Red modified microsphere immunochromatography test strip for detecting ochratoxin A in wine and application of red modified microsphere immunochromatography test strip

Abstract

The invention discloses a red modified microsphere immunochromatography test strip for detecting ochratoxin A in wine and application of the red modified microsphere immunochromatography test strip, and relates to the technical field of mycotoxin detection. The test strip consists of a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC (Polyvinyl Chloride) rubber plate. When the test strip is used for detection, an object to be detected and an ochratoxin A-BSA antigen on the nitrocellulose membrane can compete for a red modified microsphere-ochratoxin A monoclonal antibody conjugate which is more tightly combined with each other due to modification on the combination pad together, and whether a sample contains ochratoxin A or not can be judged according to color reaction. The test strip can also be combined with a color microsphere immunoassay analyzer for quantitative detection of the ochratoxin content in a wine sample. The test strip disclosed by the invention is high in sensitivity, strong in specificity, simple and convenient to operate and visual in result, and can be used for detecting 1-20mu g / mL ochratoxin A in wine within 10min.

Description

Technical Field

[0001] The present application belongs to the technical field of mycotoxin detection, and specifically relates to a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and an application thereof. Background Art

[0002] During the production, storage and sales of wine, its quality and safety have always attracted much attention. Ochratoxin A, a toxic secondary metabolite produced by Aspergillus and Penicillium, is potentially harmful to human health and can cause adverse effects such as kidney toxicity, liver toxicity, immunotoxicity and even carcinogenicity. Grapes, the raw materials for winemaking, are easily contaminated by toxin-producing fungi during field growth, harvesting and subsequent processing, which leads to the risk of ochratoxin A contamination in wine.

[0003] At present, there are many methods for detecting ochratoxin A, including traditional laboratory detection technologies such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Although these methods have high accuracy and sensitivity, they often require complex and expensive instruments and equipment, professional operators, and cumbersome sample pretreatment steps. The detection cycle is long and cannot meet the needs of rapid detection at the wine production site and preliminary screening of a large number of samples. The immunochromatographic technology, which has the advantages of simple operation, no need for large instruments, and fast detection speed, has gradually attracted widespread attention. However, this immunochromatographic test strip is mainly used for qualitative detection (determining whether there is ochratoxin A in the sample), or can only perform semi-quantitative detection (roughly judging the content range by the color depth of the strip).

[0004] The Chinese patent application with publication number CN106908599 A discloses a method based on silver nanoparticles and Ru(phen)3 2+ An immunochromatographic test strip based on the extinction effect between fluorescent microspheres is used for the rapid detection of ochratoxin A in wine and grape juice. 2+ Fluorescent microspheres are used as fluorescent background signals, and their maximum excitation wavelength is 450nm. They are mixed with OTA detection antigens and sprayed on the detection area of ​​the test strip; a complex of spherical silver nanoparticles with an SPR absorption peak of 450nm and OTA monoclonal antibodies is used as an extinction probe. When testing positive samples, free OTA in the sample competes with the detection antigen fixed on the test line and binds to the silver probe. The OTA content in the sample is inversely proportional to the number of silver probes bound to the test line, and is proportional to the intensity of the fluorescence signal. This technical solution is more sensitive in qualitative detection of OTA.

[0005] The above-mentioned immunochromatographic test strips can only be used for qualitative detection, not quantitative detection, and there is still room for improvement in performance such as sensitivity and specificity. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present application provides a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application. The test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, absorbent paper and a PVC sheet. When the test strip is used for detection, the analyte and the ochratoxin A-BSA antigen on the nitrocellulose membrane will compete for the ochratoxin A monoclonal antibody-red modified microsphere conjugate on the conjugate pad, and the color reaction can be used to determine whether the sample contains ochratoxin A. The test strip can also be used in conjunction with a color microsphere immunoassay analyzer to quantitatively detect the ochratoxin content in a wine sample.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions: In a first aspect, the present application provides a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine. The red modified microsphere immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, absorbent paper and a PVC sheet, wherein a red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad, and a T1 test line, a T2 test line and a quality control line are arranged from left to right on the nitrocellulose membrane, wherein the T1 test line and the T2 test line are fixed with ochratoxin A-BSA antigens of different concentrations, and a goat anti-mouse polyclonal antibody is fixed on the quality control line.

[0008] In a second aspect, the present application provides an application of a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine, comprising the following steps: Putting the prepared red modified microsphere immunochromatographic test strip on a card shell to make a finished test strip; Then, the ochratoxin A solutions of different concentrations are diluted with carbonate solutions to prepare ochratoxin A standard test solutions of different concentrations; Subsequently, the ochratoxin A standard test solution is added dropwise to the sample addition holes of the finished test strips, and then the test strips are placed in an incubator for chromatography reaction; After the chromatography reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer from low concentration to high concentration, and the absorbance values ​​of the T1 test line, T2 test line and quality control line are measured; The absorbance ratio of the test line and the quality control line of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; The sample to be tested is diluted and mixed with the carbonate solution in the same way, and then dripped into the sample adding hole of the test strip, and then the test strip is placed in an incubator for chromatography reaction; After the reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer to measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of the ochratoxin A sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0009] Beneficial technical effects: Compared with the immunochromatographic test strips of the prior art, which are mainly used for qualitative detection (determining whether there is ochratoxin A in the sample), or can only perform semi-quantitative detection (roughly judging the content range by the color depth of the strip), the red modified microsphere immunochromatographic test strips provided by the present application can achieve quantitative detection of ochratoxin A in red wine and white wine within 10 minutes, and have the characteristics of high precision and high accuracy. In addition, the red microspheres used in the immunochromatographic test strips have been surface-modified, so that the surface carboxyl groups are increased, which can better form a conjugate with the ochratoxin A monoclonal antibody, and at the same time make it more specific.

[0010] The method provided in this application has high sensitivity to ochratoxin A in wine, with a minimum detection limit of 1 μg / mL and a quantitative detection range of 1-20 μg / mL, which meets the domestic and EU limit standards for ochratoxin A in wine and provides an important tool for the rapid quantitative detection of ochratoxin A in wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic diagram of the structure of the red modified microsphere immunochromatographic test strip.

[0012] Figure 2 It is a schematic diagram of preparing red modified microspheres by reaction of red microspheres.

[0013] Figure 3 It is a schematic diagram of the structure of the finished product of the red modified microsphere immunochromatographic test strip.

[0014] Figure 4 It is the standard relationship curve and compliance of the red modified microsphere immunochromatographic test strip.

[0015] Among them: 1. Sample pad, 2. Binding pad, 3. T1 detection line, 4. T2 detection line, 5. Quality control line, 6. Nitrocellulose membrane, 7. Absorbent paper, 8. PVC sheet, 9. Sample addition hole, 10. Card. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer, the present application is further described in detail below in conjunction with the embodiments. However, this should not be understood as the scope of the present application being limited to the following examples. Without departing from the above-mentioned method ideas of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0017] In this application, the terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit the application.

[0018] As used in this application, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0019] The terms “first” and “second”, if used, are used for descriptive purposes only and should not be understood as indicating or implying relative importance.

[0020] In a first aspect, the present application provides a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine, the structure of which is as follows: Figure 1 The red modified microsphere immunochromatographic test strip comprises a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 6, a blotting paper 7 and a PVC sheet 8, wherein the conjugate pad 2 is fixed with a red modified microsphere-ochratoxin A monoclonal antibody conjugate, and the nitrocellulose membrane 6 is provided with a T1 detection line 3, a T2 detection line 4 and a quality control line 5 from left to right, wherein the T1 detection line 3 and the T2 detection line 4 are fixed with ochratoxin A-BSA antigens of different concentrations, and the quality control line 5 is fixed with a goat anti-mouse polyclonal antibody.

[0021] In a possible implementation, the method for preparing the red modified microsphere-ochratoxin A monoclonal antibody conjugate comprises the following steps: The red microspheres are dispersed in an emulsion system containing monomers, emulsifiers and initiators, and a polymerization reaction is carried out at a set temperature. After the reaction is completed, the red modified microspheres are obtained by demulsification and washing. After surface modification, the carboxyl groups on the surface of the red microspheres are increased, which can better form a conjugate with the ochratoxin A monoclonal antibody, thereby ultimately improving the specificity of the test strip. The reactions occurring during the surface modification process are as follows: Figure 2 As shown; The red modified microspheres are mixed with the first buffer solution, dispersed by ultrasonication, and activated by adding carbodiimide for 30 to 60 minutes to obtain a first mixed solution; The first mixed solution is centrifuged, re-dissolved with the first buffer, and then ultrasonically dispersed and added with ochratoxin A monoclonal antibody for coupling for 30 to 45 minutes to obtain a second mixed solution; The second mixed solution was centrifuged, re-dissolved with a blocking solution, and then ultrasonically dispersed and blocked for 30 to 60 minutes to obtain a third mixed solution; The third mixed solution is centrifuged, re-dissolved with the second buffer solution, and ultrasonically dispersed to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate.

[0022] In a possible implementation, the red microspheres include polystyrene microspheres containing carboxyl groups on the surface, and the average particle size is 50~400nm; the monomer includes one or more of acrylic acid, methacrylic acid, and itaconic acid; the emulsifier includes one or two of sodium dodecyl sulfate and sodium hexadecyl sulfate; the initiator includes one or more of potassium persulfate, diisopropylbenzene peroxide, and azobisisobutyronitrile; the set temperature is 60~80℃; the mass ratio of the red microspheres, monomer, emulsifier, and initiator is (10~20):(70~90):(1~5):(0.5~2).

[0023] In a possible implementation, the ultrasonic dispersion time after the red modified microspheres are mixed with the first buffer is 1 to 5 minutes; the centrifugation conditions of the first mixed solution are 8000 to 20000 r / min, the centrifugation time is 5 to 20 minutes, and the ultrasonic dispersion time is 1 to 5 minutes; the centrifugation conditions of the second mixed solution are 8000 to 20000 r / min, the centrifugation time is 5 to 20 minutes, and the ultrasonic dispersion time is 1 to 5 minutes; the centrifugation conditions of the third mixed solution are 8000 to 20000 r / min, the centrifugation time is 5 to 20 minutes, and the ultrasonic dispersion time is 1 to 5 minutes.

[0024] In a possible implementation, in the method for preparing the red modified microsphere-ochratoxin A monoclonal antibody conjugate, the mass ratio of the red modified microsphere, the first buffer, and the carbodiimide is (40-50): (40-60): (1-5); the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is (10-20): (20-30): (10-20): (20-30): (20-30).

[0025] In a possible implementation, in the method for preparing the red modified microsphere-ochratoxin A monoclonal antibody conjugate, the ion concentration of the first buffer is 15~30mM and the pH is 8.0~10.0; the components of the first buffer include sucrose, BSA and boric acid buffer; the mass ratio of sucrose, BSA and boric acid buffer is (5~10):(1~3):(87~94).

[0026] In a possible implementation, in the method for preparing the red modified microsphere-ochratoxin A monoclonal antibody conjugate, the ion concentration of the blocking solution is 15~30mM, and the pH is 7.0~9.0; the components of the blocking solution include BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is (0.5~3): (0.1~1): (96~99.4).

[0027] In a possible implementation, in the preparation method of the red modified microsphere-ochratoxin A monoclonal antibody conjugate, the ion concentration of the second buffer is 10~30mM and the pH is 8.0~10.0; the components of the second buffer include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is (5~10):(1~3):(1~3):(84~94).

[0028] In a possible implementation, the concentration of ochratoxin A-BSA antigen on the T1 test line 3 is 0.5~1.5 mg / mL; the concentration of ochratoxin A-BSA antigen on the T2 test line 4 is 0.3~1.2 mg / mL; the concentration of goat anti-mouse antibody on the quality control line 5 is 0.05~0.3 mg / mL; and the intervals between the T1 test line 3, the T2 test line 4 and the quality control line 5 are 0.5~1.5 cm.

[0029] In a possible implementation, the red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 4-10 μL / cm.

[0030] In a second aspect, the present application provides an application of a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine, comprising the following steps: The prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip, and its structure is as follows: Figure 3 As shown; Then, the ochratoxin A solutions of different concentrations are diluted with carbonate solutions to prepare ochratoxin A standard test solutions of different concentrations; Then, the ochratoxin A standard test solution is added dropwise to the sample addition well 9 of the finished test strip, and then the test strip is placed in an incubator for chromatography reaction; After the chromatography reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer from low concentration to high concentration, and the absorbance values ​​of the T1 test line 3, the T2 test line 4 and the quality control line 5 are measured; The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain the standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value, and the compliance can be verified by the standard relationship curve, such as Figure 4 As shown; The sample to be tested is diluted and mixed with the carbonate solution in the same way, and then dripped into the sample adding hole 9 of the test strip, and then the test strip is placed in an incubator for chromatography reaction; After the reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer to measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of the ochratoxin A sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0031] In a possible implementation, the concentration of the ochratoxin A solution is 0-20 μg / mL; and the carbonate solution is a carbonate solution containing 1-5% Tween-20, 30-100 mM.

[0032] In a possible implementation, the amount of the ochratoxin A standard test solution added to the sample addition hole 9 of the finished test strip is 100 μL.

[0033] In a possible implementation, the chromatography reaction is carried out in the incubator at a temperature of 30-50° C. for 6-15 min.

[0034] In a possible implementation, the intervals between the T1 detection line 3 , the T2 detection line 4 and the quality control line 5 are 0.5-1.5 cm.

[0035] The following will specifically describe a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application provided by the present application in combination with different embodiments.

[0036] The materials and reagents used in the following examples were all produced by Nanjing Microtest Biological Company.

[0037] Embodiment 1: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 50 nm in an emulsion system containing acrylic acid, sodium dodecyl sulfate and potassium persulfate, and perform polymerization reaction at 60°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 10:85:4:1; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 20 mM and a pH of 8.0, and ultrasonically dispersed for 3 min, and activated with carbodiimide for 60 min to obtain a first mixed solution; wherein the first buffer solution comprises sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution is 5:1:94; the mass ratio of the red modified microspheres, the first buffer solution and carbodiimide is 45:50:5; 3. The first mixed solution was centrifuged at 8000 r / min for 5 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min, and then ochratoxin A monoclonal antibody was added to carry out coupling for 30 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 8000 r / min for 5 min, and then re-dissolved in a blocking solution with an ion concentration of 30 mM and a pH of 9.0, and then ultrasonically dispersed for 3 min and blocked for 60 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 3:1:96; 5. The third mixed solution was centrifuged at 8000 r / min for 5 min, re-dissolved in a second buffer solution with an ion concentration of 30 mM and a pH of 8.0, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 5:3:3:89; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 10:20:10:30:30; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 5 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 2% Tween-20 and 100 mM to prepare ochratoxin A standard test solutions of 0, 1, 2, 5, 10, 15, and 20 μg / mL respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the finished test strip, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 40° C. for 10 min. 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with a carbonate solution containing 2% Tween-20 and 100 mM in the same way to prepare a 5 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip. The amount of addition is 100 μL. Then, the test strip is placed in an incubator for chromatography reaction. The reaction temperature is 40°C and the reaction time is 10 min. 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0038] Embodiment 2:

[0039] A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Dispersing red microspheres with an average particle size of 100 nm in an emulsion system containing methacrylic acid, sodium hexadecyl sulfate and dicumyl peroxide, and performing polymerization reaction at 70°C. After the reaction, demulsification and washing are performed to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 15:80:3:2; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 20 mM and a pH of 10.0, and ultrasonically dispersed for 3 min. Carbodiimide was added for activation for 45 min to obtain a first mixed solution. The first buffer solution contained sucrose, BSA and boric acid buffer solution. The mass ratio of sucrose, BSA and boric acid buffer solution was 6:2:92. The mass ratio of the red modified microspheres, the first buffer solution and carbodiimide was 48:50:2. 3. The first mixed solution was centrifuged at 10000 r / min for 10 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min, and then ochratoxin A monoclonal antibody was added for coupling. The coupling time was 35 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 10000 r / min for 10 min, and then re-dissolved in a blocking solution with an ion concentration of 20 mM and a pH of 8.0, and then ultrasonically dispersed for 3 min, and blocked for 40 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 2:1:97; 5. The third mixed solution was centrifuged at 10000 r / min for 10 min, re-dissolved in a second buffer solution with an ion concentration of 20 mM and a pH of 9.0, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 6:2:2:90; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 15:30:15:20:20; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 8 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with 1% Tween-20, 80 mM carbonate solution to prepare ochratoxin A standard test solutions of 0, 1, 2, 5, 10, 15, and 20 μg / mL concentrations, respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the test strip product, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 50° C. and a reaction time of 6 min. 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with 1% Tween-20, 80 mM carbonate solution in the same way to prepare 1 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip, with a drop volume of 100 μL. Then put the test strip into the incubator for chromatography reaction, the reaction temperature is 50°C, and the reaction time is 6 min; 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0040] Embodiment 3: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 150 nm in an emulsion system containing itaconic acid, sodium dodecyl sulfate and azobisisobutyronitrile, and perform polymerization reaction at 80°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 20:75:3.5:1.5; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 15 mM and a pH of 8.0, and ultrasonically dispersed for 3 min. Carbodiimide was added for activation for 60 min to obtain a first mixed solution. The first buffer solution included sucrose, BSA and boric acid buffer solution. The mass ratio of sucrose, BSA and boric acid buffer solution was 8:3:89. The mass ratio of the red modified microspheres, the first buffer solution and carbodiimide was 50:47:3. 3. The first mixed solution was centrifuged at 12000 r / min for 15 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min, and then ochratoxin A monoclonal antibody was added for coupling for 45 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 12000 r / min for 15 min, and then re-dissolved in a blocking solution with an ion concentration of 15 mM and a pH of 7.0, and then ultrasonically dispersed for 3 min, and blocked for 60 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 1.5:0.5:98; 5. The third mixed solution was centrifuged at 12000 r / min for 15 min, re-dissolved in a second buffer solution with an ion concentration of 25 mM and a pH of 8.0, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 7:2:1:90; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 20:20:20:20:20; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 10 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. The structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 3% Tween-20 and 60 mM to prepare ochratoxin A standard test solutions of 0, 1, 2, 5, 10, 15, and 20 μg / mL respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the finished test strip, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 45° C. for 8 min; 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with 3% Tween-20 and 60 mM carbonate solution in the same way to prepare a 2 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip. The amount of addition is 100 μL. Then, the test strip is placed in an incubator for chromatography reaction. The reaction temperature is 45°C and the reaction time is 8 min. 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0041] Embodiment 4: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 200 nm in an emulsion system containing methacrylic acid, sodium hexadecyl sulfate and potassium persulfate, and perform polymerization reaction at 65°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 12:85:2:1; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 15 mM and a pH of 8.5, and ultrasonically dispersed for 3 min, and activated with carbodiimide for 50 min to obtain a first mixed solution; wherein the first buffer solution comprises sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution is 6:1:93; the mass ratio of the red modified microspheres, the first buffer solution and carbodiimide is 42:54:4; 3. The first mixed solution was centrifuged at 15000 r / min for 8 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min before adding ochratoxin A monoclonal antibody for coupling for 35 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 15000 r / min for 8 min, and then re-dissolved in a blocking solution with an ion concentration of 20 mM and a pH of 8.5, and then ultrasonically dispersed for 3 min, and blocked for 50 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 2:1:97; 5. The third mixed solution was centrifuged at 15000 r / min for 8 min, re-dissolved in a second buffer solution with an ion concentration of 15 mM and a pH of 10.0, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 7:1:3:89; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 12:26:12:25:25; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 6 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 4% Tween-20 and 50 mM to prepare ochratoxin A standard test solutions with concentrations of 0, 1, 2, 5, 10, 15, and 20 μg / mL, respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the finished test strip, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 40° C. for 10 min. 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with a carbonate solution containing 4% Tween-20 and 50 mM in the same way to prepare a 10 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip. The amount of addition is 100 μL. Then, the test strip is placed in an incubator for chromatography reaction. The reaction temperature is 40°C and the reaction time is 10 min. 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0042] Embodiment 5: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 300 nm in an emulsion system containing methacrylic acid, sodium hexadecyl sulfate and dicumyl peroxide, and carry out polymerization reaction at 70°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 15:82:1:2; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 30 mM and a pH of 9.0, and ultrasonically dispersed for 3 min, and activated with carbodiimide for 50 min to obtain a first mixed solution; wherein the first buffer solution comprises sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution is 10:2:88; the mass ratio of the red modified microspheres, the first buffer solution and carbodiimide is 40:57:3; 3. The first mixed solution was centrifuged at 8000-20000 r / min for 5-20 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min before adding ochratoxin A monoclonal antibody for coupling for 30-45 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 20000r / min for 5min, and then re-dissolved in a blocking solution with an ion concentration of 25mM and a pH of 9.0, and then ultrasonically dispersed for 3min and blocked for 35min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 0.5:1:98.5; 5. The third mixed solution was centrifuged at 20000r / min for 5min, re-dissolved in a second buffer solution with an ion concentration of 25mM and a pH of 10.0, and ultrasonically dispersed for 3min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of sucrose, trehalose, BSA and boric acid buffer is 10:3:3:84; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 15:30:15:20:20; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 4 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 5% Tween-20 and 30 mM to prepare ochratoxin A standard test solutions with concentrations of 0, 1, 2, 5, 10, 15, and 20 μg / mL, respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the test strip product, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 50° C. and a reaction time of 6 min. 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with 5% Tween-20, 30 mM carbonate solution in the same way to prepare 15 μg / mL ochratoxin A sample to be tested, add it dropwise to the sample well 9 of the test strip, the amount of addition is 100 μL, and then put the test strip into the incubator for chromatography reaction, the reaction temperature is 50°C, and the reaction time is 6 min; 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0043] Embodiment 6: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 400 nm in an emulsion system containing itaconic acid, sodium dodecyl sulfate and potassium persulfate, and perform polymerization reaction at 75°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 18:78:2.5:1.5; 2. The red modified microspheres were mixed with a first buffer solution with an ion concentration of 15 mM and a pH of 8.0, and ultrasonically dispersed for 3 minutes. Carbodiimide was added for activation for 30 minutes to obtain a first mixed solution; wherein the first buffer solution included sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution was 5:3:92; the mass ratio of the red modified microspheres, the first buffer solution and carbodiimide was 46:52:2; 3. The first mixed solution was centrifuged at 10000 r / min for 10 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min, and then ochratoxin A monoclonal antibody was added for coupling for 40 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 10000 r / min for 10 min, and then re-dissolved in a blocking solution with an ion concentration of 30 mM and a pH of 7.5, and then ultrasonically dispersed for 3 min and blocked for 40 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 2.5:0.1:97.4; 5. The third mixed solution was centrifuged at 10000 r / min for 10 min, re-dissolved in a second buffer solution with an ion concentration of 20 mM and a pH of 8.5, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 9:2:2:87; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 16:28:16:20:20; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 7 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 2.5% Tween-20 and 70 mM to prepare ochratoxin A standard test solutions of 0, 1, 2, 5, 10, 15, and 20 μg / mL respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the finished test strip, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 35° C. for 12 min. 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the test samples with 2.5% Tween-20 and 70 mM carbonate solution in the same way to prepare 20 μg / mL ochratoxin A test samples, add them dropwise to the sample well 9 of the test strip, and add 100 μL of them. Then put the test strip into the incubator for chromatography reaction at 35°C for 12 min. 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0044] Comparative Example 1: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Mix the red microspheres with a first buffer solution with an ion concentration of 20 mM and a pH of 8.0, perform ultrasonic dispersion for 3 minutes, add carbodiimide for activation for 60 minutes, and obtain a first mixed solution; wherein the first buffer solution includes sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution is 5:1:94; the mass ratio of the red microspheres, the first buffer solution and carbodiimide is 45:50:5; 2. The first mixed solution was centrifuged at 8000 r / min for 5 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min before adding ochratoxin A monoclonal antibody for coupling for 30 min to obtain a second mixed solution; 3. The second mixed solution was centrifuged at 8000 r / min for 5 min, and then re-dissolved in a blocking solution with an ion concentration of 30 mM and a pH of 9.0, and then ultrasonically dispersed for 3 min, and blocked for 60 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 3:1:96; 4. The third mixed solution was centrifuged at 8000 r / min for 5 min, re-dissolved in a second buffer solution with an ion concentration of 30 mM and a pH of 8.0, and ultrasonically dispersed for 3 min to obtain the red microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 5:3:3:89; In the above steps 1 to 4, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 10:20:10:30:30; 5. The prepared red microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 5 μL / cm. Then, the prepared red microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 6. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 2% Tween-20 and 100 mM to prepare ochratoxin A standard test solutions with concentrations of 0, 1, 2, 5, 10, 15, and 20 μg / mL, respectively; 7. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the test strip product, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 40° C. and a reaction time of 10 min. 8. After the chromatography reaction is completed, insert the test strip into the red microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 9. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 10. Dilute and mix the sample to be tested with a carbonate solution containing 2% Tween-20 and 100 mM in the same way to prepare a 5 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip. The amount of addition is 100 μL. Then, the test strip is placed in an incubator for chromatography reaction. The reaction temperature is 40°C and the reaction time is 10 min. 11. After the reaction is completed, insert the test strip into the red microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of the ochratoxin A sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the test solution can be calculated.

[0045] Comparative Example 2: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Disperse red microspheres with an average particle size of 150 nm in an emulsion system containing itaconic acid, sodium dodecyl sulfate and azobisisobutyronitrile, and perform polymerization reaction at 80°C. After the reaction, demulsify and wash to obtain red modified microspheres; the mass ratio of the red microspheres, monomer, emulsifier and initiator is 20:75:3.5:1.5; 2. The red modified microspheres were mixed with a first buffer solution having an ion concentration of 15 mM and a pH of 8.0, and ultrasonically dispersed for 3 min to obtain a first mixed solution; wherein the first buffer solution comprises sucrose, BSA and a boric acid buffer solution; the mass ratio of the sucrose, BSA and the boric acid buffer solution is 8:3:89; the mass ratio of the red modified microspheres to the first buffer solution is 50:50; 3. The first mixed solution was centrifuged at 12000 r / min for 15 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min, and then ochratoxin A monoclonal antibody was added for coupling for 45 min to obtain a second mixed solution; 4. The second mixed solution was centrifuged at 12000 r / min for 15 min, and then re-dissolved in a blocking solution with an ion concentration of 15 mM and a pH of 7.0, and then ultrasonically dispersed for 3 min, and blocked for 60 min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 1.5:0.5:98; 5. The third mixed solution was centrifuged at 12000 r / min for 15 min, re-dissolved in a second buffer solution with an ion concentration of 25 mM and a pH of 8.0, and ultrasonically dispersed for 3 min to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 7:2:1:90; In the above steps 2 to 5, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 20:20:20:20:20; 6. The prepared red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 10 μL / cm. Then, the prepared red modified microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. The structure is as follows: Figure 3 As shown; 7. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 3% Tween-20 and 60 mM to prepare ochratoxin A standard test solutions of 0, 1, 2, 5, 10, 15, and 20 μg / mL respectively; 8. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the finished test strip, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 45° C. for 8 min; 9. After the chromatography reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 10. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 11. Dilute and mix the sample to be tested with 3% Tween-20 and 60 mM carbonate solution in the same way to prepare a 2 μg / mL ochratoxin A sample to be tested, and add it dropwise to the sample well 9 of the test strip. The amount of addition is 100 μL. Then, the test strip is placed in an incubator for chromatography reaction. The reaction temperature is 45°C and the reaction time is 8 min. 12. After the reaction is completed, insert the test strip into the red modified microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of ochratoxin A in the sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the sample to be tested can be calculated.

[0046] Comparative Example 3: A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine and its application, comprising the following steps: 1. Mix the red microspheres with a first buffer solution with an ion concentration of 30 mM and a pH of 9.0, and disperse them by ultrasonication for 3 minutes to obtain a first mixed solution; wherein the first buffer solution includes sucrose, BSA and boric acid buffer solution; the mass ratio of the sucrose, BSA and boric acid buffer solution is 10:2:88; the mass ratio of the red microspheres to the first buffer solution is 40:60; 2. The first mixed solution is centrifuged at 8000-20000 r / min for 5-20 min, re-dissolved with the same first buffer, and then ultrasonically dispersed for 3 min before adding ochratoxin A monoclonal antibody for coupling for 30-45 min to obtain a second mixed solution; 3. The second mixed solution was centrifuged at 20000r / min for 5min, and then re-dissolved in a blocking solution with an ion concentration of 25mM and a pH of 9.0, and then ultrasonically dispersed for 3min, and blocked for 35min to obtain a third mixed solution; wherein the blocking solution comprises BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is 0.5:1:98.5; 4. The third mixed solution was centrifuged at 20000r / min for 5min, re-dissolved in a second buffer solution with an ion concentration of 25mM and a pH of 10.0, and ultrasonically dispersed for 3min to obtain the red microsphere-ochratoxin A monoclonal antibody conjugate; wherein the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer is 10:3:3:84; In the above steps 1 to 4, the mass ratio of the first mixed solution, the first buffer, the ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is 15:30:15:20:20; 5. The prepared red microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad 2 by spraying, and the spraying amount is 4 μL / cm. Then, the prepared red microsphere immunochromatographic test strip is put on the card shell 10 to make a finished test strip. Its structure is as follows: Figure 3 As shown; 6. Then, the ochratoxin A solutions of different concentrations were diluted with a carbonate solution containing 5% Tween-20 and 30 mM to prepare ochratoxin A standard test solutions with concentrations of 0, 1, 2, 5, 10, 15, and 20 μg / mL, respectively; 7. Then, the ochratoxin A standard test solution was added dropwise to the sample well 9 of the test strip product, with the amount of 100 μL, and then the test strip was placed in an incubator for chromatography reaction at a reaction temperature of 50° C. and a reaction time of 6 min. 8. After the chromatography reaction is completed, insert the test strip into the red microsphere immunoassay analyzer to measure the absorbance values ​​of T1 test line 3, T2 test line 4 and quality control line 5; 9. The absorbance ratio of the test line and the quality control line 5 of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; 10. Dilute and mix the sample to be tested with 5% Tween-20, 30 mM carbonate solution in the same way to prepare 15 μg / mL ochratoxin A sample to be tested, add it dropwise to the sample well 9 of the test strip, the amount of addition is 100 μL, and then put the test strip into the incubator for chromatography reaction, the reaction temperature is 50°C, and the reaction time is 6 min; 11. After the reaction is completed, insert the test strip into the red microsphere immunoassay analyzer and measure the (T1+T2) / C value. According to the standard relationship curve between the concentration of the ochratoxin A sample to be tested and the (T1+T2) / C value, the content of ochratoxin A in the test solution can be calculated.

[0047] The effect of the red modified microspheres was verified by testing the specificity and accuracy of the test results of the test strips prepared in the examples and comparative examples for ochratoxin A test samples of different concentrations and other mycotoxin test samples (OTB). The results are shown in Table 1.

[0048] Table 1 Test results of test strips prepared in Examples and Comparative Examples

[0049] As can be seen from Table 1, the accuracy of the detection results of Examples 1 to 6 for ochratoxin A test samples of different concentrations is very high, and the accuracy results are significantly compared with the accuracy results of other mycotoxin test samples (OTB) of different concentrations; it illustrates the specificity and accuracy of Examples 1 to 6 for the detection of ochratoxin A. At the same time, whether it is for ochratoxin A test samples of different concentrations or other mycotoxin test samples (OTB) of different concentrations, the accuracy of Examples 1 to 6 is higher than that of Comparative Examples 1 to 3. This is because the red microspheres used in the immunochromatographic test strips prepared in Examples 1 to 6 have been surface-modified, so that the number of surface carboxyl groups is increased, which can better form a conjugate with the ochratoxin A monoclonal antibody, making it more specific and more accurate at the same time.

[0050] In Comparative Examples 1 to 3, the red microspheres used in some immunochromatographic test strips have not been surface-modified (Comparative Example 1); the red microspheres used in some immunochromatographic test strips have been surface-modified but the surface carboxyl groups have not been activated (Comparative Example 2); and the red microspheres used in some immunochromatographic test strips have neither been surface-modified nor have the surface carboxyl groups activated, so the specificity and accuracy are poor.

[0051] In addition, the immunochromatographic test strip prepared in Example 1 was further tested to further demonstrate its performance. The results include the following: The samples to be tested were prepared with negative wine into samples of ochratoxin A with concentrations of 1, 2, and 4 μg / mL, respectively, and tested with test strips, each concentration was repeated 5 times, and the intra-batch precision of the test strips was calculated. Different batches of test strips were used to test the samples to be tested for ochratoxin A, each concentration was repeated 5 times, and the inter-batch precision of the test strips was calculated. As shown in Table 2, the intra-batch coefficient of variation of the ochratoxin A immune test strips obtained by the coefficient of variation calculation formula was 4.14%, and the inter-batch coefficient of variation of the ochratoxin A immune test strips was 4.32%, respectively, indicating that the intra-batch and inter-batch coefficients of variation of the test strips were small, the precision was high, the accuracy was good, and it basically met the requirements of the quantitative detection test strips.

[0052] Table 2 Precision of ochratoxin A immunoassay strips

[0053] Weigh 30 ochratoxin A positive wine samples (15 red wines and 15 white wines), add 500 μL sample diluent to prepare a test solution, and use the ochratoxin A immune test strip developed by the present invention to detect, and repeat 3 times for each concentration. At the same time, high performance liquid chromatography is used as a control to calculate the accuracy of the method. As shown in Table 3, the accuracy of red wine is between 92.13% and 109.92%, and the accuracy of white wine is between 86.67% and 106.29%. It shows that the test strip has high precision and good accuracy, which meets the requirements of quantitative detection test strips.

[0054] Table 3 Accuracy of ochratoxin A immunoassay strips

[0055] Analogues with similar structures to ochratoxin A (OTB, OTC) and three common mycotoxins (AFB1, DON and ZEN) were selected, and negative wine samples were spiked to 10 μg / mL to test the specificity of the ochratoxin A immunoassay strip. As can be seen from Table 4, ochratoxin A has a slight cross-reaction with its structural analogues (OTB, OTC), but no cross-reaction with the other three mycotoxins, indicating that after surface modification, the carboxyl groups on the surface of the red microspheres are increased, which can better form a conjugate with the ochratoxin A monoclonal antibody, thereby ultimately improving the specificity of the test strip, and being able to quickly, stably and accurately detect ochratoxin A contained in wine samples.

[0056] Table 4 Specificity of ochratoxin A immunoassay strips

[0057] The above results show and describe the basic principles and main features of the present application as well as the advantages of the present application.

[0058] Those skilled in the art should understand that the present application is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present application. Without departing from the spirit and scope of the present application, the present application may have various changes and improvements, and these changes and improvements fall within the scope of the present application for which protection is sought. The scope of protection sought in the present application is defined by the equivalents of the attached claims.

Claims

1. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine, characterized in that: The red modified microsphere immunochromatographic test strip comprises a sample pad (1), a conjugate pad (2), a nitrocellulose membrane (6), absorbent paper (7) and a PVC sheet (8), wherein the conjugate pad (2) is fixed with a red modified microsphere-ochratoxin A monoclonal antibody conjugate, and the nitrocellulose membrane (6) is provided with a T1 test line (3), a T2 test line (4) and a quality control line (5) from left to right, wherein the T1 test line (3) and the T2 test line (4) are fixed with ochratoxin A-BSA antigens of different concentrations, and the quality control line (5) is fixed with a goat anti-mouse polyclonal antibody.

2. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 1, characterized in that: The method for preparing the red modified microsphere-ochratoxin A monoclonal antibody conjugate comprises the following steps: The red microspheres are dispersed in an emulsion system containing a monomer, an emulsifier and an initiator, and a polymerization reaction is carried out at a set temperature. After the reaction is completed, the red modified microspheres are obtained by demulsification and washing. The red modified microspheres are mixed with the first buffer solution, dispersed by ultrasonication, and activated by adding carbodiimide for 30 to 60 minutes to obtain a first mixed solution; The first mixed solution is centrifuged, re-dissolved with the first buffer, and then ultrasonically dispersed and then added with ochratoxin A monoclonal antibody for coupling for 30 to 45 minutes to obtain a second mixed solution; The second mixed solution was centrifuged, re-dissolved with a blocking solution, and then ultrasonically dispersed and blocked for 30 to 60 minutes to obtain a third mixed solution; The third mixed solution is centrifuged, re-dissolved with the second buffer solution, and ultrasonically dispersed to obtain the red modified microsphere-ochratoxin A monoclonal antibody conjugate.

3. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 2, characterized in that: In the process of preparing red modified microspheres, the red microspheres include polystyrene microspheres containing carboxyl groups on the surface, and the average particle size is 50-400 nm; the monomer includes one or more of acrylic acid, methacrylic acid, and itaconic acid; the emulsifier includes one or two of sodium dodecyl sulfate and sodium hexadecyl sulfate; the initiator includes one or more of potassium persulfate, diisopropylbenzene peroxide, and azobisisobutyronitrile; the set temperature is 60-80° C.; the mass ratio of the red microspheres, monomer, emulsifier, and initiator is (10-20): (70-90): (1-5): (0.5-2).

4. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 2, characterized in that: The mass ratio of the red modified microspheres, the first buffer, and carbodiimide is (40-50): (40-60): (1-5); the mass ratio of the first mixed solution, the first buffer, ochratoxin A monoclonal antibody, the blocking solution, and the second buffer is (10-20): (20-30): (10-20): (20-30): (20-30).

5. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 2, characterized in that: The ion concentration of the first buffer solution is 15-30 mM, and the pH is 8.0-10.0; the components of the first buffer solution include sucrose, BSA and boric acid buffer; the mass ratio of the sucrose, BSA and boric acid buffer solution is (5-10): (1-3): (87-94).

6. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 2, characterized in that: The ion concentration of the blocking solution is 15-30 mM, and the pH is 7.0-9.0; the components of the blocking solution include BSA, glycine and boric acid buffer; the mass ratio of BSA, glycine and boric acid buffer is (0.5-3): (0.1-1): (96-99.4).

7. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 2, characterized in that: The ion concentration of the second buffer solution is 10-30 mM, and the pH is 8.0-10.0; the components of the second buffer solution include sucrose, trehalose, BSA and boric acid buffer; the mass ratio of the sucrose, trehalose, BSA and boric acid buffer solution is (5-10): (1-3): (1-3): (84-94).

8. The red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 1, characterized in that: The concentration of ochratoxin A-BSA antigen on the T1 test line (3) is 0.5-1.5 mg / mL; the concentration of ochratoxin A-BSA antigen on the T2 test line (4) is 0.3-1.2 mg / mL; the concentration of goat anti-mouse antibody on the quality control line (5) is 0.05-0.3 mg / mL; and the intervals between the T1 test line (3), the T2 test line (4) and the quality control line (5) are 0.5-1.5 cm.

9. A red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 1, characterized in that: The red modified microsphere-ochratoxin A monoclonal antibody conjugate is fixed on the conjugate pad (2) by spraying, and the spraying amount is 4-10 μL / cm.

10. Use of a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to any one of claims 1 to 9, characterized in that: The following steps are involved: Putting the prepared red modified microsphere immunochromatographic test strip into a card shell (10) to make a finished test strip; Then, the ochratoxin A solutions of different concentrations are diluted with carbonate solutions to prepare ochratoxin A standard test solutions of different concentrations; Subsequently, the ochratoxin A standard test solution is added dropwise to the sample addition holes (9) of the finished test strip, and then the test strip is placed in an incubator for chromatography reaction; After the chromatography reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer from low concentration to high concentration, and the absorbance values ​​of the T1 test line (3), the T2 test line (4) and the quality control line (5) are measured; The absorbance ratio of the test line and the quality control line of the ochratoxin A standard test solution of different concentrations is recorded as (T1+T2) / C value, and is fitted with the ochratoxin A standard test solution of different concentrations to obtain a standard relationship curve between the concentration of the ochratoxin A standard test solution and the (T1+T2) / C value; The sample to be tested is diluted and mixed with the carbonate solution in the same way, and then dripped into the sample adding hole of the test strip, and then the test strip is placed in an incubator for chromatography reaction; After the reaction is completed, the test strip is inserted into the red modified microsphere immunoassay analyzer to measure the (T1+T2) / C value. The ochratoxin A content in the sample to be tested is calculated based on the standard relationship curve between the concentration of the sample to be tested and the (T1+T2) / C value.

11. The use of a red modified microsphere immunochromatographic test strip for detecting ochratoxin A in wine according to claim 10, characterized in that: The concentration of the ochratoxin A solution is 0-20 μg / mL; the carbonate solution is a carbonate solution containing 1-5% Tween-20 and 30-100 mM; the amount of the ochratoxin A standard test solution added to the sample well of the test strip is 100 μL; the chromatography reaction in the incubator is carried out at a temperature of 30-50° C. for 6-15 minutes; the intervals between the T1 detection line (3), the T2 detection line (4) and the quality control line (5) are 0.5-1.5 cm.

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