidemic diarrhea virus kit, characterized in that, It includes a sample pad, a conjugate pad, a chromatography membrane, an absorbent pad, a PVC carrier, and a polypropylene shell. The conjugate pad is coated with colloidal gold-labeled mouse anti-PEDV monoclonal antibody 1. The chromatography membrane has a detection line composed of mouse anti-PEDV monoclonal antibody 2 and a control line composed of goat anti-mouse IgG polyclonal antibody.

2. The porcine epidemic diarrhea virus kit according to claim 1, characterized in that, The method for preparing the conjugate pad is as follows: A1. Mix mouse anti-PEDV monoclonal antibody 1 and stabilizer to obtain a mixture. Vortex the colloidal gold solution and the mixture, and let it stand at room temperature for 1 hour. Then add 1-2% PBS-BSA solution, vortex, and let it stand at room temperature for 1 hour. Centrifuge at low temperature for 15-25 minutes, discard the supernatant, and resuspend the precipitate in 2-3% PBS-BSA solution to obtain a gold-labeled antibody solution of mouse anti-PEDV monoclonal antibody 1 labeled with colloidal gold particles. A2. Use a pipette to draw 8-12 µL of gold-labeled antibody solution onto a glass fiber membrane, and then dry it at 30-40℃ for 1-2 hours to obtain the conjugate pad.

3. The porcine epidemic diarrhea virus kit according to claim 2, characterized in that, In step A1, the stabilizer is a mixture of alginate and pullulan in a mass ratio of 0.4-1:3-7; the mass ratio of mouse anti-PEDV monoclonal antibody 1 to the stabilizer is 1-2:3-5; and the mass ratio of the mixture, colloidal gold solution, 1-2% PBS-BSA solution, and 2-3% PBS-BSA solution is 2-3:10-12:4-6:1.5-2.

5.

4. The porcine epidemic diarrhea virus kit according to claim 2, characterized in that, The preparation of the colloidal gold solution in step A1 is as follows: B1. Heat double-distilled water to boiling, add 1-2% tetrachloroauric acid solution, stir and mix, then add 1-2% trisodium citrate solution, stir and heat to boiling for 10-20 minutes, cool to room temperature to obtain mixture 1. B2. Add polyethylene glycol dithiol to mixture 1 and stir to react for 0.5-1.5 hours to obtain mixture 2; B3. Stir and mix 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and dicarboxylated polyethylene glycol for 10-20 min to obtain activated dicarboxylated polyethylene glycol; mix the activated dicarboxylated polyethylene glycol with mixture 2 and react for 2-4 h to obtain colloidal gold solution.

5. The porcine epidemic diarrhea virus kit according to claim 4, characterized in that, In step B1, the mass ratio of double-distilled water, tetrachloroauric acid solution, and trisodium citrate solution is 220-260:2-3:6-8.

6. The porcine epidemic diarrhea virus kit according to claim 4, characterized in that, In step B2, the mass ratio of polyethylene glycol dithiol to mixture 1 is 1-3:8000-10000; the molecular weight of polyethylene glycol dithiol is 900-1100.

7. The porcine epidemic diarrhea virus kit according to claim 4, characterized in that, In step B3, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxysuccinimide solution, and dicarboxylated polyethylene glycol is 2-3:0.4-1:8-12; the molecular weight of dicarboxylated polyethylene glycol is 1800-2200; the mass ratio of activated dicarboxylated polyethylene glycol to mixture 2 is 2-4:8000-10000; and the particle size of colloidal gold particles in the colloidal gold solution is 10-20 nm.

8. The porcine epidemic diarrhea virus kit according to claim 1, characterized in that, The method for preparing the chromatography membrane is as follows: The mouse anti-PEDV monoclonal antibody 2 was streaked onto the nitrocellulose membrane at a rate of 1-2 µL / cm using a three-dimensional planar streaking apparatus to obtain the test line; the goat anti-mouse IgG polyclonal antibody was streaked onto the nitrocellulose membrane at a rate of 1-2 µL / cm to obtain the control line; the distance between the test line and the control line was 4-6 mm. The streaked nitrocellulose membrane was then dried at 30-40℃ for 16-20 h to obtain the desired chromatographic membrane.

9. The porcine epidemic diarrhea virus kit according to claim 8, characterized in that, The concentration of the mouse anti-PEDV monoclonal antibody 2 was 1-2 mg / mL; the concentration of the goat anti-mouse IgG polyclonal antibody was 1-2 mg / mL.

10. A method for preparing a porcine epidemic diarrhea virus kit according to any one of claims 1-9, characterized in that, Includes the following steps: The sample pad, conjugate pad, chromatography membrane, and absorbent pad are sequentially attached to the PVC carrier, with adjacent components overlapping by 2-3 mm to obtain the test strip. The test strip is then cut into 3-5 mm wide strips using a strip cutter. A polypropylene shell is attached to the outer end of the test strip, with the sample well from the sample pad and the observation window from the nitrocellulose membrane on the polypropylene shell, thus obtaining the required porcine epidemic diarrhea virus kit.

Images