Vomitoxin detection method based on quantum detection technology

Abstract

The invention belongs to the technical field of toxin detection, and particularly relates to a vomitoxin detection method based on a quantum detection technology. Based on a QBs fluorescence labeling technology and a competitive immunochromatography principle, a high-sensitivity quantitative detection system for DON in grain and oil crops is successfully constructed. By systematically optimizing the technical parameters of an antigen-antibody reaction system and a detection device, trace detection of DON (the detection limit reaches 10 ng / mL) is realized. A high-performance monoclonal antibody is screened based on a hybridoma cell technology, indirect ELISA detection shows that the half inhibitory concentration (IC50) to DON is lower than 40 ng / mL, and the affinity advantage of the antibody is verified.

Description

Technical Field

[0001] The present invention belongs to the technical field of toxin detection, and in particular relates to a method for detecting vomitoxin based on quantum detection technology. Background Art

[0002] The main component of vomitoxin is deoxynivalenol (DON), a compound from the trichothecenes family of mycotoxins and a secondary metabolite of Fusarium. DON is chemically stable and widespread in nature. Its toxin-producing strains thrive in cool, humid conditions, making it a common mycotoxin in grain and oil crop contamination. DON is highly cytotoxic and immunosuppressive. Once it enters the human and animal food chain, it poses a significant threat to human and animal health, as well as the agricultural economy. Consumption of DON-contaminated food can cause acute gastrointestinal symptoms, often leading to acute poisoning symptoms such as anorexia, vomiting, diarrhea, fever, unresponsiveness, and impaired immune function. In severe cases, it can damage the hematopoietic system and cause death.

[0003] Currently, commonly used DON detection methods fall into three main categories: the first is based on spectroscopic chemical analysis techniques such as spectroscopy, chromatography, and mass spectrometry, including liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), gas chromatography-mass spectrometry (GC / MS), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC); the second is rapid screening methods based on immunoadsorption and nucleic acid aptamer technology; and the third is convenient and rapid methods based on immunochromatographic technology. Spectroscopic chemical analysis techniques are highly sensitive and can accurately quantify, but they require lengthy testing times, expensive equipment, require specialized technicians, and are not suitable for large-scale, rapid screening of field samples. Immunoadsorption techniques offer strong specificity, but the activity of their immunoenzyme preparations is unstable and prone to false-positive results. Nucleic acid aptamer technology is low-cost and highly sensitive, but the relevant database is incomplete and still under research and development. Immunochromatographic technology offers the advantages of low cost, short testing times, simple operation, and portability. Traditional immunochromatography technology uses colloidal gold particles as a marker. Colloidal gold has poor stability, resulting in low detection sensitivity, difficulty in quantification, and inaccurate results. Therefore, exploring the introduction of alternative marker materials to improve its sensitivity has become a focus of immunochromatography technology in recent years.

[0004] Quantum dots (QDs) are nano-semiconductor crystals with quantum effects, possessing unique optical and electronic properties. Chemically synthesizing QDs with polymer materials, they form quantum dot nanobeads (QBs). These particles range in size from nanometers to micrometers, exhibiting higher brightness and greater stability. As markers, they can significantly improve detection sensitivity and accuracy. However, no technology has yet been reported for the detection of vomitoxin using QDs. Summary of the Invention

[0005] This invention uses QBs as labeling materials, based on the principle of competitive immunoassay, and utilizes the quantitative mechanical properties of lateral flow chromatography to establish a quantum detection system for vomitoxin. It successfully constructs a rapid and simple quantitative detection method for DON in grain and oil crops, and realizes rapid and efficient on-site quantitative screening of DON in grain and oil crops. It can provide a reference for the development of rapid detection technology of fungi and the research on the application of QDs in the field of fungal toxin detection.

[0006] In one aspect, the present invention provides a method for constructing a DON detection system, comprising: Step (1), using mercaptopropionic acid to prepare DON complete antigen DON-BSA; Step (2), inoculating mice with the complete DON antigen DON-BSA to prepare DON monoclonal antibodies through animal immunization; Step (3), coupling the DON monoclonal antibody to the QBs surface using the EDC / NHS method to obtain the antibody QBs probe; Step (4): dilute the antibody QBs probe and spray it on the conjugate pad, streak-coat DON-BSA on the NC membrane as the T line, and immobilize and coat 0.1 mg / mL goat anti-mouse IgG on the NC membrane as the C line. Assemble the absorbent pad, conjugate pad, sample pad and NC membrane, and dry them in a drying oven at 37°C to obtain a DON detection system.

[0007] In some embodiments, in step (1), 5 mg of DON standard is weighed and dissolved in 1 mL of acetonitrile, 200 μL of acetic acid is added to completely dissolve the DON standard, and then 10 mg of mercaptopropionic acid is added. The DON standard is stirred at 80° C. for 12 h, and then 20 μL of water is added to stop the reaction. The pH is adjusted to 13. The standard is then extracted with 2 mL of ethyl acetate, the organic phase is distilled and dried, and the crystals are washed with petroleum ether to obtain the DON-derivatized hapten DON-MPA. Weigh 2 mg of DON-MPA and dissolve it in 0.5 mL of DMF. Add 1.65 mg of DCC and 1.21 mg of NHS and activate it at room temperature in the dark for 60 min. Weigh 6 mg of BSA and dissolve it in 1 mL of PBS. Add the activated DON-MPA dropwise to the BSA solution and react at room temperature in the dark for 2 h. The reaction product is dialyzed at 4°C with stirring for 3 days to obtain the complete DON antigen DON-BSA.

[0008] In some embodiments, in step (3), 5 μg EDC, 5 μg NHS, 0.5 mg QBs and 1000 μL of 0.5 mg / mL DON monoclonal antibody are added to 1.0 mL PB buffer, the PB buffer is 0.01 M, pH 8.0; after being placed on a magnetic stirrer for 2 h, 500 μL of 10% BSA solution is added and the reaction is continued for 1 h; the reaction product is centrifuged at 12000 rpm for 10 min, the precipitate is washed with 0.01 M PBS buffer and redissolved in 0.5 mL of the redissolved solution to obtain the antibody QBs probe.

[0009] In some embodiments, in step (4), the antibody QBs probe is diluted 15 times.

[0010] In some embodiments, in step (4), the coating amount of DON-BSA is 0.1 mg / mL.

[0011] In another aspect, the present invention provides a DON detection system obtained by the above construction method.

[0012] The present invention is based on the principle of competitive immunoassay, that is, the antibody concentration in the sample is inversely proportional to the fluorescence signal value of the T line within a certain range. The developed detection system uses a fluorescence reader to read the fluorescence signal values of the T line and C line, and establishes a standard curve through the difference in relative fluorescence intensity (T / C) values at different concentrations of the target, thereby quantitatively detecting the target content.

[0013] In yet another aspect, the present invention provides a method for detecting DON, comprising: The sample to be tested was crushed and passed through a 20-mesh sieve. The sample was weighed and mixed with extraction buffer at a ratio of 1:5 (w / v). Then, 100 μL of the diluted extract was taken into a 1.5 mL centrifuge tube and tested using the DON detection system. After 15 minutes, the fluorescence signal values of the T line and C line were captured and read using an HF6500 fluorescence reader.

[0014] In some embodiments, the sample to be tested is selected from soybean, corn, wheat, brown rice, rice, sesame, mung bean, and peanut.

[0015] In some embodiments, the extraction buffer is an ethanol-water solution. When the sample to be tested is a grain crop, since it contains relatively little oil, a 0.04% ethanol solution is used as the extraction buffer; when the sample to be tested is a grain or oil crop, since it contains a relatively high oil content, a 40% ethanol solution is used as the extraction buffer.

[0016] In some embodiments, the test sample further contains a mycotoxin selected from 3-AC-DON, 15-AC-DON, OTA, ZEN, AFM1, AFB1, AFG1, FB1, and T-2.

[0017] The beneficial effects of the present invention are: Based on the fluorescent labeling technology of QBs and the principle of competitive immunochromatography, the present invention successfully constructed a highly sensitive quantitative detection system for DON in grain and oil crops. By systematically optimizing the technical parameters of the antigen-antibody reaction system and the detection device, trace detection of DON was achieved (detection limit of 10 ng / mL). Based on hybridoma cell technology, high-performance monoclonal antibodies were screened, and their indirect ELISA test showed that the half-inhibitory concentration (IC50) for DON was less than 40 ng / mL, verifying the antibody affinity advantage. Further studies have shown that the system can effectively avoid the interference of coexisting toxins such as 3-AC-DON and 15-AC-DON in the grain and oil matrix, and the correlation coefficient (R 2 ) reached 0.9961, showing good specificity and quantitative reliability. In the spike recovery test, the average recovery rates of typical matrices such as corn and wheat ranged from (80.0 to 114.0)%, with a CV ≤ 15%, which fully met the national mycotoxin detection standards. In addition, the CV value decreased significantly as the DON concentration approached the national limit (1000 μg / kg), proving that it is more suitable for accurate determination of DON detection results. In addition, in view of the complexity of grain and oil crop matrices, the commonly used extraction liquids in China are 50%-80% methanol aqueous solution or ethanol aqueous solution. The present invention explored the use of a non-organic solvent liquid-solid extraction method (organic solvent proportion <0.05%). This method avoids the hazards of traditional volatile solvents while having the advantages of efficient phase separation (operation time <3 min) and stable recovery (CV ≤ 15%). It provides a practical example for green pretreatment technology. However, when extracting matrices with high oil content such as peanuts and sesame, the proportion of organic solvents needs to be increased accordingly.

[0018] This study uses the mercaptopropionic acid method to achieve directed coupling of DON haptens with carrier proteins, successfully preparing a highly immunogenic complete antigen and screening high-affinity anti-DON monoclonal antibodies using hybridoma cells. Based on QBs labeling technology, a novel competitive fluorescence immunochromatographic detection system was constructed. This system utilizes a portable fluorescence reader for quantitative analysis. It is simple to operate, sensitive, and highly specific. It can be used for rapid on-site screening of grain and oil crops, and is particularly suitable for rapid risk warnings by grassroots regulatory agencies and storage and procurement processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the UV spectrum of the antigen DON-BSA in Example 1 of the present invention; Figure 2The T / C results of different DON-BSA coating concentrations and antibody QBs probe dilution ratios in Example 1 of the present invention are shown; Figure 3 A linear graph showing the sensitivity results of the DON detection system in Example 2 of the present invention is shown; Figure 4 The quantitative linear fitting diagram of the coarse screening of the DON detection system in Example 2 of the present invention is shown; Figure 5 The accurate quantitative linear fitting diagram of the DON detection system in Example 2 of the present invention is shown; Figure 6 The standard curve of the DON detection system in Example 2 of the present invention is shown.

[0020] Specific embodiment In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0021] Materials and instruments Example 1 Chemical reagents and materials DON, 3-acetyl-deoxynivalenol (3-AC-DON), 15-acetyl-deoxynivalenol (15-AC-DON), ochratoxin A (OTA), zearalenone (ZEN), aflatoxin M1 (AFM1), aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), fumonisin B1 (FB1), and trichothecenes (T-2) standards, Zhengzhou Junyue Kelaite Health Technology Co., Ltd. Acetonitrile, acetic acid, mercaptopropionic acid, ethyl acetate, petroleum ether, N,N-dimethylformamide (DMF), N,N-dicyclohexylcarbodiimide hydrochloride (DCC), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS), phosphate buffer (PB), ammonium sulfate ((NH4)2SO4), paraffin oil, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) were purchased from Henan Huafeng Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) and goat anti-mouse IgG (immunoglobulin G, IgG) were purchased from Sigma Aldrich (Shanghai). Water-soluble carboxyl CdSe / ZnS quantum dots (QBs, emission wavelength 620 nm ± 10 nm), Beijing Beida Jubang Technology Co., Ltd. Polyvinyl chloride (PVC) adhesive base, sample pad, conjugate pad, nitrocellulose membrane (NC), and absorbent pad were purchased from Hebei Yilisha Biotechnology Co., Ltd.

[0022] Experimental animals, reagents and materials 6- to 8-week-old female BALB / C mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Myeloma cells SP2 / 0, cell bank of the Chinese Academy of Sciences, Shanghai; Freund's complete adjuvant, Freund's incomplete adjuvant, reconstitution solution (0.02 M Na2HPO4, 0.4% Tween-20, 5% sucrose (w / v), 3% trehalose (w / v), 0.1% NaN3, 0.05% PEG20000), and extraction buffer were provided by Sinopharm Chemical Reagent Co., Ltd. Cell culture plates, Costar, USA; 96-well ELISA plate, Corning, USA.

[0023] Preparation and identification of complete DON antigen 5 mg of DON standard was weighed and dissolved in 1 mL of acetonitrile. 200 μL of acetic acid was added to completely dissolve the DON standard, followed by 10 mg of mercaptopropionic acid. The reaction was stirred at 80°C for 12 h, and then 20 μL of water was added to stop the reaction and adjust the pH to 13. The DON-derivatized hapten, DON-MPA, was then extracted with 2 mL of ethyl acetate. The organic phase was distilled to dryness and the crystals were washed with petroleum ether to obtain the DON-derivatized hapten. 2 mg of DON-MPA was weighed and dissolved in 0.5 mL of DMF. 1.65 mg of DCC and 1.21 mg of NHS were added for activation at room temperature in the dark for 60 min. 6 mg of BSA was weighed and dissolved in 1 mL of PBS. The activated DON-MPA was added dropwise to the BSA solution and allowed to react at room temperature in the dark for 2 h. The reaction product was dialyzed at 4°C for 3 days to obtain the complete DON antigen, DON-BSA, which was aliquoted and frozen for future use.

[0024] The complete antibody was identified by UV-visible scanning spectroscopy. Figure 1 As shown in the figure, the maximum absorption peak of BSA is 278nm, the peak of DON is 213nm, and the maximum absorption peak of the artificial antigen DON-BSA is 275nm. The shift of the absorption peaks proves that the coupling of the artificial antigen is successful.

[0025] Preparation and identification of DON monoclonal antibody 1) Animal immunization DON-BSA was diluted to 1 mg / mL, emulsified with an equal volume of Freund's adjuvant, and inoculated into BALB / c mice. The first immunization involved multiple subcutaneous injections of a mixture of DON-BSA and complete Freund's adjuvant (100 μg / mouse). For the second, third, and fifth immunizations, 50 μg / mouse were administered subcutaneously with incomplete Freund's adjuvant (50 μg / mouse), with an interval of 14 days between immunizations. Starting with the third immunization, tail tip blood was collected 7 days after each immunization to measure titer and inhibition by ELISA. One week after the fifth immunization, tail tip blood was collected to measure titer by ELISA. Mice with the highest serum titer were selected for booster immunizations, and spleen cells were harvested 3 days later for fusion with SP2 / 0 cells.

[0026] 2) Collection, purification and identification of monoclonal antibodies Monoclonal antibodies were prepared using hybridoma cell inoculation within the peritoneal cavity of mice, followed by collection of ascites fluid. Two-month-old female BALB / C mice were injected intraperitoneally with 500 μL of paraffin oil. Within 7 to 14 days, 300 μL of fused hybridoma SP2 / 0 cells (1×10⁶ cells / mL) were inoculated intraperitoneally. Seven days later, ascites fluid was collected and centrifuged at 10,000 rpm for 10 minutes at 4°C to remove impurities. The fluid was then frozen at -20°C until further use. After rethawing, the antibody was purified using (NH₄)₂SO₄ precipitation to obtain the DON monoclonal antibody.

[0027] Absorbance (A) values at 280 nm and 260 nm were measured using a UV spectrophotometer. Antibody concentration was calculated using the following formula: C (mg / mL) = (1.45 × A1 - 0.74 × A2) × N, where N is the dilution factor, A1 is the absorbance at 280 nm, and A2 is the absorbance at 260 nm. Absorbance (A) values were measured using a microplate reader at 450 nm. The antibody titer was determined by determining the dilution factor at which the antibody's A450 value was ≥ 2.1 times that of the negative control well and the A450 value was greater than 1.0.

[0028] Dilute the mycotoxin standard with PBS to different concentrations of standard solution, determine it according to the ELISA method, establish a standard curve, and calculate the IC 50 The results of the four parallel experiments are shown in Table 1.

[0029] Table 1 Preparation of Antibody QBs Probes The purified DON monoclonal antibody was directly coupled to the surface of QBs using the EDC / NHS method: 5 μg EDC, 5 μg NHS, 0.5 mg QBs and 1000 μL DON monoclonal antibody (0.5 mg / mL) were added to 1.0 mL PB buffer (0.01 M, pH 8.0) and placed on a magnetic stirrer for 2 h. After that, 500 μL 10% BSA solution (w / v) was added and the reaction continued for 1 h. The reaction product was centrifuged at 12000 rpm for 10 min, the precipitate was washed with 0.01 M PBS buffer, redissolved in 0.5 mL of the reconstitution solution, and stored at 4°C until use.

[0030] Determination of DON-BSA and Antibody QBs Probe Dilution Factors The antibody QBs probe was diluted at a dilution factor of 1:5, 1:10, 1:15, and 1:20 and then sprayed onto the conjugate pad. DON-BSA was coated on the NC membrane at a coating amount of 0.05, 0.07, 0.1, and 0.15 mg / mL as the test line (T line). 0.1 mg / mL goat anti-mouse IgG was fixed and coated on the NC membrane as the control line (C line). The absorbent pad, conjugate pad, sample pad, and NC membrane were sequentially attached to the PVC backing according to a certain assembly process to assemble the detection system. 100 uL PBS was added to each sample well of the detection system. After ten minutes, the fluorescence intensity was read using a fluorescence test strip reader. The results are shown in Table 2 and Figure 2 shown.

[0031] Table 2 Selection of antigen coating concentration and antibody QBs probe dilution multiple Taking into account the fluorescence signal intensities of the T line and C line, and the fluorescence signal intensity of the T line was slightly greater than that of the C line (i.e., T / C>1), the optimal coating amount of DON-BSA was finally determined to be 0.1 mg / mL, and the dilution ratio of the antibody QBs probe was 1:15.

[0032] Construction of DON detection system The absorbent pad, NC membrane, conjugate pad, and sample pad were assembled according to the above process. 0.1 mg / mL goat anti-mouse secondary antibody and optimized concentration of DON-BSA were sprayed on the quality control line (C line) and test line (T line), respectively. The samples were placed in a 37°C constant temperature drying oven for drying. The optimized antibody QBs probe was sprayed on the conjugate pad, cut into 4 mm wide strips, and stored in a refrigerator at 4°C for later use.

[0033] Example 2 The sample to be tested was crushed and passed through a 20-mesh sieve. The sample was weighed and mixed with 1X extraction buffer at a ratio of 1:5 (w / v). Then 100 μL of the diluted extract was taken into a 1.5 mL centrifuge tube. The assembled and cut test strips were placed vertically into the centrifuge tube. After 15 minutes, the fluorescence signal values of the T line and C line were captured and read using an HF6500 fluorescence reader.

[0034] Detection system performance evaluation 1) Sensitivity and linearity The sensitivity and linear range of the detection system were evaluated by diluting DON standard solutions at different concentrations of 5, 10, 20, 40, 80, 100, 200, 400, 800, 1600, and 2000 ng / mL. Each concentration was repeated three times, and PBS buffer was used as a blank control. After extraction, the reaction was carried out under the optimized optimal conditions for 15 minutes, and the fluorescence signal values of the T line and C line were captured using an HF6500 fluorescence reader. The results are shown in Table 3. Figure 4 、 Figure 5 shown.

[0035] Table 3 Sensitivity results of DON detection system When the T / C value is ≤1, the maximum concentration of DON is 10 ng / mL, i.e. 0.01 ppm, which is the detection limit of the method. 2 The lowest concentration of 40 ng / mL when the concentration was ≥0.98 was the coarse screening limit of quantification of the method, and the range of 40 to 1600 ng / mL was the coarse screening linear range of quantification. 2 The method's accurate quantification limit is 80 ng / mL, with a minimum concentration of ≥0.99, and the linear range is 80–1600 ng / mL. This meets the requirements of national standard GB2761-2017.

[0036] 2) Specificity DON and other common mycotoxins that contaminate grain and oil crops (3-AC-DON, 15-AC-DON, OTA, ZEN, AFM1, AFB1, AFG1, FB1, T-2) standards were diluted to concentrations of 320, 160, 80, 40, 20, 10, 5, and 1 ng / mL, respectively, for reaction and the results were read, as shown in Table 4.

[0037] Table 4 Specificity results The DON standard solution showed a positive reaction at concentrations above the detection limit, while the other nine interfering samples all showed T / C values greater than 1 and were negative, indicating no cross-reaction. This demonstrates that the detection system has good specificity for DON detection.

[0038] 3) Establishment of standard curve According to GB 2761-2017, the limit for DON in food is ≤1000 μg / kg. A series of standard working solutions (40, 80, 160, 320, 640, and 1280 ng / mL) were prepared using a DON standard according to GB5009.111. These standard solutions were tested sequentially from low to high concentration to obtain T / C values. Three independent parallel tests were performed, with the concentration of the DON standard working solution as the horizontal axis and the average T / C value as the vertical axis to construct a standard curve.

[0039] like Figure 5 and Figure 6 As shown, the standard curve of DON mass concentration was obtained by nonlinear fitting, and the standard curve equation was Y=0.0059x 2 - 0.197x + 1.135, correlation coefficient R 2 = 0.9922.

[0040] 4) Add recycling test The method's systematic measurement error was assessed using a spike-recovery test. Method accuracy was evaluated by calculating the recovery rate, and the dispersion of replicate results was evaluated by calculating the coefficient of variation (CV), thereby measuring the method's precision. Samples from various matrices, including soybeans, corn, wheat, brown rice, rice, sesame, mung beans, and peanuts, were collected. DON was not detected using the GB5009.111-2016 method. Based on the aforementioned limit of quantification, standard solutions of 40, 200, and 1000 ng / mL were spiked with the samples. Six replicates were performed at each spike level, and the recovery and CV values were calculated. The results were calculated using Equation (4) according to GB5009.111-2016, as shown in Table 5.

[0041] Table 5 Recovery results of different matrix additions Calculation formula: , Where: X—DON content in food, unit: μg / kg; ρ x —DON concentration in the test solution, in ng / mL; V—the volume of the extract, in mL; f—the dilution factor during the pretreatment process; m—the sample weight, in g.

[0042] Based on the fluorescent labeling technology of QBs and the principle of competitive immunochromatography, the present invention successfully constructed a highly sensitive quantitative detection system for DON in grain and oil crops. By systematically optimizing the technical parameters of the antigen-antibody reaction system and the detection device, trace detection of DON was achieved (detection limit of 10 ng / mL). Based on hybridoma cell technology, high-performance monoclonal antibodies were screened, and their indirect ELISA test showed that the half-inhibitory concentration (IC50) for DON was less than 40 ng / mL, verifying the antibody affinity advantage. Further studies have shown that the system can effectively avoid the interference of coexisting toxins such as 3-AC-DON and 15-AC-DON in the grain and oil matrix, and the correlation coefficient (R 2 ) reached 0.9961, showing good specificity and quantitative reliability. In the spike recovery test, the average recovery rates of typical matrices such as corn and wheat ranged from (80.0 to 114.0)%, with a CV ≤ 15%, which fully met the national mycotoxin detection standards. In addition, the CV value decreased significantly as the DON concentration approached the national limit (1000 μg / kg), proving that it is more suitable for accurate determination of DON detection results. In addition, in response to the complexity of grain and oil crop matrices, the present invention explored the use of non-organic solvent liquid-solid extraction (organic solvent proportion <0.05%). This method avoids the hazards of traditional volatile solvents while having the advantages of efficient phase separation (operation time <3 min) and stable recovery (CV ≤ 15%). It provides a practical example for green pretreatment technology. However, when extracting matrices with high oil content such as peanuts and sesame, the proportion of organic solvents needs to be increased accordingly.

[0043] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for constructing a DON detection system, comprising: Step (1), using mercaptopropionic acid to prepare DON complete antigen DON-BSA; Step (2), inoculating mice with the complete DON antigen DON-BSA to prepare DON monoclonal antibodies through animal immunization; Step (3), coupling the DON monoclonal antibody to the QBs surface using the EDC / NHS method to obtain the antibody QBs probe; Step (4): dilute the antibody QBs probe and spray it on the conjugate pad, streak-coat DON-BSA on the NC membrane as the T line, and immobilize and coat 0.1 mg / mL goat anti-mouse IgG on the NC membrane as the C line. Assemble the absorbent pad, conjugate pad, sample pad and NC membrane, and dry them in a drying oven at 37°C to obtain a DON detection system.

2. The construction method according to claim 1, wherein In step (1), 5 mg of DON standard was weighed and dissolved in 1 mL of acetonitrile. 200 μL of acetic acid was added to completely dissolve the DON standard, and then 10 mg of mercaptopropionic acid was added. The reaction was stirred at 80°C for 12 h, and then 20 μL of water was added to stop the reaction. The pH was adjusted to 13. The DON-derivatized hapten DON-MPA was obtained after extraction with 2 mL of ethyl acetate, and the organic phase was distilled and dried. The crystals were washed with petroleum ether to obtain the DON-MPA. Weigh 2 mg of DON-MPA and dissolve it in 0.5 mL of DMF. Add 1.65 mg of DCC and 1.21 mg of NHS and activate it at room temperature in the dark for 60 min. Weigh 6 mg of BSA and dissolve it in 1 mL of PBS. Add the activated DON-MPA dropwise to the BSA solution and react at room temperature in the dark for 2 h. The reaction product is dialyzed at 4°C with stirring for 3 days to obtain the complete DON antigen DON-BSA.

3. The construction method according to claim 1, wherein: In step (3), 5 μg EDC, 5 μg NHS, 0.5 mg QBs and 1000 μL 0.5 mg / mL DON monoclonal antibody were added to 1.0 mL PB buffer (0.01 M, pH 8.0); the mixture was placed on a magnetic stirrer for 2 h and then 500 μL 10% BSA solution was added to continue the reaction for 1 h; the reaction product was centrifuged at 12000 rpm for 10 min, the precipitate was washed with 0.01 M PBS buffer and redissolved in 0.5 mL of the solution to obtain the antibody QBs probe.

4. The construction method according to claim 1, wherein: In step (4), the antibody QBs probe is diluted 15 times.

5. The construction method according to claim 1, wherein: In step (4), the coating amount of DON-BSA is 0.1 mg / mL.

6. A DON detection system obtained by the construction method according to any one of claims 1 to 5.

7. A method for detecting DON, comprising: The sample to be tested was crushed and passed through a 20-mesh sieve. The sample was weighed and mixed with the extraction buffer at a ratio of 1:5 (w / v). Then, 100 μL of the diluted extract was taken into a 1.5 mL centrifuge tube and tested using the DON detection system described in claim 6. After 15 minutes, the fluorescence signal values of the T line and C line were captured and read using an HF6500 fluorescence reader.

8. The DON detection method according to claim 7, wherein: The sample to be tested is selected from soybean, corn, wheat, brown rice, rice, sesame, mung bean, and peanut.

9. The DON detection method according to claim 7, wherein: The extraction buffer is an ethanol aqueous solution.

10. The DON detection method according to claim 7, wherein: The sample to be tested also contains mycotoxins selected from 3-AC-DON, 15-AC-DON, OTA, ZEN, AFM1, AFB1, AFG1, FB1, and T-2.

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