Method for preparing hapten for detecting oxadone in tobacco and application of the obtained hapten

Abstract

The present application discloses a method for preparing a hapten for detecting oxadone in tobacco and the application of the obtained hapten. The molecular formula of the hapten is: When the obtained antibody is made into a test paper for detecting oxadone in fresh tobacco leaves and first-cured tobacco leaves, the detection limit is 0.1 mg / kg, the detection result has a high accuracy rate, and the problem of false positive and false negative can be effectively solved. In particular, in the specificity experiment, it shows significant specificity for oxadone, and the detection result is not interfered by carbendazim, metalaxyl, imidacloprid and carbofuran added in the sample.

Description

Technical Field

[0001] The present application relates to the technical field of detection of oxadone residues in tobacco leaves (harvested tobacco leaves to be cured and first-cured tobacco leaves), and in particular to a method for preparing a hapten for detecting oxadone in tobacco and the application of the obtained hapten. Background Art

[0002] Oxychlorothiazide (also known as oxychlorothiazide) is a type of systemic agricultural fungicide with a broad spectrum of fungicides. It has a high preventive effect on pathogenic bacteria of the Peronosporales, has protective and therapeutic effects, and has a long lasting effect. It is mainly used to prevent and control diseases of vegetables, fruits, etc. Because pesticides are commonly present during use and will cause pollution to the environment.

[0003] GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" sets the maximum residue limit in vegetables at 0.5 mg / kg; YQ50-2014 "Maximum Residue Limits of Pesticides in Tobacco Leaves" issued by China National Tobacco Corporation sets the maximum residue limit of carbendazim in tobacco leaves at 0.1 mg / kg.

[0004] Existing methods for detecting oxadone include gas chromatography, gas chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry, liquid chromatography-mass spectrometry / mass spectrometry, etc. Compared with the above methods, immunochemical detection has the advantages of rapid, specific, sensitive, accurate, batch monitoring, simple sample processing, and automated operation for drug detection in samples.

[0005] For example, CN202110285110.7 discloses a hybridoma cell line secreting a monoclonal antibody against oxadone and its application. The monoclonal antibody secreted by the hybridoma cell line AAB2 has good specificity and detection sensitivity for oxadone (oxadone IC 50 =3.68μg / L, IC of metalaxyl, dimethomorph, and procymidone 50 However, the antibody was only used in the cucumber sample addition recovery test in the examples. Since the main chemical components in cucumber are different from those in tobacco, the antibody is not necessarily suitable for the detection of oxadone contained in tobacco plants. At the same time, the use of hybridoma cells to obtain antibodies has the risk of decreasing and losing the amount of cell analysis antibodies, making it difficult to achieve stable antibody production. Summary of the invention

[0006] In response to the above technical problems, the present application provides a method for preparing a hapten for detecting oxadone in tobacco and the application of the obtained hapten. The hapten is prepared by a nucleophilic substitution reaction between compound a and mercaptopropionic acid, has a stable structure, a short preparation reaction cycle, and high antibody stability; and the antibody prepared with the hapten can react specifically with oxadone, has high sensitivity to oxadone, is simple to operate, has low cost, and short detection time.

[0007] The present application provides a method for preparing a hapten for detecting oxadixyl in tobacco, comprising the following steps: a compound a is subjected to a nucleophilic substitution reaction with mercaptopropionic acid to prepare the hapten for detecting oxadixyl in tobacco;

[0008] Compound a molecular formula: C 13 H 15 ClN2O4, compound a has the following structural formula:

[0009] Formula (II);

[0010] The molecular formula of the prepared hapten for detecting oxadone in tobacco is: C 16 H 20 N2O6S, the structural formula is as follows:

[0011] Formula (I).

[0012] After the hapten of the molecular formula is used to prepare monoclonal antibodies according to the existing method, the colloidal gold test strip can be assembled. The obtained test strip has significant specificity for oxadrenol, and the detection limit in tobacco leaves is 0.1 mg / kg, which can realize the rapid detection of oxadrenol in tobacco, and is particularly suitable for the rapid detection of large batches of samples. The nuclear magnetic resonance results of the hapten are shown in Figure 1 .

[0013] The hapten is obtained by the following reaction:

[0014]

[0015] Preferably, the method comprises the following steps:

[0016] 1) After compound a is dissolved, add mercaptopropionic acid, potassium tert-butoxide, lead acetate, and ligand (R)-(-)-1-[(S)-2-(dicyclohexylphosphino)ferrocene]ethyl di-tert-butylphosphine, and stir to obtain a reaction solution;

[0017] 2) The reaction solution obtained in step 1) undergoes a nucleophilic substitution reaction to obtain a solution system;

[0018] 3) Post-treating the solution system to obtain the hapten.

[0019] Preferably, the nucleophilic substitution reaction conditions are to heat to 90-120° C. for 3-5 hours to produce a solution system. The nucleophilic substitution reaction can occur under these conditions.

[0020] Preferably, the post-treatment comprises the following steps of sequentially performing the following steps on the solution system: removing the solvent by rotary evaporation, adjusting the pH value, extracting, evaporating the organic phase to dryness, and eluting through a silica gel column.

[0021] Preferably, the pH value is adjusted to 6; ethyl acetate is added during extraction; and the elution solvent is a mixture of dichloromethane and methanol in a volume ratio of 10:1.

[0022] Specifically, the preparation of the hapten comprises the following steps:

[0023] Take 2.98g of compound a, add 100ml of toluene to dissolve, add 2.24g of potassium tert-butoxide, add 1.27g of mercaptopropionic acid, stir thoroughly, add 0.3g of lead acetate, add 0.5g of ligand (R)-(-)-1-[(S)-2-(dicyclohexylphosphino)ferrocene]ethyl di-tert-butylphosphine, stir, heat to 110℃ to react for 4h, stop the reaction, remove toluene by rotary evaporation, add 200ml of water, add 1mol / L hydrochloric acid to adjust the pH value to 6, add 200ml of ethyl acetate to extract, evaporate the organic phase to dryness, put it on a silica gel column, and elute and separate with dichloromethane / methanol (v / v, 10 / 1) to obtain 1.21g of compound b, which is the hapten product, with a yield of 32.88%.

[0024] Another aspect of the present application provides an antigen for detecting oxadone in tobacco, which is a conjugate obtained by coupling the hapten prepared by the above method with a carrier protein.

[0025] The carrier protein used for the antigen can be bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.

[0026] Preferably, the carrier protein is hemocyanin.

[0027] Specifically, the antigen preparation method includes: taking 12.26 mg of the above-mentioned hapten, adding 1 ml of DMF to dissolve and clarify, adding 6.77 mg of NHS and 9.57 mg of EDC, fully dissolving and mixing, reacting at room temperature for 2 hours, and obtaining a hapten activation liquid A; taking 50 mg of keyhole limpet hemoalbumin (KLH), adding 4 ml of 0.1M CB9.5 buffer to dissolve, obtaining a liquid B, adding the A liquid dropwise into the B liquid, reacting at room temperature for 6 hours, stopping the reaction, dialyzing and purifying with 0.02M PBS for 3 days, changing the liquid 3 times a day, centrifuging and packaging, and obtaining a oxadiazine-KLH conjugate, which is the antigen.

[0028] Another aspect of the present application provides an antibody for detecting oxadone in tobacco, which is obtained by immunizing the above antigen with animals, cell fusion and cloning.

[0029] The antibodies prepared from the above antigens may be prepared by animal immunization, cell fusion, and cloning methods commonly used in the art. After obtaining a hybridoma cell line that stably secretes monoclonal antibodies, the antibody is secreted by the cell line.

[0030] Specifically, the method for preparing the antibody comprises the following steps:

[0031] (1) Animal immunization

[0032] The immunogen obtained in step 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce antiserum.

[0033] (2) Cell fusion and cloning

[0034] Spleen cells of immune Balb / c mice were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). The cell supernatant was determined by indirect competitive ELISA to screen positive wells. The positive wells were cloned by limiting dilution until a hybridoma cell line that stably secreted monoclonal antibodies was obtained.

[0035] (3) Cell cryopreservation and recovery

[0036] Hybridoma cells were prepared into a cell suspension of 1×106 cells / ml using cryopreservation solution and stored in liquid nitrogen for a long time. When thawing, the cryopreservation tube was taken out and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to a culture bottle for culture.

[0037] (4) Preparation and purification of monoclonal antibodies

[0038] Incremental culture method: hybridoma cells are placed in a cell culture medium, cultured at 37°C, and the obtained culture fluid is purified by the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which are stored at -20°C. The cell culture medium is prepared by adding calf serum and sodium bicarbonate to the RPMI1640 culture medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium is 7.4.

[0039] Another aspect of the present application provides a colloidal gold test strip for detecting oxadone in tobacco, and the antibody used is the above-mentioned antibody.

[0040] The test paper is prepared by the existing colloidal gold test paper preparation method. The test paper strip preparation method is the same as the existing commonly used preparation method. The prepared test paper strip can realize the rapid detection of oxadone contained in tobacco, the detection result has a high accuracy rate, and is suitable for the detection of large quantities of samples.

[0041] Preferably, the colloidal gold test paper is prepared by the following operation:

[0042] 1) Preparation of antibody-colloidal gold marker for detection of oxadone in tobacco;

[0043] 2) preparing the conjugate release pad, reaction membrane and sample absorption pad;

[0044] 3) Assemble the test strips.

[0045] Specifically, the preparation of the antibody-colloidal gold marker for detecting oxadone in tobacco in step 1) comprises the following steps:

[0046] Under magnetic stirring, adjust the pH value of colloidal gold to 7.0 with 0.2mol / L potassium carbonate solution, add the antibody to the colloidal gold solution according to the standard of adding 20~50μg per ml of colloidal gold solution, continue stirring and mixing for 30 minutes, add 10% BSA to make the final concentration in the colloidal gold solution 1% (volume fraction), and let it stand for 10 minutes. Centrifuge at 12000r / min and 4℃ for 40min, discard the supernatant, wash the precipitate twice with resolubilization buffer, resuspend the precipitate with resolubilization buffer with a volume of 1 / 10 of the initial colloidal gold volume, and place it at 4℃ for use.

[0047] Preferably, the colloidal gold test strip has a false positive rate of 0, a false negative rate of 0, a detection limit of 0.1 mg / kg, good specificity for oxadone, and a cross-reaction rate of up to 100%.

[0048] The beneficial effects of this application include:

[0049] 1) The present application provides a method for preparing a hapten for detecting oxadone in tobacco and the application of the obtained hapten. The hapten uses compound a as a starting material, undergoes a nucleophilic substitution reaction with mercaptopropionic acid, and obtains compound b. Compound b is used as a hapten to couple with a carrier protein, and an animal is immunized to prepare a monoclonal antibody prepared by immunizing an animal with a full antigen. When the obtained antibody is made into a test paper for the detection of oxadone in fresh tobacco leaves and first-cured tobacco leaves, the detection limit is 0.1 mg / kg, the detection result has a high accuracy rate, and can effectively solve the problem of false positives and false negatives. In particular, in the specificity experiment, it shows significant specificity for oxadone, and the detection result is not interfered by carbendazim, metalaxyl, imidacloprid, and carbofuran added to the sample.

[0050] 2) The present application provides a method for preparing a hapten for detecting oxadone in tobacco and the application of the obtained hapten. The antibody can react specifically with oxadone. When it is applied to a rapid test strip, the detection time is short, the operation is simple, and the cost is low. It is suitable for testing units to test a large number of samples at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 A schematic diagram of the hapten nuclear magnetic resonance results for the detection of oxadone in tobacco provided in this application; DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0053] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The technical means that are not described in detail in this application and are not used to solve the technical problems of this application are all set according to the common knowledge in the field, and can be implemented in a variety of common knowledge settings.

[0055] Example

[0056] Unless otherwise specified, the reagents and instruments used in the following examples were obtained from commercial channels.

[0057] Example 1 Preparation of Oxaloxaline Hapten

[0058] Take 2.98g of compound a, add 100ml of toluene to dissolve, add 2.24g of potassium tert-butoxide, add 1.27g of mercaptopropionic acid, stir thoroughly, add 0.3g of lead acetate, add 0.5g of ligand (R)-(-)-1-[(S)-2-(dicyclohexylphosphino)ferrocene]ethyl di-tert-butylphosphine, stir, heat to 110℃ for 4h, stop the reaction, remove toluene by rotary evaporation, add 200ml of water, add 1mol / L hydrochloric acid to adjust the pH value to 6, add 200ml of ethyl acetate to extract, evaporate the organic phase to dryness, put on a silica gel column, and elute and separate with dichloromethane / methanol (v / v, 10 / 1) to obtain 1.21g of compound b, which is the hapten product, with a yield of 32.88%. See the NMR result of the obtained hapten for details. Figure 1 .

[0059] Example 2 Preparation of immunogen (oxadone-KLH conjugate)

[0060] Take 12.26 mg of compound b, add 1 ml of DMF to dissolve and clarify, add 6.77 mg of NHS and 9.57 mg of EDC, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation liquid A; take 50 mg of keyhole limpet hemoalbumin (KLH), add 4 ml of 0.1 M CB9.5 buffer to dissolve, obtain liquid B, add liquid A drop by drop into liquid B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 M PBS for 3 days, change the liquid 3 times a day, centrifuge and package, and obtain the oxadiazine-KLH conjugate, which is the immunogen.

[0061] Example 3 Preparation of the coating source (oxadixyl-OVA conjugate)

[0062] Take 16.35 mg of compound b, add 1 ml of DMF to dissolve and clarify, add 11.2 mg of NHS and 13.57 mg of EDC, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation liquid A; take 100 mg of ovalbumin (OVA), add 8 ml of 0.1 M CB9.5 buffer to dissolve, obtain liquid B, add liquid A drop by drop into liquid B, react at room temperature for 6 hours, stop the reaction, dialyze and purify with 0.02 M PBS for 3 days, change the liquid 3 times a day, centrifuge and package, and obtain the oxadiazine-OVA conjugate, which is the coating source.

[0063] Example 4 Preparation of monoclonal antibody against oxaloxaline

[0064] (1) Animal immunization

[0065] The immunogen obtained in step 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce antiserum.

[0066] (2) Cell fusion and cloning

[0067] Spleen cells of immune Balb / c mice were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio). The cell supernatant was determined by indirect competitive ELISA to screen positive wells. The positive wells were cloned by limiting dilution until a hybridoma cell line that stably secreted monoclonal antibodies was obtained.

[0068] (3) Cell cryopreservation and recovery

[0069] Hybridoma cells were cryopreserved in 1×10 6 Cell suspension of 100 cells / ml is stored in liquid nitrogen for a long time. When thawing, take out the cryopreservation tube and immediately put it into a 37℃ water bath to thaw quickly. After centrifugation to remove the cryopreservation solution, transfer it into a culture bottle for culture.

[0070] (4) Preparation and purification of monoclonal antibodies

[0071] Incremental culture method: hybridoma cells are placed in cell culture medium and cultured at 37°C. The obtained culture fluid is purified using the caprylic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which are then stored at -20°C.

[0072] The cell culture medium is prepared by adding calf serum and sodium bicarbonate to RPMI1640 culture medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium is 7.4.

[0073] Example 5 Preparation of Oxaloxaline Monoclonal Antibody-Colloidal Gold Label

[0074] (1) Preparation of colloidal gold

[0075] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, take 100 ml and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, add 2.5 ml 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir and heat at a uniform speed until the solution turns translucent red, stop, cool to room temperature, restore to the original volume with deionized water, and store at 4°C. The prepared colloidal gold has a pure, translucent appearance, without precipitation and floating matter.

[0076] (2) Preparation of Oxysulfuron Monoclonal Antibody-Colloidal Gold Label

[0077] Under magnetic stirring, adjust the pH value of colloidal gold to 7.0 with 0.2 mol / L potassium carbonate solution, add oxadiazine monoclonal antibody to the colloidal gold solution at a standard of 20-50 μg per ml of colloidal gold solution, continue stirring and mixing for 30 minutes, add 10% BSA to make the final concentration in the colloidal gold solution 1% (volume fraction), and let it stand for 10 minutes. Centrifuge at 12000r / min and 4℃ for 40 minutes, discard the supernatant, wash the precipitate twice with resolubilization buffer, resuspend the precipitate with resolubilization buffer with a volume of 1 / 10 of the initial colloidal gold volume, and place at 4℃ for use.

[0078] Reconstitution buffer: containing 0.02%~0.1% (mass fraction) casein, 0.05%~0.2% (mass fraction) Tween-80, 0.02 mol / L phosphate buffer at pH 7.2.

[0079] Example 6 Preparation of Oxysulfuron Test Strips

[0080] The preparation method of the test strip mainly comprises the following steps:

[0081] 1) preparing a release pad sprayed with the conjugate of the oxadixyl monoclonal antibody-colloidal gold marker obtained in Example 5;

[0082] 2) preparing a reaction membrane having a detection line coated with a hapten-carrier protein conjugate of oxadone and a quality control line coated with a goat anti-mouse anti-antibody;

[0083] 3) Assemble the conjugate release pad, reaction membrane, sample absorption pad, water absorption pad and PVC bottom plate prepared in 1) and 2) into a test strip.

[0084] Preparation of goat anti-mouse antibodies used

[0085] Sheep are used as immune animals, and mouse antibodies are used as immunogens to immunize pathogen-free sheep to obtain sheep anti-mouse antibodies.

[0086] 1.1. Preparation of the conjugate release pad used in the above step 1):

[0087] The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer was 0.5%), pH 7.2, and 0.5 mol / L, and was evenly soaked for 1 hour, and then dried at 37°C for 3 hours for use. The prepared oxadixyl monoclonal antibody-colloidal gold marker was evenly sprayed on the conjugate release pad using an Isoflow film sprayer. After spraying 0.01 ml of oxadixyl monoclonal antibody-colloidal gold marker per 1 cm of the conjugate release pad, it was placed in a 37°C environment (humidity <20%) for 60 minutes, then taken out and placed in a dry environment (humidity <20%) for storage.

[0088] 2.1. Preparation of the reaction membrane in the above step 2):

[0089] The oxadiazine hapten-ovalbumin conjugate was coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody was coated on the reaction membrane to form a quality control line.

[0090] Coating process: dilute the oxadiazine hapten-ovalbumin conjugate to 10 mg / ml with phosphate buffer, and coat it on the test line (T line) on the nitrocellulose membrane with an Isoflow film dispenser, with a coating amount of 0.8 μl / cm; dilute the goat anti-mouse anti-antibody to 200 μg / ml with 0.01 mol / L, pH 7.4 phosphate buffer, and coat it on the quality control line (C line) on the nitrocellulose membrane with an Isoflow film dispenser, with a coating amount of 1.0 μl / cm. Dry the coated reaction membrane at 37°C for 2 hours and set aside.

[0091] 3.1. Preparation of the sample absorption pad used in step 3) above:

[0092] The sample absorption pad was immersed in a phosphate buffer solution containing 0.1 mol / L, pH 7.2, and 0.5% bovine serum albumin (volume fraction) for 2 h, and then dried at 37°C for 2 h for later use.

[0093] 3.2. Assembly of the test strips in step 3) above:

[0094] The sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially pasted on the PVC bottom plate; 1 / 3 of the conjugate release pad is covered by the sample absorption pad from the starting end, the end of the conjugate release pad is connected to the starting end of the reaction membrane, the end of the reaction membrane is connected to the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the end of the water absorption pad is aligned with the end of the PVC bottom plate; there are detection lines and quality control lines on the reaction membrane, and the detection line (T line) and the quality control line (C line) are strips perpendicular to the length of the test strip; the detection line is located on the side close to the end of the conjugate release pad; the quality control line is located on the side away from the end of the conjugate release pad; the test strip is cut into small strips with a width of 3 mm by a machine, and is placed in a special plastic card, which can be stored for 12 months under the condition of 4-30°C.

[0095] Example 7 Detection limit test of oxadone test strips

[0096] The method of using the oxadone test strip in the following Examples 7-8 is as follows: use a pipette to draw up the sample solution to be tested and vertically drip 3 drops into the sample addition hole. When the liquid flows, start timing. After reacting for 5-10 minutes, determine the result.

[0097] Analysis of the test results of the oxadone test strips in the following examples:

[0098] Read the results through the colloidal gold analyzer (referred to as the "analyzer"):

[0099] Negative (-): indicates that the concentration of the substance to be tested in the sample is lower than the detection limit;

[0100] Positive (+): indicates that the concentration of the substance to be tested in the sample is equal to or higher than the detection limit;

[0101] Invalid: Indicates that retesting is required.

[0102] The detection limit test consists of the following steps:

[0103] Blank tobacco leaves (harvested tobacco leaves to be cured, first-cured tobacco leaves) were used as samples for sample preparation. The sample preparation method was as follows: 0.15 g of tobacco leaves were placed in a centrifuge tube, and the leaves were rotated and squeezed several times with a grinding rod to ensure that the leaf tissue was fully ground; 2 mL of 0.02 mol / L PB buffer was added, and the grinding was repeated to mix the sample and the buffer thoroughly; after removing the grinding rod, the sample was allowed to stand for more than 1 min, and the supernatant was the sample solution to be tested.

[0104] The obtained samples of tobacco leaves to be cured and the samples of newly cured tobacco leaves were divided into three parts, and oxadone was added to each sample to a final concentration of 0.05, 0.1, and 0.2 mg / kg, respectively. Test strips were taken to test the samples, and each sample was tested three times.

[0105] Test results: When the concentration of oxadone added in the sample was 0.05 mg / kg, the analyzer displayed a negative result; when the concentration of oxadone added in the sample was 0.1 and 0.2 mg / kg, the analyzer displayed a positive result, indicating that the detection limit of the test paper for oxadone in various tobacco leaf samples is 0.1 mg / kg.

[0106] Example 8 False positive rate and false negative rate test of oxadone test strips

[0107] Take 20 samples of post-harvest flue-cured tobacco leaves and freshly-cured tobacco leaves with a 0.1 mg / kg oxadone content as positive samples; take 20 samples of post-harvest flue-cured tobacco leaves and freshly-cured tobacco leaves with a oxadone content less than 0.05 mg / kg as negative samples; use three batches of test strips to test each sample, and count the number of negative and positive results for each sample. The results are shown in Table 1.

[0108] Table 1 Test results of tobacco leaves (harvested tobacco leaves to be cured, first-cured tobacco leaves)

[0109]

[0110] The results in Table 1 show that when the test strips produced in three batches were used to test positive tobacco leaves (harvested tobacco leaves to be cured and first-cured tobacco leaves), all the results were positive, indicating that the positive sample compliance rate was 100% and the false negative rate was 0;

[0111] When testing 20 negative tobacco leaf samples (harvested tobacco leaves to be cured and newly cured tobacco leaves), all the results were negative, indicating that the negative compliance rate was 100% and the false positive rate was 0.

[0112] It is shown that the test strip for detecting oxadone of the present invention can quickly detect oxadone residues in tobacco leaves (harvested tobacco leaves to be cured and newly cured tobacco leaves), and it is shown that the detection limit of oxadone in various tobacco leaf samples by the test strip is 0.1 mg / kg.

[0113] Example 9 Specificity test of the oxadone test strip

[0114] The indirect competitive ELISA method was used to determine the cross-reaction rate of the oxadone test strip with carbendazim, metalaxyl, oxadone, imidacloprid and chlorpyrifos. The determination method was carried out according to the existing commonly used operations, with specific reference to the requirements for the filing technical information of veterinary drug residue detection kits (strips and cards) and the regulations in the review work procedures. The calculation formula is as follows: The results are shown in Table 2.

[0115]

[0116] Table 2 Specificity test

[0117]

[0118] As can be seen from Table 2, the test paper prepared with the hapten provided in the present application has good specificity for oxadixyl, and the detection result is not interfered by bacillus, metalaxyl, imidacloprid and carbofuran contained in the sample.

[0119] Example 10

[0120] The difference from Example 1 is that the nucleophilic substitution reaction condition is heating to 90° C. for 3 hours.

[0121] Embodiment 11

[0122] The difference from Example 1 is that the nucleophilic substitution reaction condition is heating to 120° C. for 5 hours.

[0123] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hapten for detecting oxadone in tobacco, characterized in that: The structural formula is as follows: Formula (I).

2. The method for preparing a hapten for detecting oxadone in tobacco according to claim 1, characterized in that: The following steps are involved: 1) After compound a is dissolved, add mercaptopropionic acid, potassium tert-butoxide, lead acetate, and ligand (R)-(-)-1-[(S)-2-(dicyclohexylphosphino)ferrocene]ethyl di-tert-butylphosphine, and stir to obtain a reaction solution; Compound a molecular formula: C 13 H 15 ClN2O4, compound a has the following structural formula: Formula (II); 2) The reaction solution undergoes a nucleophilic substitution reaction to obtain a reaction solution; 3) Post-treating the reaction solution to obtain a hapten; The nucleophilic substitution reaction conditions are as follows: heating to 90-120° C. for 3-5 hours to cause the nucleophilic substitution reaction and obtain a reaction solution.

3. The method according to claim 2, characterized in that The post-treatment comprises: performing the following steps on the reaction solution in sequence: removing the solvent by rotary evaporation, adjusting the pH value, extracting, evaporating the organic phase to dryness, and eluting through a silica gel column.

4. The method according to claim 3, characterized in that The pH value was adjusted to 6; ethyl acetate was added during extraction; the elution solvent was a mixture of dichloromethane and methanol in a volume ratio of 10:

1.

5. An antigen for detecting oxadone in tobacco, characterized in that: The conjugate is obtained by coupling the hapten for detecting oxadone in tobacco prepared by the method as described in any one of claims 2 to 4 with a carrier protein.

6. The antigen according to claim 5, characterized in that The carrier protein is hemocyanin.

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