Detection test strip, detection method and application of ochratoxin A

Abstract

According to the present invention, the quantum dot is loaded on the surface of the PS microsphere to construct the quantum dot microsphere composite signal marker, and the novel fluorescence immunochromatography detection test strip based on the PS microsphere quantum dot loading technology is successfully developed, and is used for rapid quantitative analysis of OTA in grain and oil crops and nuts. The ochratoxin A quantum dot fluorescence detection test strip constructed by the invention is high in sensitivity, good in specificity, simple and convenient to operate and rapid in analysis, is suitable for rapid and accurate quantification of ochratoxin A in grain and oil crops and nuts, and provides an effective technical means for on-site screening and monitoring of fungaltoxin in agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to an ochratoxin A test strip based on polystyrene microsphere quantum dot loading technology, a detection method, and its application in grain and oil crops and nuts. Background Technology

[0002] Ochratoxin A (OTA) is a key secondary metabolite produced by the filamentous fungus *Aspergillus ochraceus*. It is characterized by high toxicity, widespread contamination, and stable properties, making it difficult to metabolize and degrade. Due to the widespread presence of *Aspergillus ochraceus* in nature, OTA is widely distributed in various animal and plant-based foods, with the most severe contamination and significant risks found in grains, oil crops, nuts, and animal livers and blood. This toxin exhibits neurotoxicity and immunotoxicity, damages the liver and kidneys of humans and animals, and poses teratogenic, carcinogenic, and mutagenic risks. It has been assessed by the International Agency for Research on Cancer (IARC) as a Group 2(B) possible human carcinogen, posing a serious threat to food safety and public health.

[0003] To effectively control OTA (over-the-counter) contamination levels, major regulatory agencies and countries / regions worldwide have established strict limits. The World Health Organization (WHO), based on risk assessment, has established the Daily Tolerable Intake (DTI), stipulating that the maximum daily intake of OTA per kilogram of body weight should not exceed 16 ng. The Codex Alimentarius Commission (CAC) has set a uniform maximum limit of 5.0 μg / kg for unprocessed grains (such as barley, wheat, and rye). The European Union (EU) has implemented more refined classification controls, setting stringent OTA residue limits for up to 20 categories of food: 5.0 μg / kg for unprocessed grains and instant coffee; 3.0 μg / kg for finished products and roasted coffee intended for end consumers; 2.0 μg / kg for wine; and the strictest limits are for foods for special medical purposes for infants, baby food, and processed cereals for infants, not exceeding 0.5 μg / kg. μg / kg; Switzerland has also established corresponding standards in the field of feed safety, stipulating that the detection limit of OTA in pig and poultry feed shall not exceed 200 μg / kg and 1000 μg / kg, respectively; the mandatory national standard GB2761-2017 "National Food Safety Standard Limits of Mycotoxins in Food" clearly stipulates that the maximum allowable limit of OTA in grains, beans and their products is also 5.0 μg / kg.

[0004] To ensure food safety and meet increasingly stringent global OTA regulatory limits, researchers have developed a variety of OTA detection technologies, including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), aptamer technology, and immunochromatography. HPLC and LC-MS / MS methods are currently the most commonly used, suitable for high-precision quantitative analysis of complex matrix samples. However, their operation procedures are cumbersome, the detection cycle is long, and the instrument cost and maintenance costs are high, making it difficult to meet the requirements of rapid on-site detection. TLC can only achieve preliminary qualitative analysis and has low sensitivity and poor accuracy, and uses a large amount of harmful organic solvents. ELISA is simple to operate, but antibody cross-reactivity leads to insufficient specificity and a high risk of false positives. Aptamer technology screening is complex and time-consuming, and the success rate is greatly affected by the target properties, library design, and screening strategy. Immunochromatography, based on the antigen-antibody competitive inhibition binding mechanism, achieves qualitative and semi-quantitative analysis of OTA. It is simple to operate, economical and efficient, and suitable for rapid on-site screening of large batches of samples. However, the detection sensitivity is highly dependent on the performance of the labeling material. Traditional labeling materials have problems such as poor stability and uneven signal intensity, which restricts the quantitative accuracy. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the inventors, based on polystyrene (PS) microsphere quantum dot loading technology, fixed quantum dots on the surface of functionalized PS microspheres through covalent bonds, thus preparing a novel high-fluorescence quantum dot nanobead (QBs) probe; and developed a high-sensitivity OTA quantum detection system, thereby completing this invention.

[0006] This invention includes the following technical solutions: A test strip for ochratoxin A includes: a sample pad, a conjugate pad, an NC membrane, a T line, a C line, and an absorbent pad stacked sequentially on a base plate. The conjugate pad is coated with a QBs probe conjugated with an OTA monoclonal antibody. The T line is coated with an OTA-OVA-coated antigen diluent. The C line is coated with goat anti-mouse secondary antibody.

[0007] In some embodiments, the QBs probe conjugated with OTA monoclonal antibody is prepared by the following method: S1, take the carboxylated PS microsphere suspension, add MES, mix, centrifuge, discard the supernatant, wash and resuspend in MES, immediately add EDC and NHS to activate at room temperature to obtain activated PS microsphere suspension; take amino water-soluble CdSe / ZnS quantum dot solution, replace the buffer with MES, and finally resuspend in MES to obtain amino quantum dot solution, quickly add all of it to the activated PS microsphere suspension, stir at room temperature for 2 h; add Tris, quench at room temperature, centrifuge, discard the supernatant and wash to obtain QBs probe; S2, resuspend the QBs probe in MES, add EDC and NHS to activate the reaction at room temperature; centrifuge, discard the supernatant, resuspend in PBS, immediately add OTA monoclonal antibody, vortex at room temperature for 2 hours, centrifuge, discard the supernatant, wash twice with PBS; resuspend the product in MES, add mPEG-NH2, EDC and NHS, react at room temperature for 1 hour, add BSA to a final concentration of 1%, continue the reaction for 1 hour for blocking, centrifuge and wash 3 times with PBS, resuspend in PBS containing 1% BSA to obtain the QBs probe conjugated with OTA monoclonal antibody, and store at 4°C protected from light.

[0008] In some embodiments, the ochratoxin A test strip is prepared by the following method: The conjugate pad was immersed in the pretreatment solution and dried; the QBs probe solution conjugated with OTA monoclonal antibody was sprayed onto the conjugate pad and vacuum dried. The OTA-OVA-coated antigen and goat anti-mouse secondary antibody were sprayed onto the T-line C-line and dried. The sample pad, pretreated binding pad, NC membrane, and absorbent pad are sequentially attached to the base plate, pressed firmly, cut into 4mm wide strips, sealed, and stored at 4℃ for later use.

[0009] In a preferred embodiment, the concentration of the OTA-OVA-coated antigen is 0.1 mg / mL, and the dilution factor of the QBs probe solution conjugated with OTA monoclonal antibody is 1:15.

[0010] In a preferred embodiment, PBS is used as the diluent for the OTA-OVA-coated antigen.

[0011] In a preferred embodiment, the pretreatment solution used to treat the conjugate pad is selected from 0.01M PBS, 0.1% Tween-20 + 0.01M PBS, 0.1% PEG200 + 0.01M PBS, 0.1% Tween-20 + 0.1% PEG200 + 0.01M PBS, preferably 0.01M PBS.

[0012] A method for detecting ochratoxin A, comprising: Weigh the sample and place it in a sample tube containing the extraction solution. Vortex to mix. Take out a portion and centrifuge. Take the supernatant into a centrifuge tube and adjust the pH value to 6.2-7.0. Place the ochratoxin A test strip vertically into the centrifuge tube and react for 3 minutes. Take it out and read the fluorescence signal values ​​of the T line and C line with a fluorescence reader. Compare with the standard curve to obtain the sample concentration.

[0013] In some embodiments, the detection method exhibits good linearity in the range of 2-20 ng / mL; the limit of detection is 0.5 ng / mL.

[0014] The method for detecting ochratoxin A is applied to the detection of ochratoxin A in grain and oil crops and nuts.

[0015] The grain and oil crops and nuts are selected from rice, pistachios, corn, cashews, peanuts, soybeans, and walnuts.

[0016] Technical effect This invention constructs a quantum dot-microsphere composite signal marker by loading quantum dots onto the surface of PS microspheres, and successfully develops a novel fluorescent immunochromatographic test strip based on PS microsphere quantum dot loading technology for rapid quantitative analysis of OTA in grains, oil crops, and nuts. Unlike quantum dot immunochromatographic technology where the recognition element is a nucleic acid aptamer, the recognition element of this invention is a monoclonal antibody, a protein produced by animal immunization. A PS microsphere serves as the "core carrier," with a large number of quantum dots loaded on its surface to form quantum dot microspheres. Then, the monoclonal antibody is coupled to the surface of the microsphere to form a signal probe.

[0017] This design represents a significant improvement over traditional quantum dot labeling technology: a single PS microsphere can carry a large number of quantum dots, which is equivalent to integrated amplification of the fluorescence signal, thereby achieving high detection sensitivity on the immunochromatographic platform, with a detection limit of up to 0.5 ng / mL; at the end of QBs probe coupling, double blocking with mPEG-NH2 and BSA is used, which can effectively reduce nonspecific binding.

[0018] For nut samples (peanuts, walnuts, pistachios, etc.), their high fat and high protein content is the main challenge for extraction. This invention attempts to use a 60% ethanol-water system as the extraction solution to remove interfering substances such as fat and protein to the greatest extent. Through optimization of experimental parameters, the extraction solution is made compatible with the detection environment, achieving environmental protection, safety and practicality.

[0019] In summary, the OTA quantum detection test strip based on PS microsphere quantum dot loading technology established in this invention has advantages such as high sensitivity, strong specificity, rapid quantification, and simple operation. Standardized methodological evaluation verifies that this method is reliable and accurate, providing an effective technical means for on-site monitoring of OTA in grains, oil crops, and nuts. It has good application prospects and promotional value in food safety risk early warning and control. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the ochratoxin A quantum detection system in Embodiment 1 of the present invention; Figure 2 This is a graph showing the optimization results of OTA-OVA coating concentration and QBs probe dilution factor in Example 3 of the present invention; Figure 3 This is a graph showing the optimization results of the OTA-OVA diluent in Example 4 of the present invention; Figure 4 This is a diagram showing the optimization results of the binding pad treatment solution in Example 5 of the present invention; Figure 5 This is a linear fitting graph showing the results of different mass concentrations of OTA standard in Example 6 of the present invention; Figure 6 This is a linear fitting graph of OTA accurate quantitative analysis in Embodiment 6 of the present invention; Figure 7 This is an analysis diagram of the specific detection results in Example 7 of the present invention. Detailed Implementation

[0021] 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. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0022] The materials and reagents used in this invention are as follows: OTA (100ug / mL), Deoxynivalenol (DON) (1000 μg / mL), Zearalenone (ZEN) (100 μg / mL), Fumonisin B1 (FB1) (10mg / L) standards, provided by Tanmo Quality Inspection Technology Co., Ltd.; Aflatoxin B1 (AFB1) (100ug / mL), Aflatoxin B2 (AFB2) (100ug / mL), Aflatoxin G1 (AFG1) (100ug / mL), Trichothecene toxins (T-2) (100 μg / mL) standards, provided by Shanghai Anpu Cuishi Standard Technology Service Co., Ltd. Polyethylene glycol (PEG), methoxypolyethylene glycol amine (mPEG-NH2), PEG200, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS) (0.01M), phosphate buffer (PB), carbonate buffer solution (CBS), 2-morpholinoethanesulfonic acid (MES) (0.1M), tris(Hydroxymethyl)aminomethane hydrochloride (Tris(Hydroxymethyl)aminomethane) Hydrochloride (Tris-HCl) (1M), tris(hydroxymethyl)aminomethane (Tris), QBs probe dilution buffer (containing 1% sucrose, 0.2% BSA, 0.01M PBS), extraction buffer (60% ethanol-water system), Tween-20, methanol, acetonitrile, anhydrous ethanol, Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA), ovalbumin (OVA), and goat anti-mouse secondary antibody were produced by Shanghai Maclean Biotechnology Co., Ltd.; amino-water-soluble CdSe / ZnS quantum dot solution (emission wavelength 600±10nm) (5 mg / mL), OTA monoclonal antibody (prepared by animal immunization method), and OTA-OVA coating antigen were produced by Hebei Elisa Biotechnology Co., Ltd.; carboxylated polystyrene (PS) microsphere suspension (10 mg / mL) was produced by Beijing Zhongke Keyou Technology Co., Ltd., with a microsphere particle size of 150 nm.

[0023] PVC adhesive baseboard, sample pad, bonding pad (SB06 glass fiber pad), nitrocellulose membrane (NC membrane), absorbent pad, Hebei Elisa Biotechnology Co., Ltd.

[0024] Example 1 Preparation of QBs probes: QBs probes were prepared using the PS microsphere quantum dot loading method. The quantum dots were oriented and immobilized by covalent coupling between the carboxyl groups on the surface of the PS microspheres and the amino groups on the surface of the quantum dots.

[0025] Take 1.0 mL of carboxylated PS microsphere suspension (10 mg / mL), add 3.0 mL of 2-morpholine ethanesulfonic acid buffer (MES, 0.1 M, pH 6.0), mix well, centrifuge at 4000 rpm for 10 min, discard the supernatant, wash twice with MES (pH 6.0), and resuspend in 1.0 mL of MES (pH 6.0). Immediately add 5.0 μL of EDC (100 mg / mL) and 5.0 μL of NHS (50 mg / mL), and activate at room temperature for 30 min.

[0026] Take 1.0 mL of a 5 mg / mL amino-based water-soluble CdSe / ZnS quantum dot solution, replace the buffer with MES (pH 6.0), and finally resuspend in 1.0 mL of MES (pH 6.0). Quickly add the prepared amino quantum dot solution to the activated PS microsphere suspension and react at room temperature with magnetic stirring (300 rpm) for 2 h. After the reaction is complete, add 50.0 μL of Tris (pH 8.0), quench at room temperature for 10 min, centrifuge at 5000 rpm for 10 min, discard the supernatant, and wash three times with MES (pH 6.0) to obtain the QBs probe.

[0027] QBs probe conjugation: OTA monoclonal antibody was directly conjugated to the surface of the QBs probe using the EDC / NHS activation conjugation method. 0.5 mg of QBs probe was resuspended in 1.0 mL MES (pH 6.0), and 1.0 mg EDC and 0.5 mg NHS were added for activation at room temperature for 25 min. After centrifugation and discarding the supernatant, the probe was resuspended in 1.0 mL PBS (pH 7.4), and 5.0 μL of OTA monoclonal antibody (1 mg / mL) was immediately added. The conjugation was carried out with shaking at room temperature for 2 h. After centrifugation and discarding the supernatant, the probe was washed twice with PBS (pH 7.4) to remove unbound antibody. The product was resuspended in MES (pH 6.0), and mPEG-NH2 (final concentration 2 mg / mL), 10.0 μL EDC (100 mg / mL), and 10.0 μL NHS (50 mg / mL) were added. The mixture was reacted at room temperature for 1 h, followed by the addition of BSA to a final concentration of 1%, and the reaction was continued for another 1 h for blocking. The sample was then washed three times by centrifugation with PBS (pH 7.4). The product was resuspended in PBS (pH 7.4) containing 1% BSA to obtain the QBs probe conjugated with OTA monoclonal antibody, which was stored at 4°C protected from light.

[0028] The ochratoxin A test strip consists of several parts, including a PVC adhesive backing, sample pad, conjugate pad, NC membrane, absorbent pad, test line (T line), and control line (C line). Its structure is as follows: Figure 1 As shown.

[0029] The conjugate pad was immersed in pretreatment solution (0.01M PBS) for 30 min and dried at 37℃ for 2 h. A QBs probe solution (0.2 mg / mL) conjugated with OTA monoclonal antibody was uniformly sprayed onto the conjugate pad (1 μL / cm) using an X-only one-way sprayer, and vacuum dried at 37℃ for 1 h, then stored in the dark. A certain concentration of OTA-OVA-coated antigen and goat anti-mouse secondary antibody were sprayed onto the test line (T line) and control line (C line), respectively, at a spray volume of 1 μL / cm, with a line width of approximately 1 mm and a spacing of 5 mm between the T and C lines. The mixture was dried at 37℃ for 2 h and then sealed and stored in the dark. The sample pad, pretreated conjugate pad, NC membrane, and absorbent pad were sequentially glued onto a PVC base plate according to a specific assembly process, compacted, and then the laminated base plate was cut into 4 mm wide strips using a cutting machine. These strips were sealed and stored at 4℃ for later use, yielding the ochratoxin A test strip.

[0030] Example 2 Establishment of testing methods for OTA detection system (test strips) Weigh 5g of the ground sample (approximately 50% of which can pass through a 20-mesh sieve) into a sample tube containing 15.0 mL of extraction solution (60% ethanol-water system). Vortex and mix for 2 min. Transfer 1.0 mL of the mixture into a 1.5 mL centrifuge tube and centrifuge at 2000 r / min for 2 min. Transfer 100 μL of the supernatant into another clean 1.5 mL centrifuge tube and adjust the pH to 6.2-7.0. Place the test strip assembled in Example 1 vertically into the centrifuge tube and react for 3 min. Then, use an HF6500 fluorescence reader to read the fluorescence signal values ​​of the T and C lines.

[0031] Example 3 Optimization of OTA-OVA coating concentration and QBs probe dilution factor The test strips for ochratoxin A were prepared using a method similar to that in Example 1, with the following differences: OTA-OVA was diluted to different concentrations of 0.05 mg / mL, 0.07 mg / mL, 0.1 mg / mL, and 0.15 mg / mL and streaked onto the T line of the NC membrane. QBs probes were diluted at ratios of 1:5, 1:10, 1:15, 1:20, and 1:30 and sprayed onto the conjugate pad. Goat anti-mouse secondary antibody was fixed at 0.1 mg / mL and coated onto the C line. The sample pad, conjugate pad, NC membrane, and absorbent pad were assembled according to a specific process.

[0032] Following the method in Example 2, the assembled series of test systems were vertically placed into centrifuge tubes containing 100 μL PBS, reacted for 3 min, and the fluorescence intensity was read. The fluorescence signal intensity of the T line and C line was observed, and the results are shown in Table 1.

[0033] Table 1. Experimental results of OTA-OVA coating concentration and QBs probe dilution factor.

[0034] Note: "+++" indicates strong fluorescence signal; "++" indicates moderate fluorescence signal; "+" indicates fluorescence signal; "-" indicates no fluorescence signal.

[0035] The fluorescence reader reads the T / C value and analyzes it through visual charts, such as... Figure 2 As shown. Considering the fluorescence signals of the T and C lines and cost analysis, the optimal combination for OTA-OVA was determined to be 0.1 mg / mL, with the T line showing relatively strong fluorescence signals and the T line having a higher colorimetric value than the C line (T / C > 1). The QBs probe dilution factor was 1:15.

[0036] Example 4 Optimization of OTA-OVA diluent The test strips for ochratoxin A were prepared using a method similar to that in Example 1, with the following differences: PBS, CBS, PB, Tris-HCl buffer, PB containing 0.1% Tween-20, and PB containing 1% BSA were selected as OTA-OVA diluents. OTA-OVA was diluted to 0.05 mg / mL and then streaked onto an NC membrane with 0.1 mg / mL goat anti-mouse secondary antibody to assemble the test strip.

[0037] Following the method in Example 2, the assembled test strips were vertically placed one by one into centrifuge tubes containing 100 μL of OTA standard (0.5 ng / mL). After the reaction, the clarity of the T and C lines and the fluorescence signal were observed. The results are as follows: Figure 3 As shown.

[0038] Depend on Figure 3 It can be seen that, by comprehensively comparing the fluorescence signals and clarity of the T-line and C-line on various detection systems within the same time period, PBS was selected as the original diluent for coating.

[0039] Example 5 Optimization of combined pad treatment fluid The test strips for ochratoxin A were prepared using a method similar to that in Example 1, with the following differences: SB06 glass fiber pads were used as conjugate pads and were soaked overnight in different treatment solutions (0.01M PBS, 0.1% Tween-20 + 0.01M PBS, 0.1% PEG200 + 0.01M PBS, 0.1% Tween-20 + 0.1% PEG200 + 0.01M PBS). The soaked conjugate pads were then removed, dried at 60℃ for 1 h, and used to assemble ochratoxin A test strips.

[0040] Following the method in Example 2, the assembled test strips were vertically placed one by one into centrifuge tubes containing 100 uLOTA standard (0.5 ng / mL). After the reaction was completed, the color development, clarity, and chromatography speed of the T and C lines were observed.

[0041] The fluorescence signal intensity and reaction sensitivity of the treated conjugate pad under the above optimal conditions are shown in Table 2. The clarity of the T-line and C-line and the T / C value are shown in Table 2. Figure 4 As shown, item 1 is 0.01M PBS, item 2 is 0.1% Tween-20 + 0.01M PBS, item 3 is 0.1% PEG200 + 0.01M PBS, and item 4 is 0.1% Tween-20 + 0.1% PEG200 + 0.01M PBS.

[0042] Table 2. Fluorescence signal intensity and reaction sensitivity under different conjugate pad treatment conditions

[0043] Note: "+++" indicates strong fluorescence signal; "++" indicates moderate fluorescence signal; "+" indicates fluorescence signal; "-" indicates no fluorescence signal.

[0044] The results above show that after treating the binding pad with 0.01M PBS, the fluorescence signal of the detection results is more intuitive and the brightness is close (i.e., the T / C value is close to 1), the chromatography speed is the fastest, and the two lines are clearer.

[0045] Example 6 OTA Quantum Test Strip Performance Evaluation Establishing a standard curve Accurately weigh a certain amount of OTA standard, dissolve it in methanol-acetonitrile (1:1) to prepare a 1 mg / mL standard stock solution. Referring to GB 5009.96-2016 "National Food Safety Standard - Determination of Ochratoxin A in Food", independently prepare different OTA mass concentrations of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL. Take 100 μL of the above OTA standard dilution and PBS (blank control) and add them dropwise to centrifuge tubes. Place the OTA quantum detection test strip assembled in Example 1 vertically in the centrifuge tube at room temperature for 3 min. Perform three parallel independent tests. Measure the fluorescence signal values ​​of the T line and C line with a fluorescence reader. Plot the average T / C ratio as the ordinate and the OTA standard mass concentration as the abscissa. Fit the curve to obtain the linear regression equation and determine the linear range, limit of detection (LOD), and limit of quantification (LOQ) of the method. The nine sets of valid data are shown in Table 3.

[0046] Table 3 Test results of various mass concentrations of OTA standard products

[0047] Plotting the average T / C ratio on the ordinate and the OTA standard concentration on the abscissa, a linear fit was obtained. Figure 5 The linear regression equation is Y = 0.0012. X 2 –0.0692 X +1.077, correlation coefficient r 2 =0.9898; Discarding the deviation data, only retaining 6 sets of continuous calibration point data, the linear fitting standard curve is as follows. Figure 6 As shown, the linear regression equation is Y=0.0019 X 2 –0.0833X +1.1118, correlation coefficient r 2 =0.9996, exhibiting good linearity in the range of 2-20 ng / mL, meeting the accurate quantification requirements in GB / T 27417-2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods".

[0048] When the concentration of OTA standard is 0.5 ng / mL, it can be partially detected and the average T / C value is close to 1, so the LOD is evaluated as 0.5 ng / mL; when the initial concentration of the standard curve is 2 ng / mL, the T / C value is stable and <1, so the LOQ is evaluated as 2 ng / mL.

[0049] Example 7 Using the method of Example 2, high, medium, and low concentrations of OTA (20 ng / mL, 8 ng / mL, 2 ng / mL) standard solutions and a high concentration (20 ng / mL) of other standards (eight common mycotoxins in grain and oil crops and nuts: DON, ZEN, FB1, AFB1, AFB2, AFG1, T-2) were subjected to extraction reactions, and the results were read. Each mass concentration was tested in triplicate independently. The results are as follows: Figure 7 As shown.

[0050] Depend on Figure 7 The results showed that when detecting the other seven fungal toxins at high concentrations (20 ng / mL), the T / C values ​​were all between 1.2 and 1.5, which was close to the results of the blank control. However, the T / C values ​​of the OTA positive samples (20 ng / mL, 8 ng / mL, and 2 ng / mL) were all less than 1, proving that the OTA quantum detection system has excellent selectivity for OTA and no cross-reaction with other common fungal toxins.

[0051] Example 8 Add recycling test evaluation Recovery tests were designed according to LS / T 6142-2023. Different matrix samples (rice, pistachios, corn, cashews, peanuts, soybeans, and walnuts) were collected, pulverized, and divided into three groups, each group further divided into three portions. 5 g of each portion was weighed into a 50 mL centrifuge tube. High, medium, and low concentration standard solutions were prepared, and 100 μL of each solution was added to the corresponding 50 mL centrifuge tubes. After mixing, extraction and detection were performed according to the method in Example 2. Each group was independently tested by three evaluators.

[0052] The test results for the high, medium, and low content groups are shown in Table 4. The recovery rate and relative standard deviation (RSD) values ​​were calculated with reference to LS / T 6142-2023.

[0053] Table 4. Results and Analysis of the Recovery Test

[0054] The recoveries of all three groups were between 94% and 105%, and the RSD values ​​were all less than 15%. The accuracy and precision at the three different evaluation contents all met the requirements of LS / T 6142-2023, indicating that the OTA quantum detection system has a stable response value to each evaluation content and is suitable for rapid detection of mycotoxins in grain and oil crops and nuts.

Claims

1. A test strip for ochratoxin A, comprising: The sample pad, conjugate pad, NC membrane, T line, C line and absorbent pad are stacked in sequence on the base plate. The conjugate pad is sprayed with QBs probe coupled with OTA monoclonal antibody, the T line is sprayed with OTA-OVA coated antigen diluent, and the C line is sprayed with goat anti-mouse secondary antibody.

2. The test strip for ochratoxin A as described in claim 1, wherein, The QBs probe conjugated with OTA monoclonal antibody was prepared by the following method: S1, take the carboxylated PS microsphere suspension, add MES, mix, centrifuge, discard the supernatant, wash and resuspend in MES, immediately add EDC and NHS to activate at room temperature to obtain activated PS microsphere suspension; take amino water-soluble CdSe / ZnS quantum dot solution, replace the buffer with MES, and finally resuspend in MES to obtain amino quantum dot solution, quickly add all of it to the activated PS microsphere suspension, stir at room temperature for 2 h; add Tris, quench at room temperature, centrifuge, discard the supernatant and wash to obtain QBs probe; S2, resuspend the QBs probe in MES, add EDC and NHS to activate the reaction at room temperature; centrifuge, discard the supernatant, resuspend in PBS, immediately add OTA monoclonal antibody, vortex at room temperature for 2 hours, centrifuge, discard the supernatant, wash twice with PBS; resuspend the product in MES, add mPEG-NH2, EDC and NHS, react at room temperature for 1 hour, add BSA to a final concentration of 1%, continue the reaction for 1 hour for blocking, centrifuge and wash 3 times with PBS, resuspend in PBS containing 1% BSA to obtain the QBs probe conjugated with OTA monoclonal antibody, and store at 4°C protected from light.

3. The test strip for ochratoxin A as described in claim 1 or 2, wherein, The test strip for ochratoxin A is prepared by the following method: The conjugate pad was immersed in the pretreatment solution and dried; the QBs probe solution conjugated with OTA monoclonal antibody was sprayed onto the conjugate pad and vacuum dried. The OTA-OVA-coated antigen and goat anti-mouse secondary antibody were sprayed onto the T line and C line, respectively, and then dried. The sample pad, pretreated binding pad, NC membrane, and absorbent pad are sequentially attached to the base plate, pressed firmly, cut into 4 mm wide strips, sealed, and stored at 4°C for later use.

4. The test strip for ochratoxin A as described in claim 3, wherein, The concentration of the OTA-OVA-coated antigen was 0.1 mg / mL, and the dilution factor of the QBs probe solution conjugated with OTA monoclonal antibody was 1:

15.

5. The test strip for ochratoxin A as described in claim 3, wherein, PBS was used as the diluent for the OTA-OVA-coated antigen.

6. The test strip for ochratoxin A as described in claim 3, wherein, The pretreatment solutions used to treat the conjugate pads were selected from 0.01M PBS, 0.1% Tween-20 + 0.01M PBS, 0.1% PEG200 + 0.01M PBS, and 0.1% Tween-20 + 0.1% PEG200 + 0.01M PBS.

7. A method for detecting ochratoxin A, comprising: Weigh the sample and place it in a sample tube containing the extraction solution. Vortex to mix. Take out a portion and centrifuge. Take the supernatant into a centrifuge tube and adjust the pH to 6.2-7.

0. Place the ochratoxin A test strip of any one of claims 1-6 vertically into the centrifuge tube and react for 3 minutes. Take it out and read the fluorescence signal values ​​of the T line and C line with a fluorescence reader. Compare the values ​​with the standard curve to obtain the sample concentration.

8. The method for detecting ochratoxin A as described in claim 7, wherein, The detection method exhibits good linearity in the range of 2-20 ng / mL; the limit of detection is 0.5 ng / mL.

9. The application of the method for detecting ochratoxin A according to claim 7 or 8 in the detection of ochratoxin A in grain and oil crops and nuts.

10. The application as described in claim 9, wherein, The grain and oil crops and nuts are selected from rice, pistachios, corn, cashews, peanuts, soybeans, and walnuts.

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