A test strip and method for detecting phorate and its metabolites

Abstract

The application discloses a test strip and a method for detecting phorate and metabolites thereof. The test strip comprises a test paper and a micropore reagent, the test paper comprises a reaction film, a sample absorption pad, a water absorption pad and a bottom plate, the reaction film is provided with a detection line coated with a phorate hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody, and the micropore reagent is freeze-dried with a phorate monoclonal antibody-gold label. The method for detecting phorate and metabolites thereof by using the test strip is simple, rapid, intuitive, accurate, portable, widely applicable, low in cost and easy to popularize and use.

Description

TECHNICAL FIELD

[0001] The present application relates to a test strip and method for detecting phorate and its metabolites, in particular to a colloidal gold test strip for detecting phorate and its metabolites, which is particularly suitable for detecting phorate and its metabolites in fruits and vegetables. BACKGROUND

[0002] Phorate is a highly toxic and broad-spectrum organophosphorus insecticide, mainly used for preventing and controlling underground pests, aphids, etc. It is widely used in the process of growing agricultural products because of its high insecticidal activity and low price. Direct consumption of agricultural products containing phorate and its metabolites will inhibit cholinesterase activity, causing physiological function disorder. Phorate that has not been absorbed by plants is transferred to surface water, rivers and lakes with water, forming secondary pollution. Phorate will be metabolized to produce phorate sulfone and phorate sulfoxide, which are more toxic and more difficult to degrade, causing greater harm to the ecological environment and human health. GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Foods" stipulates that the maximum residue content of phorate, phorate sulfoxide and phorate sulfone in vegetables and fruits shall not exceed 0.01 mg / kg. Therefore, it is necessary to establish an efficient, sensitive and accurate method for detecting phorate and its metabolites in fruits and vegetables.

[0003] The methods reported so far for detecting phorate and its metabolites mainly include fluorescence spectrophotometry, gas chromatography, gas chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, liquid chromatography, liquid chromatography-tandem mass spectrometry, etc. These methods all need to be operated under laboratory conditions, and the sample pretreatment is complicated and time-consuming. In addition, expensive instruments and equipment are required, the detection cost is high, the time consumption is long, and the operation is complex. In the actual application process, there are great limitations, and it is difficult to meet the needs of rapid detection of a large number of samples and on-site samples. Therefore, the development of a simple and rapid colloidal gold test strip suitable for detecting phorate and its metabolites in fruits and vegetables can meet the needs of on-site screening and monitoring of a large number of samples, and can better meet the detection work of food supervision departments and the like in China. SUMMARY

[0004] The purpose of the present application is to provide a colloidal gold test strip capable of detecting phorate and its metabolite residues in fruits and vegetables, and to provide a detection method that is efficient, accurate, simple, suitable for on-site monitoring and large sample screening.

[0005] The test strip for detecting phorate and its metabolites provided by the present application comprises a test paper and a micropore reagent; the test paper comprises a bottom plate, a sample absorption pad, a reaction membrane and a water absorption pad, which are connected in sequence, the reaction membrane has a detection line coated with a phorate hapten-carrier protein conjugate and a quality control line coated with a sheep anti-mouse antibody; the micropore reagent has a phorate monoclonal antibody-colloidal gold label freeze-dried thereon, and has a micropore plug.

[0006] The monoclonal antibody of phorate is prepared by taking the phorate hapten-carrier protein conjugate as an immunogen.

[0007] The phorate hapten-carrier protein conjugate is obtained by coupling the phorate hapten and a carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin or human serum albumin.

[0008]

[0009] The bottom plate is a PVC bottom plate or other hard non-water-absorbing material; the sample absorption pad is made of polyester fiber or glass fiber material; the water absorption pad is water absorption filter paper; and the reaction membrane is nitrocellulose membrane or cellulose acetate membrane.

[0010] Another object of the present application is to provide a method for preparing the test strip, which comprises the following steps:

[0011] 1) preparing a freeze-dried microporous reagent with phorate monoclonal antibody-gold label;

[0012] 2) preparing a reaction membrane with a detection line coated with phorate hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse antibody;

[0013] 3) assembling the reaction membrane prepared in 2) with a sample absorption pad, a water absorption pad and a bottom plate into a test paper;

[0014] 4) assembling the freeze-dried microporous reagent with phorate monoclonal antibody-gold label prepared in 1) and the test paper prepared in 3) into a test strip.

[0015] Specifically, the steps include:

[0016] 1) condensation reaction of O-ethyl S-[(ethylthio)methyl] ester and N-(2-aminopropyl) maleimide under catalysis of pyridine to prepare the phorate hapten;

[0017] 2) coupling of the phorate hapten and the carrier protein to prepare the phorate hapten-carrier protein conjugate;

[0018] 3) immunize mice with phorate hapten-carrier protein conjugate, fuse mouse spleen cells and mouse myeloma cells, screen to obtain a hybridoma cell strain secreting phorate monoclonal antibody;

[0019] 4) extract mouse IgG, immunize healthy goats, and obtain goat anti-mouse antibody;

[0020] 5) coat phorate hapten-carrier protein conjugate and goat anti-mouse antibody on the test line (T) and the control line (C) of the reaction membrane, respectively;

[0021] 6) prepare colloidal gold by reacting sodium citrate with chloroauric acid;

[0022] 7) add the prepared phorate monoclonal antibody to the prepared colloidal gold to obtain phorate monoclonal antibody-colloidal gold label;

[0023] 8) freeze-dry the phorate monoclonal antibody-colloidal gold label in the micropore reagent, and then add a micropore plug to the micropore reagent;

[0024] 9) soak the sample absorption pad in 0.2 mol / L phosphate buffer containing 1% bovine serum albumin and having a pH of 7.2 for 2 h, and dry at 37℃ for 2 h for standby;

[0025] 10) sequentially paste the sample absorption pad, the reaction membrane, and the water absorption pad on the bottom plate, and finally cut into small strips with a width of 3.95 mm, add a plastic box, and vacuum package;

[0026] 11) assemble the prepared micropore reagent and test paper into a test strip, and store under the condition of 2-8℃ for 12 months.

[0027] Another object of the present application is to provide a method for detecting phorate and its metabolites in fruits and vegetables by using the above test strip, which comprises the steps of:

[0028] (1) sample pretreatment;

[0029] (2) detection by using the test strip;

[0030] (3) analysis of the detection result.

[0031] The phorate metabolites are phorate sulfoxide and phorate sulfone.

[0032] The rapid detection test strip of phorate and its metabolites of the present application adopts highly specific antigen-antibody reaction and immunochromatographic analysis technology, and the phorate monoclonal antibody-colloidal gold label is freeze-dried in the micropore reagent, the phorate and its metabolites in the sample are first fully reacted with the same, to form drug-antibody-colloidal gold label; then, the sample is dropped into the card hole of the test strip, and the drug in the sample competes with the phorate hapten-carrier protein conjugate on the detection line of the reaction membrane to bind the phorate monoclonal antibody-colloidal gold label in the flow process, and whether the sample liquid contains phorate and its metabolite residues is determined according to the color depth of the red band on the detection line.

[0033] During detection, the sample is treated and then dropped into the micropore reagent, and after full reaction, the test strip card hole is added, when the concentration of phorate and its metabolites in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold label will bind with the phorate hapten-carrier protein conjugate fixed on the reaction membrane in the chromatographic process, and each of the detection line (T) and the quality control line (C) appears a red band, and the color of the T line is deeper than that of the C line or consistent with that of the C line; if the concentration of phorate and its metabolites in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold label will bind with phorate and its metabolites, so that no red band appears at the T line due to the competition reaction, or the color is shallower than that of the C line. As shown in the following table. Figure 5

[0034] Negative: when the quality control line (C) shows a red band, the detection line (T) also shows a red band, and the color of the (T) line is close to or deeper than that of the (C) line, it is judged as negative.

[0035] Positive: when the quality control line (C) shows a red band, and the detection line (T) does not show color or the color of the (T) line is shallower than that of the (C) line, it is judged as positive.

[0036] Invalid: when the quality control line (C) does not show a red band, whether the detection line (T) shows a red band or not, the test strip is judged as invalid.

[0037] The test strip of the present application has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for use by various units, simple storage, long shelf life. Among them, the highly specific phorate monoclonal antibody is used to ensure the reliability of the detection result; the gold-labeled antibody is freeze-dried in the micropore reagent, which can make the gold-labeled antibody fully contact with the sample liquid to be detected and fully react, thereby reducing the error and increasing the reaction sensitivity of the whole system. The method for detecting phorate and its metabolites by using the test strip of the present application is simple, rapid, intuitive, accurate, widely applicable, low in cost and easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0038] ​Figure 1 Synthesis chart of phosphamidon hapten.

[0039] Figure 2 Nuclear magnetic resonance hydrogen spectrum chart of phosphamidon hapten.

[0040] Figure 3 Schematic diagram of test paper section structure.

[0041] Figure 4 Micro-pore reagent chart.

[0042] Figure 5 Test paper strip detection result determination chart. DETAILED DESCRIPTION

[0043] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application, and are not used to limit the scope of the application.

[0044] Preparation of test paper strip for detecting phosphamidon and its metabolites in Example 1

[0045] The preparation method of the test paper strip mainly includes the following steps:

[0046] 1) Preparation of micro-pore reagent with freeze-dried phosphamidon monoclonal antibody-gold label;

[0047] 2) Preparation of reaction membrane with detection line coated with phosphamidon hapten-carrier protein conjugate and quality control line coated with goat anti-mouse antibody;

[0048] 3) Assembly of the reaction membrane prepared in 2) with sample absorption pad, water absorption pad and bottom plate into a test paper;

[0049] 4) Assembly of the micro-pore reagent with freeze-dried phosphamidon monoclonal antibody-gold label prepared in 1) and the test paper prepared in 3) into a test paper strip.

[0050] The following is described in detail in steps:

[0051] 1. Synthesis of phosphamidon hapten (synthesis route see attached Figure 1 )

[0052] Take O-ethyl S-[(ethylthio) methyl] ester (CAS number: 181257-76-1) 2.5 g, dissolve in pyridine 150 mL, add N-(2-aminopropyl) maleimide (CAS number: 110008-25-8) 2.31 g, stir thoroughly, install a reflux condenser, react at 80℃ for 4 h, stop the reaction, rotary evaporation, remove the organic solvent, and separate by silica gel column elution with a mixture of dichloromethane-methanol (volume ratio 10:1) to obtain phosphamidon hapten.

[0053] Hydrogen nuclear magnetic resonance (1H NMR) spectrum of the hapten is shown in the following figure: 1H-NMR) determination, results are as follows Figure 2 As shown: 1 ¹H NMR (500MHz, Chloroform-d): δ (ppm) 6.71 (s, 2H), 4.42 (t, J = 4.6 Hz, 1H), 4.26 (dq, J = 8.4, 7.0 Hz, 2H), 3.92 (qt, J = 16.9, 5.1 Hz, 2H), 3.80 (s, 2H), 3.20 (q, J = 4.3 Hz, 2H), 2.57 (q, J = 5.9 Hz, 2H), 2.00–1.93 (m, 2H), 1.32 (td, J = 7.0, 0.7 Hz, 3H), 1.22 (t, J = 5.8 Hz, 3H). The chemical shift δ = 6.71 (s, 2H) is the absorption peak of the double-bonded hydrogen on the spacer arm. The presence of this peak proves successful coupling and the correct hapten structure.

[0054] 2. Preparation of immunogens

[0055] Dissolve 100 mg of bovine serum albumin (BSA) in 10 mL of PB buffer (pH 8.0), add 1 mL of an aqueous solution containing 7.2 mg of tris(2-carboxyethyl)phosphonic acid hydrochloride, and react at room temperature for 2 h to obtain solution A. Dissolve 9.34 mg of phorate hapten in 0.5 mL of N,N-dimethylformamide, add it to solution A, and react at 4 °C for 12 h. Purify by dialysis with 0.02 mol / L PB buffer to obtain the phorate hapten-BSA conjugate, which is the immunogen.

[0056] 3. Preparation of the coating agent

[0057] Dissolve 100 mg of ovalbumin (OVA) in 10 mL of pH 8.0 PB buffer, add 1 mL of aqueous solution containing 3.17 mg of tris(2-carboxyethyl)phosphonic acid hydrochloride, and react at room temperature for 2 h to obtain solution A; dissolve 4.08 mg of phorate hapten in 0.5 mL of N,N-dimethylformamide, add it to solution A, react at 4 °C for 12 h, and purify by dialysis with 0.02 mol / L PB buffer to obtain the phorate hapten-OVA conjugate, which is the coating antigen.

[0058] 4. Preparation of phorate monoclonal antibody

[0059] (1) Animal Immunization

[0060] The immunogen obtained in step 2 was injected into Balb / c mice at a dose of 150 μg per mouse to induce the production of antiserum.

[0061] (2) Cell fusion and cloning

[0062] Take the immune Balb / c mouse spleen cells, according to 8:1 (quantity ratio) proportion and SP2 / 0 myeloma cells fusion, adopt indirect competition ELISA determination cell supernatant, screening positive hole. Use the limited dilution method to the positive hole and clone, until get the hybridoma cell strain of stable secretion monoclonal antibody.

[0063] (3) Cell freezing and recovery

[0064] The hybridoma cells are made into 1×10 6 Individuals / mL cell suspension with freezing solution, long-term preservation in liquid nitrogen. When recovering, take out the freezing tube, immediately put into 37℃ water bath for rapid thawing, centrifugal removal freezing solution, move into the culture bottle and cultivate.

[0065] (4) Preparation and purification of monoclonal antibody

[0066] Incremental culture method: the hybridoma cells are placed in cell culture medium and cultured at 37℃, and the obtained culture solution is purified by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, which is stored at -20℃.

[0067] The cell culture medium is RPMI1640 medium added with calf serum and sodium bicarbonate, 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.

[0068] 5, Preparation of goat anti-mouse antibody

[0069] The goat is used as an immunized animal, and the pathogen-free goat is immunized with the mouse-derived antibody as an immunogen to obtain the goat anti-mouse antibody.

[0070] 6, Preparation of phorate monoclonal antibody-colloidal gold marker

[0071] (1) Preparation of colloidal gold

[0072] Dilute 1% chloroauric acid with double-distilled deionized water to 0.01% (mass fraction), take 100mL and place in a conical flask, heat to boiling with a constant temperature electromagnetic stirrer, add 1.5mL 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir uniformly and heat until the solution is transparent red, stop cooling to room temperature, restore to the original volume with deionized water, and store at 4℃. The prepared colloidal gold is pure, transparent, without sediment and floating material, and the color is wine red under sunlight.

[0073] (2) Preparation of phorate monoclonal antibody-colloidal gold marker

[0074] Under magnetic stirring, the pH value of the colloidal gold was adjusted to 7.2 with 0.2 mol / L potassium carbonate solution, and the monoclonal antibody of phorate was added to the colloidal gold solution at 5-50 μg per milliliter of the colloidal gold solution, and stirring was continued for 30 min; after standing for 10 min, 10% BSA was added to make the final concentration of BSA in the colloidal gold solution 1%, and standing was continued for 10 min. Centrifugation was carried out at 12000 r / min and 4°C for 40 min, the supernatant was discarded, and the precipitate was washed twice with the resuspension buffer, and the precipitate was resuspended with the resuspension buffer with a volume of 1 / 10 of the initial volume of the colloidal gold, and was stored at 4°C.

[0075] The resuspension buffer: 0.02 mol / L phosphate buffer containing 0.1%-0.5% BSA and 2%-4% sucrose, and having a pH value of 7.2.

[0076] 7. Preparation of the micropore reagent

[0077] 100 μL of the monoclonal antibody of phorate-colloidal gold label was added to the micropore reagent micropore plate, and was placed in a freeze dryer, and was pre-frozen at a cold trap temperature of -50°C for 3 h, and then was vacuum dried for 15 h, and was taken out, to obtain the micropore reagent with the monoclonal antibody of phorate-colloidal gold label, which was sealed and stored.

[0078] 8. Preparation of the sample absorption pad

[0079] The sample absorption pad was soaked in 0.2 mol / L phosphate buffer containing 1% bovine serum albumin and having a pH value of 7.2 for 2 h, and was dried at 37°C for 2 h for standby use.

[0080] 9. Preparation of the reaction membrane

[0081] The phorate hapten-OVA 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.

[0082] Coating process: the phorate hapten-OVA conjugate was diluted to 1 mg / mL with 0.01 mol / L phosphate buffer having a pH value of 7.2, and was coated on the detection line (T line) of the nitrocellulose membrane with a Bio dot membrane coating instrument, and the coating amount was 1.0 μL / cm; the goat anti-mouse antibody was diluted to 200 μg / mL with 0.01 mol / L phosphate buffer having a pH value of 7.2, and was coated on the quality control line (C line) of the nitrocellulose membrane with a Bio dot membrane coating instrument, and the coating amount was 1.0 μL / cm. The coated reaction membrane was dried at 37°C for 16 h for standby use.

[0083] 10. Assembly of the test strip

[0084] According to the attached drawings Figure 3The sample absorption pad (1), the reaction membrane (2) and the water absorption pad (3) are sequentially adhered to the PVC base plate (6) according to the profile structure of the test paper shown in the figure; the end of the sample absorption pad is connected with the start end of the reaction membrane, the end of the reaction membrane is connected with the start end of the water absorption pad, the start end of the sample absorption pad is aligned with the start end of the PVC base plate, and the end of the water absorption pad is aligned with the end of the PVC base plate; the reaction membrane has a detection line (4) and a quality control line (5), and the detection line (T line) and the quality control line (C line) are both strip-shaped bands perpendicular to the length of the test paper; the detection line is located on one side close to the end of the sample absorption pad; and the quality control line is located on one side away from the end of the sample absorption pad; the test paper is cut into small strips with a width of 3.95 mm by a machine and is loaded into a specially-made plastic card.

[0085] According to the attached drawings Figure 4 The microporous reagent diagram shown in the figure, the microporous reagent 7 has a microporous plug 8.

[0086] The test paper and the microporous reagent are assembled into a test paper strip, which is stored in an environment of 2-8°C, and the effective period is 12 months.

[0087] Example 2: Detection of phorate and its metabolites in fruits and vegetables

[0088] 1. Sample pretreatment

[0089] The fresh sample is wiped off the soil and cut into small pieces less than 1 cm square; (2.00±0.05) g of the sample is weighed into a 15 mL centrifuge tube, 6 mL of extraction solution is added, the cap is covered, and the vortex instrument is vortexed or manually shaken up and down for 1 min, and then it is left to stand for 1 min [if there is no supernatant after standing, it can be centrifuged at 3500 r / min at room temperature (20-25°C) for 3 min]; thus, the sample solution to be tested is obtained.

[0090] 2. Detection by test paper strip

[0091] 100 μL of the sample solution to be tested is taken by a micropipette into a micropore, and slowly sucked and mixed with the reagent in the micropore; after incubation at room temperature (20-25°C) for 3 min, 100 μL of the mixed solution is vertically dropped into the sample addition hole; the liquid flow starts timing, and the reaction is carried out for 10 min, and the result is determined.

[0092] 3. Analysis of detection results

[0093] Negative (-): the color development of the T line is higher than or equal to that of the C line, indicating that the concentration of phorate and its metabolites in the sample is lower than the detection limit, such as Figure 5 a, 5b.

[0094] Positive (+): the color development of the T line is lower than that of the C line, or the T line does not develop color, indicating that the concentration of phorate and its metabolites in the sample is equal to or higher than the detection limit, such as Figure 5 c, 5d.

[0095] Invalid: no C line appears, indicating incorrect operation process or that the test strip has deteriorated, such as Figure 5 e、5f.

[0096] Example 3 sample detection example

[0097] 1. Detection limit test

[0098] Take blank pear, apple, orange, tangerine, cowpea, celery samples, add phorate to a final concentration of 0.005 mg / kg, 0.01 mg / kg, 0.02 mg / kg, respectively, take test strips for detection, and repeat the determination three times for each sample.

[0099] When the pear, apple, orange, tangerine, cowpea, celery samples are detected by the test strip, when there is no phorate and the added concentration of phorate is 0.005 mg / kg, the T line on the test strip shows color higher than or equal to the color of the C line, which is negative; when the added concentration of phorate is 0.01 mg / kg, 0.02 mg / kg, the T line on the test strip shows color lower than the color of the C line or the T line does not show color, which is positive, indicating that the detection limit of the test strip for phorate in fruits and vegetables is 0.01 mg / kg, which meets the maximum residue limit specified in the national standard GB 2763.

[0100] 2. False positive rate and false negative rate test

[0101] Take 20 blank pear, apple, orange, tangerine, cowpea, celery samples and 20 positive pear, apple, orange, tangerine, cowpea, celery samples added with phorate to a final concentration of 0.01 mg / kg, and detect them with 3 batches of test strips, respectively, to calculate the positive and negative rates.

[0102] The results show that when the positive pear, apple, orange, tangerine, cowpea, celery samples are detected by the test strips of 3 batches, the results are all positive, indicating that the positive coincidence rate is 100% and the false negative rate is 0; when the blank pear, apple, orange, tangerine, cowpea, celery samples are detected, the results are all negative, indicating that the negative coincidence rate is 100% and the false positive rate is 0. It shows that the test strip of the present application can rapidly detect phorate residues in fruits and vegetables.

[0103] 3. Specificity test

[0104] When the test strip is used to detect 0.02 mg / kg phorate sulfoxide and phorate sulfoxide, the T line on the test strip shows positive color lower than the C line or no color, indicating that the test strip can detect phorate metabolites (phorate sulfoxide, phorate sulfoxide); when detecting 10 mg / kg glyphosate, chlorpyrifos, acephate, phenthoate, methyl isopropyl lysine, parathion, methyl parathion, fenthion and other similar substances, the T line on the test strip shows negative color deeper than the C line, indicating that the test strip has less cross reaction to these structural analogs and good specificity.

Claims

1. A test strip for detecting phorate and its metabolites, comprising a test paper and a microporous reagent, the test paper comprising a reaction membrane, a sample absorption pad, a water absorption pad and a base plate, the reaction membrane having a detection line coated with a phorate hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody, and the microporous reagent having a phorate monoclonal antibody-colloidal gold label lyophilized thereon; the phorate monoclonal antibody is prepared by using the phorate hapten-carrier protein conjugate as an immunogen; the phorate hapten-carrier protein conjugate is obtained by coupling a phorate hapten with a carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin or human serum albumin; characterized in that The synthesis method of the phorate hapten is as follows: 2.5 g of a compound having the structure shown in formula (I) is dissolved in 150 mL of pyridine, 2.31 g of N-(2-aminopropyl)maleimide is added, and the mixture is stirred thoroughly, a reflux condenser is installed, and the mixture is reacted at 80°C for 4 h, the reaction is stopped, and the organic solvent is removed by rotary evaporation, and the product is separated by silica gel column chromatography using a mixture of dichloromethane and methanol (volume ratio 10:1) as eluent to obtain the phorate hapten having the structure shown in formula (II): ； （Ⅰ） （Ⅱ） The phorate metabolite is phorate sulfoxide and phorate sulfone.

2. The test strip of claim 1, wherein The test paper is composed of a sample absorption pad, a reaction membrane, and a water absorption pad which are sequentially pasted on a base plate, and the microporous reagent has a microporous plug.

3. A method for preparing the test paper strip according to any one of claims 1-2, comprising the steps of: 1) preparing a microporous reagent with phorate monoclonal antibody-gold label by freeze-drying; 2) preparing a reaction membrane with a detection line coated with a phorate hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody; 3) assembling the reaction membrane prepared in 2), a sample absorption pad, a water absorption pad, and a base plate into a test paper; 4) assembling the microporous reagent with phorate monoclonal antibody-gold label prepared in 1) and the test paper prepared in 3) into a test paper strip.

4. A method for detecting phorate residues in fruits and vegetables, comprising the steps of: 1) sample pretreatment; 2) detection using the test paper strip according to any one of claims 1-2; 3) analysis of the detection results.

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