CN112067814B - Test strip and method for detecting difenoconazole - Google Patents

Abstract

The invention discloses a test strip and a method for detecting difenoconazole. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a difenoconazole monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting difenoconazole in the fruit and vegetable samples by using the test strip. The test strip provided by the invention has the advantages of simplicity in operation, high sensitivity, high detection speed, low cost, suitability for large-batch sample screening and the like, and can meet the requirements of the food supervision departments in China for on-site monitoring and detection.

Description

Test strip and method for detecting difenoconazole

Technical Field

The invention relates to a test strip and a method for detecting difenoconazole, in particular to a colloidal gold test strip for detecting difenoconazole, which is particularly suitable for detecting difenoconazole residues in fruits and vegetables.

Background

Difenoconazole (Difenoconazole), also known as Difenoconazole, is a broad-spectrum efficient triazole systemic fungicide, belongs to a 14 alpha-sterol demethylation inhibitor, can interfere hypha growth, inhibits pathogen spore germination, and finally inhibits fungal growth. The difenoconazole has the protection and treatment effects, is widely applied to crops such as grains, oil, fruits, vegetables, peanuts, beet, tea, ginseng and the like to control plant diseases and insect pests, is mainly used for controlling gray rot, downy mildew and powdery mildew, and can also inhibit strawberry and pepper anthracnose. However, as the difenoconazole is directly sprayed on crops, people are easily damaged by the difenoconazole, and the residue limit of difenoconazole in different foods is regulated in national standard GB 2763-2019 of maximum residue limit of pesticides in food safety national standard foods.

The reported methods for detecting difenoconazole mainly comprise a gas chromatography method, a gas chromatography-mass spectrometry method, a high performance liquid chromatography method and other instrument methods. The methods are operated under laboratory conditions, the pretreatment of the samples is tedious and time-consuming, expensive instruments and equipment are needed to be equipped, the detection cost is high, the time consumption is long, the operation is complex, the method has great limitation in the practical application process, and the method is difficult to meet the requirements of rapid detection of a large number of samples and on-site samples. Therefore, the colloidal gold test strip which is simple and rapid and is suitable for the difenoconazole residue in fruits and vegetables is developed, a large number of samples can be screened and monitored on site, and the detection work of food supervision departments in China and the like can be better met.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip capable of detecting difenoconazole residues in fruits and vegetables, and provides a detection method which is efficient, accurate, simple and convenient and suitable for on-site monitoring and screening of a large number of samples.

The test strip for detecting difenoconazole provided by the invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody; the difenoconazole monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad.

The difenoconazole monoclonal antibody is prepared by taking a difenoconazole hapten-carrier protein conjugate as an immunogen.

The difenoconazole hapten-carrier protein conjugate is obtained by coupling difenoconazole hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, the difenoconazole hapten is obtained by brominating difenoconazole impurity 1 serving as a starting raw material to obtain intermediate bromo-compound and then carrying out nucleophilic substitution reaction with aminopropionic acid, and the molecular structural formula is as follows:

the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, and 1/3-1/2 of the conjugate releasing pad is covered under the sample absorbing pad.

The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.

Another object of the present invention is to provide a method for preparing the above test strip, comprising the steps of:

1) Preparing a conjugate release pad sprayed with a difenoconazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction film with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.

Specifically, the method comprises the following steps:

1) The difenoconazole impurity 1 is taken as a starting material, an intermediate bromo-compound is obtained through bromination reaction, and nucleophilic substitution reaction is carried out with aminopropionic acid to prepare the difenoconazole hapten;

2) Coupling the difenoconazole hapten with carrier protein to prepare a difenoconazole hapten-carrier protein conjugate;

3) Immunizing a mouse by using a difenoconazole hapten-carrier protein conjugate, and fusing and screening spleen cells of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting a difenoconazole monoclonal antibody;

4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;

5) Coating difenoconazole hapten-carrier protein conjugate and goat anti-mouse antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;

6) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;

7) Adding the prepared difenoconazole monoclonal antibody into the prepared colloidal gold to obtain a difenoconazole monoclonal antibody-colloidal gold marker;

8) Spraying a difenoconazole monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 1h, taking out, and storing in a dry environment for standby;

9) Soaking the sample absorption pad in phosphate buffer solution containing 0.5% bovine serum albumin and having pH of 7.2 and 0.1mol/L for 2h, and drying at 37 ℃ for 2h;

10 A sample absorbing pad, a conjugate releasing pad, a reaction membrane, and a water absorbing pad are sequentially stuck on the bottom plate, and 1/3 area of the conjugate releasing pad from the initial end is covered by the sample absorbing pad. Finally cutting into small strips with the width of 3mm, adding a plastic box, vacuum packaging, and preserving for 12 months at the temperature of 4-30 ℃.

The invention also provides a method for detecting difenoconazole residues in fruits and vegetables by using the test strip, which comprises the following steps:

(1) Sample pretreatment;

(2) Detecting by using a test strip;

(3) And analyzing the detection result.

The difenoconazole rapid detection test strip adopts a highly specific antibody antigen reaction and immunochromatographic analysis technology, a difenoconazole monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and difenoconazole in a sample is combined with the difenoconazole monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a medicine-antibody-colloidal gold marker. The medicine in the sample competes with the difenoconazole hapten-carrier protein conjugate on the reaction film detection line for combining with the difenoconazole monoclonal antibody-colloidal gold marker, and whether the difenoconazole residue is contained in the sample liquid to be detected is judged according to the red stripe depth of the detection line.

During detection, after the sample is treated, dripping into a test strip clamping hole, when the concentration of difenoconazole in the sample is lower than a detection limit or zero, combining a monoclonal antibody-colloidal gold marker with a difenoconazole hapten-carrier protein conjugate fixed on a reaction membrane in a chromatography process, wherein a red strip appears on a detection line (T) and a quality control line (C), and the color development of the T line is deeper than that of the C line or consistent with that of the C line; if the concentration of difenoconazole in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold marker can be fully combined with the difenoconazole, so that red stripes are not generated or the color development is lighter than that of C line at the T line because of the competition reaction and the difenoconazole hapten-carrier protein conjugate can not be combined. As shown in fig. 3.

Negative: and when the quality control line (C) shows red stripes, the detection line (T) also shows red stripes, and the color of the (T) line is close to or deeper than that of the (C), the judgment is negative.

Positive: and judging positive when the quality control line (C) shows red stripes and the detection line (T) does not develop color or the color of the detection line (T) is lighter than that of the detection line (C).

Invalidation: when the quality control line (C) does not display red stripes, whether the detection line (T) displays red stripes or not, the test strip is judged to be invalid.

The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long shelf life. The method for detecting difenoconazole residue by using the test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

FIG. 1 is a diagram showing the synthesis of difenoconazole hapten.

Fig. 2 is a schematic diagram of a cross-sectional structure of the test strip.

Fig. 3 is a test strip detection result judgment chart.

Detailed Description

The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.

Example 1 preparation of test strip for detecting difenoconazole

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

1) Preparing a conjugate release pad sprayed with a difenoconazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction film with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.

The following is a stepwise detailed description:

1. synthesis of difenoconazole hapten (synthetic route see figure 1)

1) Taking 4.2g of difenoconazole impurity 1, adding 100mL of 1, 2-dichloroethane for dissolution, adding 0.12g of p-toluenesulfonic acid, fully stirring, dropwise adding 1.61g of liquid bromine at room temperature, dropwise adding in batches, firstly dropwise adding half of the liquid bromine, dropwise adding the other half of the liquid bromine after the color becomes light, and continuing to react for 2 hours; after the reaction is finished, adding 100mL of saturated saline water, sufficiently oscillating, standing, separating out the water phase, adding 100mL of water, oscillating, standing, separating out the water phase, concentrating and evaporating the organic phase to dryness to obtain an intermediate 1;

2) Dissolving all intermediate 1 in 100mL of N, N-dimethylformamide, adding 2.14g of KOH, fully stirring, adding 0.47g of anhydrous sodium iodide, dropwise adding 2mL of aqueous solution containing 1.8g of aminopropionic acid, heating in an oil bath, and reacting at 90 ℃ for 6 hours; after the reaction is finished, adding 200mL of water, adding 6mol/L of dilute hydrochloric acid to adjust the pH to 7, extracting with 300mL of ethyl acetate, oscillating, standing, and separating out the water phase; adding 100mL of saturated saline, washing, standing, separating the water phase, concentrating the organic phase, loading on a silica gel column, eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1, and obtaining the difenoconazole hapten.

2. Preparation of immunogens

Taking 19mg of difenoconazole hapten, adding 1mL of N, N-Dimethylformamide (DMF) for dissolution, adding 13.1mg of N-hydroxysuccinimide (NHS) and 27mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), and adding 0.05mol/LPB buffer solution for dissolution to obtain solution B; and (3) dropwise adding the solution A into the solution B, reacting for 12 hours at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/LPBS, changing the solution 3 times a day to obtain the difenoconazole hapten-BSA conjugate, namely the immunogen, sub-packaging and preserving at-20 ℃.

3. Preparation of coating Material

Taking 12mg of difenoconazole hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolution, adding 11mg of 1-Hydroxybenzotriazole (HOBT) and 11mg of EDC, and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of Ovalbumin (OVA), and adding 0.05mol/L PB buffer solution for dissolution to obtain solution B; and (3) dropwise adding the solution A into the solution B, reacting for 12 hours at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times a day to obtain the difenoconazole hapten-OVA conjugate, namely, the difenoconazole hapten-OVA conjugate is taken as a coating source, and subpackaging and preserving at-20 ℃.

4. Preparation of difenoconazole monoclonal antibody

(1) Immunization of animals

The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.

(2) Cell fusion and cloning

Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.

(3) Cell cryopreservation and resuscitation

The hybridoma cells were prepared into 1X 10 by using a frozen stock solution ⁶ Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.

(4) Preparation and purification of monoclonal antibodies

Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.

The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, wherein the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.

5. Preparation of goat anti-mouse antibody

Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.

6. Preparation of difenoconazole monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5mL1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering to original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight.

(2) Preparation of difenoconazole monoclonal antibody-colloidal gold marker

Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution, adding the difenoconazole monoclonal antibody into the colloidal gold solution according to the standard of adding 20-50 mug antibody into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30min; after 10min of standing, 10% BSA was added to give a final concentration of 1% in the colloidal gold solution, and the mixture was left for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.

Reconstitution buffer: 0.1 to 0.3 percent of BSA, 0.05 to 0.2 percent of Tween-80 and 0.02mol/L of phosphate buffer solution with pH value of 7.2.

7. Preparation of conjugate release pads

The conjugate release pad was soaked in phosphate buffer containing 0.5% BSA, pH 7.2, 0.5mol/L, soaked uniformly for 1h, and baked at 37℃for 3 h. And uniformly spraying the prepared difenoconazole monoclonal antibody-colloidal gold marker on a conjugate release pad by using an isolow film spraying instrument, spraying 0.01mL of difenoconazole monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.

8. Preparation of sample absorbent pad

The sample absorption pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin with pH of 7.2 and 0.1mol/L for soaking for 2 hours, and is dried at 37 ℃ for 2 hours for standby.

9. Preparation of reaction film

The difenoconazole hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse anti-antibody is coated on the reaction membrane to form a quality control line.

The coating process comprises the following steps: diluting the difenoconazole hapten-ovalbumin conjugate to 1mg/mL by using a phosphate buffer solution, and coating the difenoconazole hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an isolow spot membrane tester, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/mL with 0.01mol/L, pH 7.4.7.4 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an Isoflow spot film instrument in an amount of 1.0. Mu.L/cm. And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.

10. Assembly of test strips

According to the cross-section structure of the test strip shown in figure 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially stuck on a PVC bottom plate (7); the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3mm by a machine, putting the test paper strip into a special plastic card, and storing the test paper strip in an environment of 4-30 ℃ for 12 months.

Example 2 detection of difenoconazole in fruits and vegetables

1. Sample pretreatment

Wiping off soil from a fresh sample, and shearing the fresh sample into fragments smaller than 1cm square; weighing (2.00+/-0.05) g of a sample into a 15mL centrifuge tube, adding 6mL of phosphate buffer solution, covering a cover, manually oscillating for 30s, standing for 1min, and taking supernatant as a sample liquid to be detected.

2. Detection by test strips

Sucking 70 mu L of sample liquid to be detected by a micropipette and vertically dripping the 70 mu L of sample liquid into a sample adding hole; the flow of the liquid was started to time, the reaction was continued for 10 minutes, and the result was judged.

3. Analyzing the detection result

Negative (-). The color development of the T line is deeper than or consistent with that of the C line, which indicates that the concentration of difenoconazole in the sample is lower than the detection limit, as shown in figures 3a and 3b.

Positive (+): the color development of the T line is lighter than that of the C line or the T line does not develop, which indicates that the concentration of difenoconazole in the sample is equal to or higher than the detection limit, as shown in figures 3C and 3d.

Invalidation: the absence of line C indicates an incorrect procedure or that the test strip has failed due to deterioration, as shown in fig. 3e, 3f.

Example 3 sample detection example

1. Limit of detection test

Taking blank onion, tomato, kidney bean, grape, pear and apple samples, respectively adding difenoconazole to the samples with final concentration of 0.25mg/kg, 0.5mg/kg and 1.0mg/kg, taking test strips for detection, and repeatedly measuring each sample for three times.

When the test strip is used for detecting samples of onion, tomato, kidney bean, grape, pear and apple, when difenoconazole is not contained in the samples and the adding concentration of the difenoconazole is 0.25mg/kg, the test strip shows that the color development of the T line is deeper than or consistent with that of the C line, and the test strip is negative; when the adding concentration of the difenoconazole is 0.5mg/kg and 1.0mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the color development of the T line is not positive, which indicates that the detection limit of the test strip to the difenoconazole in fruits and vegetables is 0.5mg/kg.

2. False positive rate and false negative rate test

Taking blank samples of green onion, tomatoes, beans, grapes, pears and apples, adding 20 parts of positive samples of green onion, tomatoes, beans, grapes, pears and apples with the final concentration of 0.5mg/kg, respectively detecting the positive samples by using test strips produced in 3 batches, and calculating the negative positive rate.

The results show that: when positive samples are detected by using test strips produced in 3 batches, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when a negative sample was detected, the result was all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting difenoconazole can be used for rapidly detecting difenoconazole residues in fruit and vegetable samples.

3. Specificity test

When the test strip is used for detecting 10mg/kg of myclobutanil, triadimenol, tebuconazole, bistriadimenol, triadimefon, diniconazole, epoxiconazole, penconazole, flusilazole, flutriafol, propiconazole and other triazole pesticides, the test strip shows that the color development of the T line is deeper than or consistent with that of the C line, and is negative, so that the test strip has no cross reaction to the drugs.

Claims (5)

1. The test strip for detecting difenoconazole comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a difenoconazole monoclonal antibody-colloidal gold marker; the difenoconazole monoclonal antibody is prepared by taking a difenoconazole hapten-carrier protein conjugate as an immunogen; the difenoconazole hapten-carrier protein conjugate is obtained by coupling difenoconazole hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin;

the synthesis method of the difenoconazole hapten is characterized by comprising the following steps of: 1) Taking 4.2g of difenoconazole impurity 1, adding 100ml of 1, 2-dichloroethane for dissolution, adding 0.12g of p-toluenesulfonic acid, fully stirring, dropwise adding 1.61g of liquid bromine at room temperature, dropwise adding in batches, firstly dropwise adding half of the liquid bromine, dropwise adding the other half of the liquid bromine after the color becomes light, and continuing to react for 2 hours; after the reaction is finished, adding 100mL of saturated saline water, sufficiently oscillating, standing, separating out the water phase, adding 100mL of water, oscillating, standing, separating out the water phase, concentrating and evaporating the organic phase to dryness to obtain an intermediate 1; 2) Dissolving all intermediate 1 in 100mL of N, N-dimethylformamide, adding 2.14g of KOH, fully stirring, adding 0.47g of anhydrous sodium iodide, dropwise adding 2mL of aqueous solution containing 1.8g of aminopropionic acid, heating in an oil bath, and reacting at 90 ℃ for 6 hours; after the reaction is finished, adding 200mL of water, adding 6mol/L of dilute hydrochloric acid to adjust the pH to 7, extracting with 300mL of ethyl acetate, oscillating, standing, and separating out the water phase; adding 100mL of saturated saline solution, washing, standing, separating the water phase, concentrating the organic phase, loading on a silica gel column, eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1 to obtain the difenoconazole hapten, wherein the molecular structural formula is as follows:

2. the test strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane, and the absorbent pad are sequentially attached to the base plate.

3. The test strip of any one of claims 1-2, wherein the conjugate release pad is 1/3 to 1/2 coated under the sample absorbent pad.

4. A method of preparing the test strip of any one of claims 1-3, comprising the steps of:

1) Preparing a conjugate release pad sprayed with a difenoconazole monoclonal antibody-colloidal gold marker;

2) Preparing a reaction film with a detection line coated with a difenoconazole hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse antibody;

3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.

5. A method for detecting difenoconazole residues in fruit and vegetable samples comprises the following steps:

1) Sample pretreatment;

2) Detecting with the test strip of any one of claims 1-3;

3) And analyzing the detection result.

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