A method for detecting ochratoxin A in agricultural products

Abstract

This invention discloses a method for detecting ochratoxin A in agricultural products, belonging to the field of agricultural technology. The method for detecting ochratoxin A in agricultural products includes the following steps: S1, coating OTA-BSA onto an ELISA plate, then adding various known concentrations of OTA to competitively bind to McAb with OTA-BSA, forming OTA-BSA@McAb and OTA@McAb complexes respectively; S2, adding ALP-IgG to bind with OTA-BSA@McAb, then adding AAP and Ce... 4+ Multiple standard solutions were obtained using OPD, and fluorescence emission peaks at 435 nm and 565 nm were detected; S3, a regression equation was obtained; S4, the concentration of ochratoxin A in agricultural products was determined. The method proposed in this invention can accurately and reliably detect the OTA content in agricultural products.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a method for detecting ochratoxin A in agricultural products. Background Technology

[0002] Ochratoxins are primarily secondary metabolites produced by *Aspergillus* and *Penicillium* species, with ochratoxin A (OTA) being the most toxic, widely distributed, and heavily contaminated. OTAs are ubiquitous in various agricultural products and everyday foods, accumulating through the food chain and causing various toxic effects on animals and humans. The International Agency for Research on Cancer (IARC) classified it as a Group 2B carcinogen in 1993. With the rapid development of agriculture, animal husbandry, and transportation, the channels for OTA transmission are constantly increasing, and contamination can occur at any stage from preservation and transportation to processing and commercialization, thereby increasing the potential risk of disease. Therefore, strictly controlling the detection levels of OTAs in food and developing sensitive detection methods are crucial for ensuring food safety and human health.

[0003] Routine methods for detecting OTA (Otamine Oxide) mainly include chromatographic analysis and immunoassay. Chromatographic analysis primarily includes thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and high-performance liquid chromatography-tandem mass spectrometry (LC-MS). Chromatographic methods offer advantages such as high sensitivity, accuracy, and reproducibility; however, complex sample pretreatment, expensive equipment, cumbersome procedures, and the need for specialized training for personnel limit their practical application. Immunoassay primarily includes lateral flow immunoassay (LFIA) and enzyme-linked immunosorbent assay (ELISA). While LFIA can achieve rapid detection in less than 20 minutes, it still suffers from low sensitivity, false positives, and the inability to achieve quantitative detection. ELISA, on the other hand, offers advantages such as high sensitivity, simple operation, speed, and high-throughput screening, and has attracted widespread attention from researchers in the detection of small molecule targets. Summary of the Invention

[0004] Building upon existing technologies, we focus on improving the accuracy of ELISA detection while reducing testing costs, thereby mitigating potential false positives. Therefore, we have designed a novel ratiometric fluorescence immunoassay for the accurate and sensitive detection of OTA content in agricultural products. This invention provides a method for detecting ochratoxin A in agricultural products, addressing the technical problem of low accuracy in existing OTA content detection techniques.

[0005] To achieve the above-mentioned technical objectives, the present invention provides a method for detecting ochratoxin A in agricultural products, comprising the following steps:

[0006] S1. OTA-BSA is coated onto an ELISA plate, and then various known concentrations of OTA are added to compete with OTA-BSA for binding to McAb, forming OTA-BSA@McAb and OTA@McAb complexes respectively.

[0007] S2. Wash away the OTA@McAb complex, add ALP-IgG to bind with OTA-BSA@McAb, then add AAP and Ce. 4+ Multiple standard solutions were obtained using OPD, and the fluorescence emission peaks of the various standard solutions at 435 nm and 565 nm were detected.

[0008] S3. Based on the known concentration of OTA and F 435 / F 565 The fluorescence ratio yielded a regression equation;

[0009] S4. Replace the known concentration of OTA in step S1 with the agricultural product to be tested and repeat steps S1-S2. Measure the fluorescence emission peaks of the agricultural product at 435 nm and 565 nm, and combine the results with the regression equation from step S3 to obtain the concentration of ochratoxin A in the agricultural product.

[0010] Further, in step S1, the concentration of OTA-BSA is 3-12 μg / mL.

[0011] Further, in step S1, the concentration of McAb is 5-20 μg / mL.

[0012] Furthermore, in step S2, the concentration of added ALP-IgG is 1-15 μg / mL.

[0013] Furthermore, in step S2, the added Ce 4+ The concentration of AAP is 1-2 mM; and / or the concentration of AAP added is 1-25 mM, preferably 13-25 mM.

[0014] Furthermore, in step S2, the concentration of added OPD is 1-3.5 mM, preferably 3 mM.

[0015] Furthermore, in step S2, the concentration of added ALP-IgG is 11-15 μg / mL.

[0016] Furthermore, in step S2, AAP and Ce are added. 4+ The reaction time with OPD is more than 20 minutes, preferably 20-30 minutes.

[0017] Furthermore, the agricultural product in question is corn.

[0018] Further, in step S3, the regression equation is y = 22.494 - 1.156x; where y and x represent F... 435 / F 565 Fluorescence ratio and OTA concentration.

[0019] Compared with existing technologies, the beneficial effects of this invention include: firstly, OTA-BSA is coated onto an ELISA plate; then, various known concentrations of OTA are added, and the OTA contained therein competitively binds to McAb with OTA-BSA, forming OTA-BSA@McAb and OTA@McAb complexes, respectively. After washing to remove the OTA@McAb complexes, ALP-IgG is added to bind with OTA-BSA@McAb. Subsequently, ALP-IgG catalyzes the dephosphorylation of ascorbic acid phosphate (AAP) to form ascorbic acid (AA), and the product AA is coated with tetravalent cerium ions (Ce). 4+ Further oxidation to dehydroascorbic acid (DHAA). DHAA catalyzes the reaction of the substrate o-phenylenediamine (OPD) to 3-(dihydroxyethyl)furan[3,4-b]quinoxalin-1-one (DFQ), which exhibits a fluorescence emission peak at 435 nm; simultaneously, Ce... 4+ Competitive oxidation of OPD to generate 2,3-diaminophenazine (DAP) produces a new fluorescence emission peak at 565 nm. Therefore, as the concentration of OTA increases, AA gradually decreases, and the emission peak of the formed DFQ at 435 nm continuously decreases, while the emission peak of DAP at 565 nm continuously increases. Based on the known concentrations of OTA and F... 435 / F 565 The fluorescence ratio can be used to obtain a regression equation. By replacing the known concentration of OTA in step S1 with the agricultural product to be tested, steps S1-S2 are repeated. The fluorescence emission peaks at 435 nm and 565 nm measured by the agricultural product to be tested are combined with the regression equation in step S3 to obtain the concentration of ochratoxin A in the agricultural product, thereby enabling accurate and reliable detection of the OTA content in the agricultural product. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the principle of the immunoassay method based on ratiometric fluorescence for the detection of ochratoxin A in maize according to the present invention.

[0021] Figure 2 This is the feasibility verification result of the detection method of ochratoxin A in agricultural products in Example 1 of the present invention; wherein, Figure 2a: fluorescence emission spectrum; Figure 2b: ultraviolet absorption spectrum, the accompanying image is a picture under natural light; Figure 2 a and Figure 2 The added components in each curve are: Curve a: OPD, Curve b: OPD + Ce 4+ Curve c: OPD + Ce 4+ +AAP, Curve d: OPD+Ce4+ +AAP+ALP.

[0022] Figure 3 This is the optimized result of the OTA-BSA and McAb reaction concentrations in the detection method of ochratoxin A in agricultural products in Example 2 of the present invention.

[0023] Figure 4 This is the optimized result of the reaction concentration of the detection method for ochratoxin A in agricultural products in Example 2 of the present invention; wherein, Figure 4 a:Ce 4+ Concentration optimization; Figure 4 b: Optimization of ALP-IgG concentration; Figure 4 c: AAP concentration optimization; Figure 4 d: OPD concentration optimization.

[0024] Figure 5 This is the optimized reaction time result of the detection method for ochratoxin A in agricultural products in Example 3 of the present invention; wherein, Figure 5 a: Optimization of reaction time for ALP and AAP; Figure 5 b: DHAA / Ce 4+ Optimize reaction time with OPD.

[0025] Figure 6 This is an evaluation result of the sensitivity and selectivity of the detection method for ochratoxin A in agricultural products in Example 3 of the present invention; Figure 6 'a' represents the concentration of ochratoxin A and F. 435 / F 565 Standard curve of fluorescence ratio; Figure 6 b represents the selective analysis results of ochratoxin A. Detailed Implementation

[0026] This specific embodiment provides a method for detecting ochratoxin A in agricultural products, including the following steps:

[0027] S1. OTA-BSA is coated onto an ELISA plate, and then various known concentrations of OTA are added to compete with OTA-BSA for binding to McAb, forming OTA-BSA@McAb and OTA@McAb complexes respectively.

[0028] S2. Wash away the OTA@McAb complex, add ALP-IgG to bind with OTA-BSA@McAb, then add AAP and Ce. 4+ Reaction with OPD for more than 20 minutes yields multiple standard solutions, and the fluorescence emission peaks of these standard solutions at 435 nm and 565 nm are detected. In some embodiments, AAP and Ce are added. 4+ The reaction time with OPD is 20-30 minutes;

[0029] S3. Based on the known concentration of OTA and F 435 / F 565 The fluorescence ratio yielded a regression equation; the regression equation was y = 22.494 - 1.156x, and the correlation coefficient (R²) was [missing value]. 2 ) = 0.982; where y and x represent F respectively 435 / F 565 Fluorescence ratio and OTA concentration;

[0030] S4. Replace the known concentration of OTA in step S1 with the agricultural product to be tested and repeat steps S1-S2. Measure the fluorescence emission peaks of the agricultural product at 435 nm and 565 nm. Combine the results with the regression equation from step S3 to obtain the concentration of ochratoxin A in the agricultural product.

[0031] In some embodiments, in step S1, the concentration of OTA-BSA is 3-12 μg / mL; the concentration of McAb is 5-20 μg / mL.

[0032] In some embodiments, in step S2, the concentration of added ALP-IgG is 1-15 μg / mL, preferably 11-15 μg / mL; the added Ce 4+ The concentration of added AAP is 1-2 mM; and / or the concentration of added AAP is 1-25 mM, preferably 13-25 mM; the concentration of added OPD is 1-3.5 mM, preferably 3 mM; and / or the concentration of added AAP is 13-25 mM.

[0033] In some embodiments, the agricultural product is corn.

[0034] In this invention, the meanings of each abbreviation are as follows:

[0035] OTA-BSA: Ochratoxin A complete antigen; OTA: Ochratoxin A; McAb: Monoclonal antibody; ALP-IgG: Alkaline phosphatase-labeled immunoglobulin G.

[0036] The principle of the detection method proposed in this invention:

[0037] First, OTA-BSA was coated onto an ELISA plate. After agricultural products were added, the OTA contained within competed with OTA-BSA for binding to McAb, forming OTA-BSA@McAb and OTA@McAb complexes, respectively. After washing to remove the OTA@McAb complexes, ALP-IgG was added and bound to OTA-BSA@McAb. Subsequently, ALP-IgG catalyzed the dephosphorylation of ascorbic acid phosphate (AAP) to form ascorbic acid (AA). The product AA was then coated with tetravalent cerium ions (Ce). 4+Further oxidation to dehydroascorbic acid (DHAA). DHAA catalyzes the reaction of the substrate o-phenylenediamine (OPD) to 3-(dihydroxyethyl)furan[3,4-b]quinoxalin-1-one (DFQ), which exhibits a fluorescence emission peak at 435 nm; simultaneously, Ce... 4+ Competitive oxidation of OPD to generate 2,3-diaminophenazine (DAP) produces a new fluorescence emission peak at 565 nm. Therefore, as OTA concentration increases, AA gradually decreases, and the emission peak of the formed DFQ at 435 nm continuously decreases, while the emission peak of DAP at 565 nm continuously increases. By comparing the fluorescence signals at F435 and F565, the constructed ratiometric fluorescence immunoassay method requires no blank control and can accurately and reliably detect the OTA content in agricultural products.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The following embodiments include the following:

[0040] Feasibility verification of ratiometric fluorescence strategy; optimization of system reaction concentration; optimization of system reaction time; sensitivity and selectivity of the method; recovery analysis of OTA content in corn spiked samples.

[0041] The agricultural product used in the following examples is corn, but it is readily understood that the detection method of the present invention is also applicable to other agricultural products; Ce in the following examples 4+ Cerium sulfate, CeS2O8, was used.

[0042] Example 1: Feasibility verification of a ratiometric fluorescence strategy;

[0043] The feasibility of a ratiometric fluorescence strategy was investigated. After reacting each sample at room temperature (25℃) for 40 minutes, 200 μL was added to a black 96-well plate, and the fluorescence emission spectrum of the samples in the 400-700 nm range was measured using a SpectraMax iD3 multi-mode microplate reader. Figure 2 As shown, no fluorescence signal was generated by OPD under fluorescence spectroscopy analysis. Figure 2 a, a). When Ce 4+ After joining, OPD was Ce 4+ The oxidation of DAP produces an emission peak at 565 nm. Figure 2 a, b). After adding AAP, AAP cannot react directly with the system. Figure 2 a, c). When ALP is introduced into the system, ALP dephosphorylates AAP to generate AA, and AA then dephosphorylates Ce. 4+ Restored to Ce3+ Simultaneously, DHAA is generated, and it reacts with OPD to form DFQ, which produces an emission peak at 435 nm, while Ce... 4+ Restored to Ce 3+ Subsequently, the loss of oxidizing properties prevents it from reacting with OPD, leading to a decrease in the emission peak of DAP at 565 nm. Figure 2 a, d), generating ratiometric fluorescence signal output. The feasibility of the ratiometric fluorescence strategy was verified by UV-Vis spectroscopy, when Ce 4+ After OPD is oxidized to form DAP, a distinct UV absorption peak is produced at 446 nm, and the solution appears bright yellow. Figure 2 b, b, c). However, OPD or DFQ did not produce absorption peaks, and the solution was colorless. Figure 2 b, a, d).

[0044] Example 2: Optimization of system reaction concentration

[0045] Standardization of detection conditions is a key factor affecting OTA detection performance. First, the reaction concentrations of the system were optimized. This was achieved by observing changes in the fluorescence ratio at different OTA-BSA and McAb concentrations. Different concentration gradients of OTA-BSA and McAb were set. After coating with OTA-BSA at room temperature for 100 min, the mixture was washed three times with PBST buffer (0.01 M, pH 7.4). McAb was then added and bound at room temperature for 60 min. The washing steps were repeated, followed by the addition of the same amount of ALP-IgG and binding at room temperature for 60 min. After another washing operation, AAP and Ce were introduced. 4+ After adding OPD to the system and reacting at room temperature in the dark for 30 min, the fluorescence emission peaks at 435 nm and 565 nm, at the maximum excitation peak of 370 nm, were analyzed using a SpectraMax iD3 multi-functional microplate reader. With increasing concentrations of OTA-BSA and McAb antigen antibodies, F... 435 / F 565 The fluorescence ratio also increases accordingly, showing a certain linear relationship (e.g. Figure 3 (As shown). When the antigen-antibody concentrations were 3 μg / mL and 5 μg / mL, respectively, the fluorescence ratio showed minimal change while still maintaining a very high level. Based on the principles of reducing experimental costs and improving sensitivity, F was selected. 435 / F 565 The highest fluorescence ratio corresponds to the lowest antigen-antibody concentration. Therefore, the optimal reaction concentrations for OTA-BSA and McAb are 3 μg / mL and 5 μg / mL, respectively.

[0046] Based on the above optimized conditions, target optimal reaction concentrations with different gradients were set, and the same amount of other reactants were added to the system. The fluorescence emission peaks at 435 nm and 565 nm, at the maximum excitation peak of 370 nm, were analyzed using a SpectraMax iD3 multi-functional microplate reader. 4+ As a key element of triggering ratiometric fluorescence strategies, this involves observing different Ce... 4+ Optimize Ce by changing fluorescence signal at different concentrations 4+ Concentration. With Ce 4+ As the concentration increases, the background fluorescence of DAP gradually rises, leading to F 435 / F 565 The fluorescence ratio decreases. When Ce 4+ The fluorescence ratio was highest at a concentration of 1 mM, therefore 1 mM Ce was chosen for the following experiments. 4+ As the optimal dosage (e.g.) Figure 4 (as shown in a). When the ALP-IgG concentration was 11 μg / mL and the AAP concentration was 13 mM, F 435 / F 565 The highest value indicates the maximum enzymatic effect; therefore, 11 μg / mL ALP-IgG and 13 mM AAP were selected as the optimal dosages in the following experiments (e.g., Figure 4 (As shown in b and 4c). OPD was used as a substrate in a ratiometric fluorescence strategy, and different concentration gradients were set for optimization. The results showed that when the concentration was 3 mM, F... 435 / F 565 To reach its maximum, 3 mM OPD was chosen as the optimal dosage in subsequent experiments (e.g., Figure 4 (as shown in d).

[0047] Example 3: Optimization of reaction time in the system;

[0048] Based on the above optimized conditions, the optimal reaction time of the system was explored by observing fluorescence changes at different reaction times. Different ALP gradients were used to catalyze the reaction time of AAP, and then the same amount of Ce was added. 4+ The OPD was added to the system and reacted for 30 min. The fluorescence emission peaks at 435 nm and 565 nm, where the maximum excitation peak was 370 nm, were analyzed using a SpectraMax iD3 multi-functional microplate reader. The results are as follows: Figure 5 As shown in Figure a, the fluorescence signal gradually increases with increasing reaction time, reaching its maximum at a reaction time of 20 min. Further time extension has no significant effect on the fluorescence signal; therefore, the optimal reaction time for ALP and AAP is determined to be 20 min. Different gradients of DHAA and Ce were then set. 4+ The reaction time of co-catalyzed OPD, the results are as follows: Figure 5As shown in b, the highest fluorescence ratio was obtained when the reaction time was 20 min. Therefore, 20 min was chosen as the optimal time for the reaction of DHAA and Ce. 4+ The optimal reaction time for co-catalyzed OPD.

[0049] Sensitivity and selectivity of the method in Example 4;

[0050] After the above reaction conditions reached their optimal levels, the concentrations of added OTA standards were set at 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, and 1 ng / mL, and detection was performed using ratiometric fluorescence immunoassay. The ratio of fluorescence intensity (F) is plotted on the x-axis as OTA concentration. 435 / F 565 Plotting the x-axis on the y-axis, a standard curve was fitted using Origin 2022, and a regression equation was established to evaluate the sensitivity of ratiometric immunoassay. The results are as follows: Figure 6 As shown in Figure a, with the increase of OTA concentration, F 435 / F 565 The fluorescence ratio gradually decreased, showing a good linear relationship between 4 and 16 ng / mL, with a regression equation of y = 22.494 - 1.156x and a correlation coefficient (R²). 2 ) = 0.982 (where y and x represent F respectively) 435 / F 565 (Fluorescence ratio and OTA concentration). Based on the formula 3×SD / S, the limit of detection (LOD) was calculated to be 1.35 ng / mL, meeting the minimum allowable detection concentration requirement for OTA in my country. The linear range was 4.00–16.00 ng / mL (SD is the standard deviation of the fluorescence ratio measured without OTA, and S is the slope of the working curve). Based on the above regression equation, the F-values ​​of the agricultural products to be tested were obtained according to the above method. 435 / F 565 The fluorescence ratio can be obtained by substituting it into the regression equation to determine the concentration of OTA in agricultural products.

[0051] To evaluate the specificity of ratiometric fluorescence immunoassay, OTA and other mycotoxins co-detected with OTA, including AFB1, DON, ZEN, T-2 toxin, and FB1, were detected using this method. The concentration of OTA was set at 10 ng / mL, and the concentration of other mycotoxins at 1000 ng / mL. The fluorescence ratio (F...) was used to measure the specificity of the results. 435 / F 565 Cross-reactivity was assessed. Results showed that other mycotoxins exhibited strong (>10) F... 435 / F 565 Fluorescence ratio, while OTA fluorescence ratio is very low (e.g. Figure 6 (As shown in b). This indicates that the ratiometric fluorescence immunoassay has good specificity.

[0052] Example 5: Recovery analysis of OTA-spiked corn samples;

[0053] Corn samples were purchased from a local supermarket in Jingzhou and simply ground. 0.4 g of the sample was placed in a 1 mL centrifuge tube, and 300, 200, and 100 ng / mL of OTA standards were added. Then, 1 mL of PBS buffer containing 30% methanol was added, and the mixture was sonicated for 10 min and allowed to stand for 30 min. The mixed sample was centrifuged at 12000 g for 10 min, and the supernatant was diluted 20-fold with PBS buffer to minimize interference from the sample matrix. Finally, ratiometric fluorescence immunoassay was used for detection. The recovery rate was calculated using the formula: (Spiked detection concentration - Unspiked detection concentration) / Spiked concentration × 100%. The relative standard deviation (RSD) was calculated using the formula: (Standard deviation / Average detection concentration) × 100%.

[0054] The results are shown in Table 1. When OTA was added to corn samples at concentrations of 15, 10, 5, and 0 ng / mL within the linear range, no OTA was detected in the samples without added OTA. The recoveries of the spiked samples ranged from 91.65% to 105.44%, meeting the standards of the American Association of Analytical Chemistry (AOAC). The RSD was less than 9.00%. These results demonstrate that the method possesses good accuracy, practicality, and reliability.

[0055] Table 1 Recovery analysis of corn spiked samples

[0056]

[0057] Other beneficial effects:

[0058] The detection system of this invention is simple and easy to obtain, improves accuracy while reducing detection costs, and reduces the possibility of false positives in ELISA detection, providing a new approach for detecting OTA residues in agricultural products.

[0059] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting ochratoxin A in agricultural products, characterized in that, Includes the following steps: S1. OTA-BSA is coated onto an ELISA plate, and then various known concentrations of OTA are added to compete with OTA-BSA for binding to McAb, forming OTA-BSA@McAb and OTA@McAb complexes respectively. S2. Wash away the OTA@McAb complex, add ALP-IgG to bind with OTA-BSA@McAb, then add AAP and Ce. 4+ Multiple standard solutions were obtained using OPD, and the fluorescence emission peaks of the various standard solutions at 435 nm and 565 nm were detected. S3. Based on the known concentration of OTA and F 435 / F 565 The fluorescence ratio yielded a regression equation; S4. Replace the known concentration of OTA in step S1 with the agricultural product to be tested and repeat steps S1-S2. Measure the fluorescence emission peaks of the agricultural product at 435 nm and 565 nm, and combine the results with the regression equation from step S3 to obtain the concentration of ochratoxin A in the agricultural product.

2. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S1, the concentration of OTA-BSA is 3-12 μg / mL.

3. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S1, the concentration of McAb is 5-20 μg / mL.

4. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S2, the concentration of added ALP-IgG is 1-15 μg / mL.

5. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S2, Ce is added 4+ The concentration of the added AAP is 1-2 mM; and / or the concentration of the added AAP is 1-25 mM.

6. The method for detecting ochratoxin A in agricultural products according to claim 5, characterized in that, In step S2, the concentration of AAP added is 13-25 mM.

7. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S2, the concentration of added OPD is 1-3.5 mM.

8. The method for detecting ochratoxin A in agricultural products according to claim 7, characterized in that, In step S2, the concentration of added OPD is 3 mM.

9. The method for detecting ochratoxin A in agricultural products according to claim 4, characterized in that, In step S2, the concentration of added ALP-IgG is 11-15 μg / mL.

10. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S2, add AAP and Ce 4+ The reaction time with OPD is more than 20 minutes.

11. The method for detecting ochratoxin A in agricultural products according to claim 10, characterized in that, In step S2, add AAP and Ce 4+ The reaction time with OPD is 20-30 minutes.

12. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, The agricultural product in question is corn.

13. The method for detecting ochratoxin A in agricultural products according to claim 1, characterized in that, In step S3, the regression equation is y = 22.494 - 1.156x; where y and x represent Fi and Fj, respectively. 435 / F 565 Fluorescence ratio and OTA concentration.

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