Triple immunochromatographic detection kit and detection method for procymidone, carbofuran and carbendazim

Abstract

The present invention provides a procymidone, carbofuran and carbendazim triple immunochromatographic detection kit and detection method. The kit comprises a sample extraction and purification tube, a reaction substrate with reaction holes and a procymidone, carbofuran and carbendazim triple immunochromatographic detection test strip. The three-in-one colloidal gold immunochromatographic detection kit provided by the present invention can realize on-site, multi-throughput, rapid and accurate simultaneous extraction, purification and detection of procymidone, carbofuran and carbendazim residual in vegetables, greatly simplify the pre-treatment process, improve the detection efficiency, and provide strong technical support for food production safety, grassroots supervision, food inspection, large-scale event security, etc.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide residue detection, in particular to a procymidone, carbofuran and carbendazim triple immunochromatographic detection kit and a detection method. Background Art

[0002] Procymidone, chemical name N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-diformyl imide, is a low-toxic fungicide that can inhibit the synthesis of triglycerides in bacteria. Carbofuran, chemical name 2,3-dihydro-2,2-dimethyl-7-benzofuranylmethylcarbamate, is a carbamate insecticide and nematicide. Carbendazin, chemical name 2-(methoxycarbamoyl)benzimidazole, is a high-efficiency, low-toxic fungicide that can be used to prevent and treat a variety of plant diseases. Procymidone, carbofuran and carbendazol are commonly used pesticides in plant cultivation. At present, the detection rate of procymidone, carbofuran and carbendazol pesticide residues in vegetables is high, the unqualified rate is also high, and the existing detection methods are relatively cumbersome.

[0003] According to national standards, the detection method for procymidone is gas chromatography-mass spectrometry, the detection method for carbofuran is liquid chromatography-post-column derivatization, and the currently effective detection method for carbendazim is liquid chromatography-tandem mass spectrometry. Although the above methods have good stability, high accuracy, and strong repeatability, they require expensive instruments and equipment, professional operators, and complex pre-treatment processes. They are not environmentally friendly and cannot meet the requirements of timely detection, on-site office, and on-site law enforcement. In addition, the pre-treatment methods and instruments used are different, so the goal of detecting several pesticide residues at the same time is even more difficult to achieve.

[0004] Among the rapid detection methods, surface enhanced Raman spectroscopy and immunochromatographic strip rapid detection methods are widely used. However, many detection objects do not have Raman signals, while immunochromatographic strip detection objects are single, and the pre-treatment methods for different detection objects are also different, which is not conducive to the simultaneous detection of several pesticide residues. Summary of the invention

[0005] The invention aims to provide a procymidone, carbofuran and carbendazim triple immunochromatographic detection kit and a detection method.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a triple immunochromatographic test strip of procymidone, carbofuran and carbendazim, comprising a sample pad, a nitrocellulose membrane, an absorbent pad and a bottom plate, wherein the nitrocellulose membrane is provided with three detection lines and a quality control line in sequence from the sample pad to the absorbent pad, the three detection lines are respectively coated with a procymidone hapten-carrier protein conjugate, a carbofuran hapten-carrier protein conjugate and a carbendazim hapten-carrier protein conjugate, and the quality control line is coated with a sheep anti-mouse polyclonal antibody.

[0007] The sample pad, nitrocellulose membrane and water absorbent pad are alternately stacked and adhered to the bottom plate at a spacing of 2-3 mm.

[0008] The concentration of the procymidone hapten-carrier protein conjugate coated on the test line is 0.10-0.20 mg / mL, preferably 0.15 mg / mL.

[0009] The concentration of the carbofuran hapten-carrier protein conjugate coated on the test line is 0.25-0.8 mg / mL, preferably 0.6 mg / mL.

[0010] The concentration of the carbendazim hapten-carrier protein conjugate coated on the test line is 0.25-0.6 mg / mL, preferably 0.5 mg / mL.

[0011] The concentration of the goat anti-mouse polyclonal antibody coated on the quality control line was 0.25 mg / mL.

[0012] The structures of the procymidone hapten, carbofuran hapten and carbendazim hapten are shown in Formula 1) to Formula 3) respectively:

[0013] Formula 1) Procymidone hapten

[0014] Formula 2) Carbofuran hapten

[0015] Formula 3) Carbendazim hapten

[0016] Preferably, the carrier protein is bovine serum albumin.

[0017] The distance between each test line on the nitrocellulose membrane and between the test line near the absorbent pad and the quality control line is 4-5 mm, preferably 4.3 mm. In the present invention, if the distance between two adjacent test lines is too close, the color development will be disturbed; if the distance is too far, the chromatography time is too long and more test solution is required.

[0018] The width of the test strip is 3-5 mm, preferably 3.8 mm. If the test strip is too narrow, it is not conducive to the judgment of the result, and if it is too wide, more antigens, antibodies and detection liquids are required, which is not conducive to cost saving.

[0019] The material of the sample pad can be nitrocellulose membrane. The material of the bottom plate can be PVC.

[0020] Figure 1 It is a schematic diagram of the structure of the immunochromatographic test strip of the present invention.

[0021] In a second aspect, the present invention provides a triple immunochromatographic detection kit for procymidone, carbofuran and carbendazim, comprising a sample extraction purification tube (including a sample extraction purification solution), a reaction substrate with reaction holes and the triple immunochromatographic detection test strip. The sample extraction purification tube, the reaction substrate and the immunochromatographic detection test strip are sealed and loaded into the kit and stored at 4°C.

[0022] The preparation method of the sample extraction and purification tube comprises: adding 200 mg PSA (N-propylethylenediamine solid phase adsorbent), 200 mg C 18 (octadecyl bonded silica gel) and 30 mg GCB (graphitized carbon black) were placed in a 5 mL centrifuge tube, and then 2 mL of 0.05 mol / L pH 7.4 PBS was added and mixed to obtain a sample extraction and purification tube containing a sample extraction and purification solution.

[0023] The reaction wells are coated with colloidal gold labeled procymidone antibodies, colloidal gold labeled carbofuran antibodies and colloidal gold labeled carbendazim antibodies. The bottom area of ​​the reaction well is 0.32 cm 2 The volume is 365μL, and a 96-well plate can be directly used as the reaction well to facilitate the preparation of the kit in large quantities.

[0024] The preparation method of the reaction well comprises: adding 2 μL of colloidal gold-labeled procymidone antibody solution, 3 μL of colloidal gold-labeled carbofuran antibody solution and 2 μL of colloidal gold-labeled carbendazim antibody solution into the reaction well, diluting to 60 μL with freeze-drying protection solution, and performing vacuum freeze drying.

[0025] Preferably, the lyophilization protection solution is: 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% Tween 20 and 0.03% ProClin 300, prepared in water.

[0026] Preferably, the preparation method of the colloidal gold-labeled procymidone antibody solution includes: taking 1 mL of colloidal gold solution, adding 5-10 μL (preferably 7 μL) of 0.2 mol / L K2CO3 solution, mixing evenly, adding 6 μg of procymidone antibody, mixing evenly, and standing at 25°C for 10 min; then adding 50 μL of 10% BSA blocking solution (mass percentage), mixing evenly, and standing at 25°C for 15 min; then centrifuging at 4°C, 11000 rpm for 15 min, discarding the supernatant, and adding 200 μL of re-solution to obtain.

[0027] Preferably, the preparation method of the colloidal gold-labeled carbofuran antibody solution includes: taking 1 mL of colloidal gold solution, adding 6-12 μL (preferably 7 μL) of 0.2 mol / L K2CO3 solution, mixing evenly, adding 5 μg of carbofuran antibody, mixing evenly, and standing at 25°C for 10 min; then adding 50 μL of 10% BSA blocking solution (mass percentage), mixing evenly, and standing at 25°C for 15 min; then centrifuging at 4°C, 11000 rpm for 15 min, discarding the supernatant, and adding 200 μL of re-solution to obtain.

[0028] Preferably, the preparation method of the colloidal gold-labeled carbendazim antibody solution comprises: taking 1 mL of colloidal gold solution into a reaction well, adding 3-7 μL (preferably 4 μL) of a 0.2 mol / L K2CO3 solution, mixing evenly, adding 6 μg of carbendazim antibody, mixing evenly, and standing at 25°C for 10 min; then adding 50 μL of a 10% BSA blocking solution (mass percentage), mixing evenly, and standing at 25°C for 15 min; then centrifuging at 4°C, 11000 rpm for 15 min, discarding the supernatant, and adding 200 μL of a reconstitution solution to obtain the product.

[0029] Among them, the preparation method of the colloidal gold solution is: first dissolve 2g of chloroauric acid (HAuCl4) powder in 100mL of ultrapure water to obtain a 2% chloroauric acid solution; then take 1L of ultrapure water and heat it to boiling, then add 10mL of a 1% trisodium citrate solution, continue heating for 8 minutes after boiling, and then add 10mL of a 2% chloroauric acid solution. After the color of the solution turns into wine red, stop heating after heating for another 10 minutes, cool naturally, and adjust the volume to 1L, which is the colloidal gold solution.

[0030] The reconstitution solution may be 0.05 mol / L PBS containing 2% trehalose and 1% BSA, pH 7.4.

[0031] The average particle size of the colloidal gold solution is 40 nm.

[0032] The colloidal gold-labeled procymidone antibody can be used to label 5-10 μg (preferably 6 μg) of procymidone antibody by adjusting the pH of 1 mL of colloidal gold with 7 μL of 0.2 mol / L potassium carbonate solution.

[0033] The colloidal gold-labeled carbofuran antibody can be used to label 5-8 μg (preferably 5 μg) of carbofuran antibody by adjusting the pH of 1 mL of colloidal gold with 7 μL of 0.2 mol / L potassium carbonate solution.

[0034] The colloidal gold-labeled carbendazim antibody can be used to label 5-12 μg (preferably 6 μg) of carbendazim antibody by adjusting the pH of 1 mL of colloidal gold with 4 μL of 0.2 mol / L potassium carbonate solution.

[0035] In the method for preparing the reaction well, the vacuum freeze-drying conditions are: -50°C for 4 hours, -25°C for 4 hours, -15°C for 4 hours, 5°C for 2 hours, and 25°C for 10 hours.

[0036] In a third aspect, the present invention provides the use of the kit in the detection of procymidone, carbofuran and carbendazim (including non-disease diagnosis and treatment purposes). The procymidone, carbofuran and carbendazim triple colloidal gold immunochromatography kit of the present invention can simultaneously extract, purify and detect procymidone, carbofuran and carbendazim residual in vegetables.

[0037] In a fourth aspect, the present invention provides a method for simultaneously extracting, purifying and detecting procymidone, carbofuran and carbendazim residues in vegetables and fruits, comprising the following steps:

[0038] (1) Sample pretreatment: Cut the vegetable sample into pieces and homogenize it, then take 2 g and put it into an extraction and purification tube, mix it evenly, centrifuge it, and collect the supernatant as the test solution;

[0039] (2) Sample detection: Add the test solution into the reaction well, mix by suction, and insert the sample pad end of the immunochromatographic test strip into the reaction well for detection;

[0040] (3) Analyze the test results.

[0041] The sample extraction purification tube, reaction well, and immunochromatographic test strip come from the immunochromatographic test kit.

[0042] The specific detection methods are as follows:

[0043] (1) Sample pretreatment: Cut the vegetable sample into pieces, homogenize it, weigh 2 g, add it to the extraction and purification tube, vortex for 1 min to mix it evenly, centrifuge it at 4000 rpm for 2 min, and the supernatant is the test solution. Take another vegetable sample, add a certain amount of procymidone, carbofuran, and carbendazim standard solution to make the procymidone content 0.2 ppm, the carbofuran content 0.02 ppm, and the carbendazim content 2 ppm, and then operate according to the pretreatment method to obtain the spiked test solution.

[0044] (2) Sample detection: Use a pipette to take 100 μL of the actual sample solution and the spiked sample solution, place them in the reaction wells, and mix them by pumping up and down. After standing for 2 minutes, insert the sample pad end of the test strip into the reaction well and time it for 5 minutes. Remove the test strip, gently scrape off the sample pad to terminate the chromatography process, and determine the result.

[0045] (3) The results are judged as follows Figure 2 As shown:

[0046] Invalid: Line C does not show color, indicating that the test strip is invalid or improperly operated. Use a newly opened test strip to retest.

[0047] Negative: The C line is colored, and all three detection lines are colored, and the color is darker than or equal to the C line, indicating that the concentration of the detection object in the sample is lower than the detection limit.

[0048] Positive: The C line is colored, and the test line is not colored or is lighter in color than the C line, which means that the concentration of the test object is greater than or equal to the detection limit. At this time, the standard method should be used for quantitative detection.

[0049] In the present invention, the procymidone hapten-carrier protein conjugate, the carbofuran hapten-carrier protein conjugate, the carbendazim hapten-carrier protein conjugate, and the procymidone antibody, the carbofuran antibody, and the carbendazim antibody can be prepared by methods known in the art, or can also be purchased.

[0050] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0051] (1) The triple colloidal gold immunochromatographic kit of procymidone, carbofuran and carbendazim of the present invention can simultaneously extract, purify and detect the residues of procymidone, carbofuran and carbendazim, which are the three pesticides that are widely used in vegetables and have serious excessive pesticide residues. The detection object is clear and the pertinence is strong.

[0052] (ii) The preparation methods of sample extraction and purification tubes, test strips, and test kits are simple and stable, and are suitable for mass production.

[0053] (III) The detection method is simple and efficient. It takes only about ten minutes from sample pretreatment to reaction completion and result determination, which is very suitable for large-scale on-site screening.

[0054] (iv) The test strips have high sensitivity, low detection limit, good specificity and stability, making them easy for primary-level testing personnel to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The schematic diagram of the structure of the immunochromatographic test strip of the present invention is shown in FIG. 1-sample pad, 2-T1 line, 3-T2 line, 4-T3 line, 5-C line, 6-nitrocellulose membrane, 7-water absorbent pad, and 8-bottom plate.

[0056] Figure 2 It is a schematic diagram for determining the test result of the test strip of the present invention. DETAILED DESCRIPTION

[0057] The present invention provides a colloidal gold immunochromatographic test strip containing procymidone, carbofuran and carbendazim. Figure 1As shown: it comprises a base plate (8), a sample pad (1), a nitrocellulose membrane (6) and a water absorbent pad (7); the nitrocellulose membrane (6) is coated in sequence with a first detection line (2) of a procymidone hapten-carrier protein conjugate, a second detection line (3) of a carbofuran hapten-carrier protein conjugate, a third detection line (4) of a carbendazim hapten-carrier protein conjugate, and a quality control line (5) combined with a sheep anti-mouse polyclonal antibody.

[0058] The present invention provides a rapid test kit, which is composed of the above-mentioned test strip, a reaction well containing a gold-labeled antibody, and a sample extraction and purification tube. The reaction well contains procymidone, carbofuran, and carbendazim antibodies labeled with colloidal gold. The bottom area of ​​the reaction well is 0.32 cm 2 The volume is 365μL, and a 96-well plate can be directly used as the reaction well to prepare the kit in large quantities.

[0059] In the present invention, the average particle size of the colloidal gold solution used is about 40 nm. Procymidone hapten-carrier protein conjugate and its antibody, carbofuran hapten-carrier protein conjugate and its antibody, carbendazim hapten-carrier protein conjugate and its antibody can be prepared by methods known in the art, or can also be purchased.

[0060] The colloidal gold immunochromatographic test strip and rapid test kit provided by the present invention can simultaneously realize the rapid detection of procymidone, carbofuran and carbendazim, with accurate results and simple operation. The specific steps include:

[0061] 1. Prepare colloidal gold-labeled antibodies to procymidone, carbofuran and carbendazim, and freeze-dry them in the reaction wells.

[0062] 2. Prepare nitrocellulose membranes coated with the first test line of the hapten-carrier protein conjugate of procymidone, the second test line of the hapten-carrier protein conjugate of chlorpyrifos, the third test line of the hapten-carrier protein conjugate of carbendazim, and the quality control line combined with sheep anti-mouse polyclonal antibodies.

[0063] 3. Assemble the nitrocellulose membrane prepared in step 2 with the sample pad, absorbent pad and base plate to form a test strip.

[0064] 4. Place 200 mg PSA, 200 mg C18, and 30 mg GCB in a 5 mL centrifuge tube, and then add 2 mL 0.05 mol / L PBS 7.4 (i.e., PBS at pH 7.4) and mix well to obtain a sample extraction and purification tube containing the sample extraction and purification solution.

[0065] 5. Place the reaction well in step 1, the test strip in step 3, and the sample extraction and purification tube in step 4 into the reagent kit and store them in a cool and dry place at 4-25°C until use.

[0066] 6. Use the sample extraction and purification tube in step 4 to pre-treat the sample to be tested into a detection solution, transfer it to the reaction well, and detect it with the prepared test strip.

[0067] Preferably, in step 1, the colloidal gold is used to label the procymidone antibody, and the pH of 1 mL of colloidal gold is adjusted with 0.2 mol / L potassium carbonate solution, and the dosage is 5-10 μL, preferably 7 μL.

[0068] Preferably, in step 1, the colloidal gold is used to label the procymidone antibody. 1 mL of colloidal gold can label 5-10 μg of the procymidone antibody, preferably 6 μg.

[0069] Preferably, in step 1, the colloidal gold is used to label the carbofuran antibody, and the pH of 1 mL of colloidal gold is adjusted with 0.2 mol / L potassium carbonate solution, and the amount used is 6-12 μL, preferably 7 μL.

[0070] Preferably, in step 1, the colloidal gold is used to label the carbofuran antibody. 1 mL of colloidal gold can label 5-8 μg of the carbofuran antibody, preferably 5 μg.

[0071] Preferably, in step 1, the colloidal gold is used to label the carbendazim antibody, and the pH of 1 mL of colloidal gold is adjusted with 0.2 mol / L potassium carbonate solution, and the amount used is 3-7 μL, preferably 4 μL.

[0072] Preferably, in step 1, the colloidal gold is used to label the carbendazim antibody, and 1 mL of colloidal gold can label 5-12 μg of carbendazim antibody, preferably 6 μg.

[0073] Among them, the preparation method of the colloidal gold solution is: first dissolve 2g of chloroauric acid (HAuCl4) powder in 100mL of ultrapure water to obtain a 2% chloroauric acid solution; then take 1L of ultrapure water and heat it to boiling, then add 10mL of a 1% trisodium citrate solution, continue heating for 8 minutes after boiling, and then add 10mL of a 2% chloroauric acid solution. After the color of the solution turns into wine red, stop heating after heating for another 10 minutes, cool naturally, and adjust the volume to 1L, which is the colloidal gold solution.

[0074] Preferably, 2 μL, 3 μL, and 2 μL of the colloidal gold-labeled procymidone antibody, colloidal gold-labeled carbofuran antibody, and colloidal gold-labeled carbendazim antibody in step 1 are respectively placed in the reaction wells, diluted to 60 μL with lyophilization protection solution, and lyophilized.

[0075] Preferably, the concentration of the procymidone hapten-carrier protein conjugate bound to the first test line in step 2 is 0.10-0.20 mg / mL, preferably 0.15 mg / mL.

[0076] Preferably, the concentration of the carbofuran hapten-carrier protein conjugate bound to the second detection line in step 2 is 0.25-0.8 mg / mL, preferably 0.6 mg / mL.

[0077] Preferably, the concentration of the carbendazim hapten-carrier protein conjugate bound to the third detection line in step 2 is 0.25-0.6 mg / mL, preferably 0.5 mg / mL.

[0078] Preferably, the concentration of the goat anti-mouse polyclonal antibody bound to the quality control line in step 2 is 0.25 mg / mL.

[0079] Preferably, in step 2, the distance between the first test line and the second test line, the distance between the second test line and the third test line, and the distance between the third test line and the quality control line are all 4-5 mm, preferably 4.3 mm. In the present invention, if the distance between two adjacent test lines is too close, the color development will be interfered; if the distance is too far, the chromatography time is too long and more test solution is required.

[0080] Preferably, the width of the test strip in step 3 is 3-5 mm, preferably 3.8 mm. If it is too narrow, it is not conducive to the judgment of the result, and if it is too wide, more antigens, antibodies and detection liquids are required, which is not conducive to cost saving.

[0081] The procymidone, carbofuran and carbendazim triple colloidal gold immunochromatography kit of the present invention can simultaneously extract, purify and detect procymidone, carbofuran and carbendazim in vegetables, the kit is easy to operate, has high result accuracy, low detection limit, good specificity, stable performance, can meet the requirements of on-site rapid determination, and is particularly suitable for large-scale screening.

[0082] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0083] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0084] The procymidone antibody used in the following examples was purchased from Guangzhou Youkangduo Biotechnology Co., Ltd. Carbofuran antibody and carbendazim antibody were purchased from the Institute of Food Safety Science of Jiangnan University. The procymidone hapten-carrier protein conjugate was synthesized by the method of reference 1 (Zou Qiang. Preparation and characteristic analysis of monoclonal antibodies against thiophanate-methyl and procymidone [D]. Huazhong Agricultural University), the carbofuran hapten-carrier protein conjugate was synthesized by the method of reference 2 (Sun Fengxia, Kang Lichao, etc. Synthesis of artificial antigen of carbofuran and its immune application [J]. Journal of Northwest Agriculture. 2018), and the carbendazim hapten-carrier protein conjugate was synthesized by the method of reference 3 (Jiang Wenhui, Wu Xiaosheng, etc. Establishment of a rapid enzyme-linked immunosorbent assay method for carbendazim [J]. Food and Machinery. 2021).

[0085] Example 1 Construction of a triple immunochromatographic detection kit for procymidone, carbofuran and carbendazim

[0086] The preparation method of the procymidone, carbofuran and carbendazim triple immunochromatographic detection kit provided in this embodiment is as follows:

[0087] 1. Preparation of colloidal gold labeled procymidone antibody

[0088] Take 1mL colloidal gold solution, add 7μL K2CO3 solution (0.2mol / L), mix well, add 6μg procymidone antibody, mix well, and let stand at 25℃ for 10min. Add 50μL BSA blocking solution (mass fraction 10%), mix well, and let stand at 25℃ for 15min. Centrifuge at 4℃, 11000rpm for 15min, carefully remove the supernatant, and add 200μL re-solution (0.05mol / L PBS7.4, containing 2% trehalose and 1% BSA).

[0089] 2. Preparation of colloidal gold labeled carbofuran antibody

[0090] Take 1mL colloidal gold solution, add 7μL K2CO3 solution (0.2mol / L), mix well, add 5μg carbofuran antibody, mix well, and let stand at 25℃ for 10min. Add 50μL BSA blocking solution (mass fraction 10%), mix well, and let stand at 25℃ for 15min. Centrifuge at 4℃, 11000rpm for 15min, carefully remove the supernatant, and add 200μL re-solution (0.05mol / L PBS7.4, containing 2% trehalose and 1% BSA).

[0091] 3. Preparation of colloidal gold labeled carbendazim antibody

[0092] Take 1mL colloidal gold solution to the reaction well, add 4μL K2CO3 solution (0.2mol / L), mix well, add 6μg carbendazim antibody, mix well, and let stand at 25℃ for 10min. Add 50μL BSA blocking solution (mass fraction 10%), mix well, and let stand at 25℃ for 15min. Centrifuge at 4℃, 11000rpm for 15min, carefully remove the supernatant, and add 200μL re-solution (0.05mol / L PBS 7.4, containing 2% trehalose and 1% BSA).

[0093] 4. Preparation of reaction wells of the kit

[0094] 2 μL, 3 μL, and 2 μL of the colloidal gold labeled procymidone antibody, colloidal gold labeled carbofuran antibody, and colloidal gold labeled carbendazim antibody prepared in the above steps were respectively placed in the reaction wells, and diluted to 60 μL with freeze-drying protection solution (containing 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% Tween 20, and 0.03% ProClin 300). The samples were sent to a vacuum freeze dryer and freeze-dried according to the procedure of -50°C for 4 hours, -25°C for 4 hours, -15°C for 4 hours, 5°C for 2 hours, and 25°C for 10 hours.

[0095] 5. Preparation of sample extraction and purification tube

[0096] Place 200 mg PSA, 200 mg C18, and 30 mg GCB in a 5 mL centrifuge tube, and then add 2 mL 0.05 mol / L PBS7.4 and mix well to obtain a sample extraction and purification tube containing the sample extraction and purification solution.

[0097] 6. Kit Assembly

[0098] The hapten-carrier protein conjugate of procymidone, the hapten-carrier protein conjugate of carbofuran, the hapten-carrier protein conjugate of carbendazim and the sheep anti-mouse polyclonal antibody are coated on the nitrocellulose membrane, which are respectively the first detection line, i.e., T1 line (2), the second detection line, i.e., T2 line (3), the third detection line, i.e., T3 line (4) and the quality control line, i.e., C line (5). The nitrocellulose membrane (6) is assembled with the sample pad (1), the water-absorbing pad (7) and the bottom plate (8) and cut into test strips. The material of the bottom plate is PVC. The specific method is as follows:

[0099] 1) Coating of procymidone hapten-carrier protein conjugate, carbofuran hapten-carrier protein conjugate, carbendazim hapten-carrier protein conjugate and goat anti-mouse polyclonal antibody

[0100] In this method, Sartorius CN95 membrane is selected as the coating bottom membrane, and the hapten-carrier protein conjugate of procymidone is diluted to 0.15 mg / mL with 0.02 mol / L PBS, pH 7.4 (containing 0.625% NaCl), the hapten-carrier protein conjugate of carbofuran is diluted to 0.6 mg / mL, the hapten-carrier protein conjugate of carbendazim is diluted to 0.5 mg / mL, and the goat anti-mouse polyclonal antibody is diluted to 0.25 mg / mL with 0.02 mol / L PBS, pH 7.4 (containing 2% sucrose). The film drawing condition during coating is 0.8 μL / cm, and the distance between each detection line is 4.3 mm. After coating, it is dried in an oven at 60°C for 2-4 hours.

[0101] 2) Preparation of test strips

[0102] The dried nitrocellulose membrane (6) is adhered to the middle position of the bottom plate (8), and then the sample pad (1) and the absorbent paper (7) are adhered to the bottom plate at both ends of the nitrocellulose membrane, and the nitrocellulose membrane is slightly pressed to ensure smooth chromatography of the detection solution and the gold-labeled antibody. After that, the whole plate is cut into 3.8 mm test strips with a strip cutter, and sealed and stored at 4°C, and then unsealed when needed.

[0103] The rapid test kit provided by the present invention is composed of the above-mentioned test strip, a reaction well containing a gold-labeled antibody, and a sample extraction and purification tube. The reaction well contains procymidone, carbofuran, and carbendazim antibodies labeled with colloidal gold. The bottom area of ​​the reaction well is 0.32 cm 2 The volume is 365μL, and a 96-well plate can be directly used as the reaction well to facilitate the preparation of the kit in large quantities.

[0104] The kit should be stored at 4-25℃ in a cool and dry condition and has a shelf life of 12 months.

[0105] Example 2 Testing of actual samples

[0106] The kit prepared in Example 1 was used to perform actual sample testing, and the specific steps were as follows:

[0107] (1) Sample pretreatment: Cut the vegetable sample into pieces and homogenize it. Weigh 2 g and add it to the sample extraction and purification tube. Vortex for 1 min to mix thoroughly. Centrifuge at 4000 rpm for 2 min. The supernatant is the test solution. Take another vegetable sample and add a certain amount of procymidone, carbofuran and carbendazim standard solution to make the procymidone content 0.2 mg / kg, the carbofuran content 0.02 mg / kg and the carbendazim content 2 mg / kg. Then follow the pretreatment method to obtain the spiked test solution.

[0108] (2) Sample detection: Use a pipette to take 100 μL of the actual sample solution and the spiked sample solution, place them in the reaction wells, and mix them by pumping up and down. After standing for 2 minutes, insert the sample pad end of the test strip into the well and time it for 5 minutes. Remove the test strip, gently scrape off the sample pad to terminate the chromatography process, and determine the result.

[0109] (3) The results are judged as follows Figure 2 As shown:

[0110] Invalid: Line C does not show color, indicating that the test strip is invalid or improperly operated. Use a newly opened test strip to retest.

[0111] Negative: The C line is colored, and all three detection lines are colored, and the color is darker than or equal to the C line, indicating that the concentration of the detection object in the sample is lower than the detection limit.

[0112] Positive: The C line develops color, and the detection line disappears or is lighter in color than the C line, indicating that the concentration of the test object is greater than or equal to the detection limit. At this time, the standard method should be used for quantitative detection.

[0113] Example 3 Detection limit of the kit

[0114] This example aims to test the detection limit of the triple immunochromatographic detection kit for procymidone, carbofuran and carbendazim.

[0115] 1. Detection limit of procymidone: 10 mg / L procymidone standard solution of different volumes was added to leek to make the spiked concentrations: 0.1 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, and the test was performed according to the method in Example 2. The results showed that when the spiked concentration was 0.1 mg / kg, the color of the T1 line was similar to the color of the C line, which was a negative result. When the concentration was 0.2 mg / kg, the color of the T1 line was significantly lighter than the C line, and the result was positive. Therefore, the detection limit of procymidone in this kit was 0.2 mg / kg.

[0116] 2. Detection limit of carbofuran: 10 mg / L carbofuran standard solution of different volumes was added to leek, so that the spiked concentration was: 5 μg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and the test was performed according to the method in Example 2. The results showed that when the spiked concentration was 5 μg / kg, the color of the T2 line was similar to the color of the C line, which was a negative result. When the concentration was 0.01 mg / kg, the color of the T2 line was significantly lighter than the C line, and the result was positive. Therefore, the detection limit of this kit for carbofuran is 0.01 mg / kg.

[0117] 3. Detection limit of carbendazim: 10 mg / L carbendazim standard solution of different volumes was added to leeks, so that the spiked concentration was: 5 μg / kg, 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and the test was performed according to the method in Example 2. The results showed that when the spiked concentration was 5 μg / kg, the color of the T2 line was similar to the color of the C line, which was a negative result. When the concentration was 0.01 mg / kg, the color of the T2 line was significantly lighter than the C line, and the result was positive. Therefore, the detection limit of this kit for carbofuran was 0.01 mg / kg.

[0118] Example 4 Specificity of the kit

[0119] This example tests the specificity of the triple immunochromatographic detection kit of procymidone, carbofuran and carbendazim, and the method is as follows:

[0120] Common pesticides such as fipronil, chlorpyrifos, acetamiprid, cypermethrin, imidacloprid, etc. were prepared into 1 mg / kg standard solutions, 100 μL was transferred to the reaction well, and mixed by suction up and down. After standing for 2 minutes, the sample pad end of the test strip was inserted into the reaction well. After 5 minutes of timing, the test strip was taken out, the sample pad was gently scraped off, and the result was determined. The results showed that the C line, T1 line, T2 line, and T3 line were all clearly colored, indicating that these pesticides would not cross-react with the antibody and the test strip had good specificity.

[0121] Example 5 Kit Performance Comparison

[0122] The kit of the present invention is compared with the kits on the market or in the disclosed patents, as follows:

[0123] 1. Comparison of detection limits is shown in Table 1:

[0124] Table 1

[0125]

[0126]

[0127] As shown in Table 1, the detection limits of the kits of the present invention are lower than or equal to those of other commercially available kits, and the kits of the present invention can simultaneously extract, purify and detect three pesticide residues, while the products currently on the market are all single detection kits. The above shows that the present kit has better detection performance for procymidone, carbofuran and carbendazim, and more importantly, it realizes the simultaneous extraction, purification and detection of three different types of pesticide residues, fills the market gap, effectively saves resources, and is more suitable for large-scale screening on site.

[0128] The above commercial kits were all purchased from Jiangsu Meizheng Biotechnology Co., Ltd. The product number of commercial kit 1 is MK107A1, the product number of commercial kit 2 is MK108A1, and the product number of commercial kit 3 is MK106C1.

[0129] 2. Comparison of pretreatment reagents is shown in Table 2:

[0130] Table 2

[0131]

[0132] As shown in Table 2, the pretreatment reagents are slightly different for different detection objects. The kit of the present invention contains 200 mg PSA, 200 mg C 18 , 30mg GCB in 2mL 0.05mol / L PBS 7.4 as the extraction and purification solution, can simultaneously extract 3 different pesticide residues and purify them into the test solution, and the addition of the purification reagent can effectively adsorb the pigment, reduce interference for the color judgment of the test strip, improve the sensitivity of the detection method, and reduce the detection limit. This method can extract 3 test objects at the same time and perform purification detection in one treatment, which greatly saves manpower and material resources, improves detection efficiency, and can provide extremely powerful technical support for grassroots food safety supervision, food inspection, and large-scale event security.

[0133] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A triple immunochromatographic test strip for procymidone, carbofuran and carbendazim, characterized in that: It comprises a sample pad, a nitrocellulose membrane, a water absorbent pad and a bottom plate, wherein the nitrocellulose membrane is provided with three detection lines and a quality control line in sequence from the sample pad to the water absorbent pad, the three detection lines are respectively coated with a procymidone hapten-carrier protein conjugate, a carbofuran hapten-carrier protein conjugate and a carbendazim hapten-carrier protein conjugate, and the quality control line is coated with a sheep anti-mouse polyclonal antibody; The sample pad, nitrocellulose membrane and water absorbent pad are alternately stacked and pasted on the bottom plate at a spacing of 2-3 mm. The concentration of the procymidone hapten-carrier protein conjugate coated on the test line is 0.10-0.20 mg / mL, the concentration of the carbofuran hapten-carrier protein conjugate coated on the test line is 0.25-0.8 mg / mL, and the concentration of the carbendazim hapten-carrier protein conjugate coated on the test line is 0.25-0.6 mg / mL; the concentration of the goat anti-mouse polyclonal antibody coated on the quality control line is 0.25 mg / mL; The structures of the procymidone hapten, carbofuran hapten and carbendazim hapten are shown in Formula 1) to Formula 3) respectively:

2. The test strip according to claim 1, characterized in that: The concentration of the hapten-carrier protein conjugate of procymidone coated on the test line is 0.15 mg / mL; the concentration of the hapten-carrier protein conjugate of carbofuran coated on the test line is 0.6 mg / mL; the concentration of the hapten-carrier protein conjugate of carbendazim coated on the test line is 0.5 mg / mL.

3. The test strip according to claim 1, characterized in that: The distances between the test lines on the nitrocellulose membrane and between the test line near the end of the absorbent pad and the quality control line are all 4-5 mm; the width of the test strip is 3-5 mm.

4. The test strip according to claim 3, characterized in that: The distances between the test lines on the nitrocellulose membrane and between the test line near the absorbent pad and the quality control line are all 4.3 mm; the width of the test strip is 3.8 mm.

5. The test strip according to any one of claims 1 to 4, characterized in that: The material of the sample pad is nitrocellulose membrane.

6. A triple immunochromatographic detection kit for procymidone, carbofuran and carbendazim, characterized in that: The invention comprises a sample extraction and purification tube, a reaction substrate with reaction holes and the test strip according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The preparation method of the sample extraction and purification tube comprises: adding 200 mg of N-propylethylenediamine solid phase adsorbent PSA, 200 mg of octadecyl bonded silica gel C 18 and 30 mg of graphitized carbon black GCB were placed in a 5 mL centrifuge tube, and then 2 mL of 0.05 mol / L pH 7.4 PBS was added and mixed to obtain a sample extraction and purification tube containing the sample extraction and purification solution.

8. The kit according to claim 6, characterized in that The reaction wells are coated with colloidal gold-labeled procymidone antibodies, colloidal gold-labeled carbofuran antibodies, and colloidal gold-labeled carbendazim antibodies; The preparation method of the reaction well comprises: adding 2 μL of colloidal gold-labeled procymidone antibody solution, 3 μL of colloidal gold-labeled carbofuran antibody solution and 2 μL of colloidal gold-labeled carbendazim antibody solution into the reaction well, diluting to 60 μL with freeze-drying protection solution, and performing vacuum freeze drying; The preparation method of the colloidal gold solution is as follows: first, dissolve 2g of chloroauric acid powder in 100mL of ultrapure water to obtain a chloroauric acid solution with a concentration of 2%; then, take 1L of ultrapure water, heat it to boiling, add 10mL of a 1% trisodium citrate solution, continue heating for 8 minutes after boiling, then add 10mL of a 2% chloroauric acid solution, and after the color of the solution changes to wine red, stop heating after heating for another 10 minutes, cool naturally, and adjust the volume to 1L to obtain the colloidal gold solution; The preparation method of the colloidal gold-labeled procymidone antibody solution comprises: taking 1 mL of colloidal gold solution, adding 5-10 μL of 0.2 mol / L K2CO3 solution, mixing evenly, adding 6 μg of procymidone antibody, mixing evenly, and standing at 25°C for 10 min; then adding 50 μL of 10% BSA blocking solution, mixing evenly, and standing at 25°C for 15 min; then centrifuging at 4°C and 11000 rpm for 15 min, discarding the supernatant, and adding 200 μL of reconstitution solution to obtain the solution; The preparation method of the colloidal gold-labeled carbofuran antibody solution comprises: taking 1 mL of colloidal gold solution, adding 6-12 μL of 0.2 mol / L K2CO3 solution, mixing evenly, adding 5 μg of carbofuran antibody, mixing evenly, and standing at 25°C for 10 minutes; then adding 50 μL of 10% BSA blocking solution, mixing evenly, and standing at 25°C for 15 minutes; then centrifuging at 4°C and 11000 rpm for 15 minutes, discarding the supernatant, and adding 200 μL of reconstitution solution to obtain the solution; The preparation method of the colloidal gold-labeled carbendazim antibody solution comprises: taking 1 mL of colloidal gold solution into a reaction well, adding 3-7 μL of a 0.2 mol / L K2CO3 solution, mixing evenly, adding 6 μg of carbendazim antibody, mixing evenly, and standing at 25°C for 10 min; then adding 50 μL of a 10% BSA blocking solution, mixing evenly, and standing at 25°C for 15 min; then centrifuging at 4°C and 11000 rpm for 15 min, discarding the supernatant, and adding 200 μL of a reconstitution solution to obtain the solution; The reconstitution solution is: 0.05 mol / L PBS containing 2% trehalose and 1% BSA, pH 7.4; The freeze-dried protective solution is: 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% Tween 20 and 0.03% ProClin 300, prepared with water.

9. The kit according to claim 8, characterized in that The vacuum freeze-drying conditions of the reaction wells are: -50°C for 4 h, -25°C for 4 h, -15°C for 4 h, 5°C for 2 h, and 25°C for 10 h.

10. Use of the kit according to any one of claims 6 to 9 in the detection of procymidone, carbofuran and carbendazim; the use is for non-disease diagnosis and treatment purposes.

11. A method for simultaneously extracting, purifying and detecting the residues of procymidone, carbofuran and carbendazim in vegetables, characterized in that: The following steps are involved: (1) Sample pretreatment: Cut and homogenize the vegetable sample, take 2 g into a sample extraction and purification tube, mix well, centrifuge, and collect the supernatant as the test solution; (2) Sample detection: Add the test solution into the reaction well, mix by suction, and insert the sample pad end of the immunochromatographic test strip into the reaction well for detection; (3) Analyze the test results; The sample extraction and purification tube, reaction well, and immunochromatographic test strip are from the kit described in any one of claims 6 to 9.

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