CN112067814A - 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 a fruit and vegetable sample by applying the test strip. The test strip provided by the invention has the advantages of simple operation, high sensitivity, high detection speed, low cost, suitability for screening large-batch samples and the like, and can meet the requirements of food supervision departments in China 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 residue in fruits and vegetables.

Background

Difenoconazole (Difenoconazole), also known as Difenoconazole, is a broad-spectrum and efficient triazole systemic fungicide, belongs to a 14 alpha-sterol demethylation inhibitor, can interfere hypha growth, inhibit pathogen spore germination and finally inhibit fungal growth. The difenoconazole has protection and treatment effects, is widely applied to crops such as grains, oil plants, fruits, vegetables, peanuts, sugar beets, tea leaves, ginseng and the like to control plant diseases and insect pests, is mainly used for preventing and treating 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 harmed by the difenoconazole, so that the residual limit of the difenoconazole in different foods is specified in the national standard GB 2763-2019 'maximum limit of pesticide in foods of national standard for food safety'.

The currently reported methods for detecting difenoconazole mainly comprise instrument methods such as gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography and the like. The methods are operated under laboratory conditions, the sample pretreatment is complicated and time-consuming, expensive instruments and equipment are required, 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 requirement for rapidly detecting a large number of samples and field samples is difficult to meet. Therefore, the colloidal gold test strip which is simple and quick 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 and the like in China can be better met.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip capable of detecting difenoconazole residue in fruits and vegetables, and provides a detection method which is efficient, accurate, simple and convenient, and suitable for field monitoring and large-scale sample screening.

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 anti-antibody; 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, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the difenoconazole hapten is obtained by taking difenoconazole impurity 1 as an initial raw material, obtaining an intermediate bromide through bromination reaction, and then carrying out nucleophilic substitution reaction with aminopropionic acid, and the molecular structural formula of the difenoconazole hapten-carrier protein conjugate is as follows:

the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.

The bottom plate can be a PVC bottom plate or other hard non-absorbent 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 absorption pad is absorbent paper; the reaction membrane can be a nitrocellulose membrane or a cellulose acetate membrane.

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

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

2) preparing a reaction membrane 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;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

Specifically, the steps include:

1) carrying out bromination reaction on the difenoconazole impurity 1 serving as an initial raw material to obtain an intermediate bromide, and carrying out nucleophilic substitution reaction on the intermediate bromide and the aminopropionic acid to prepare the difenoconazole hapten;

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

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

4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;

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

6) preparing colloidal gold by reacting trisodium citrate with 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 later use;

9) soaking the sample absorption pad in 0.5% bovine serum albumin-containing phosphate buffer solution with pH of 7.2 and 0.1mol/L for 2h, and drying at 37 deg.C for 2 h;

10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. And finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.

The invention also aims to provide a method for detecting difenoconazole residue in fruits and vegetables by using the test strip, which comprises the following steps:

(1) pretreating a sample;

(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 immunochromatography 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 drug-antibody-colloidal gold marker. Drugs in the sample compete with the difenoconazole hapten-carrier protein conjugate on the reaction membrane detection line to combine with the difenoconazole monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains difenoconazole residue is judged according to the depth of a red strip of the detection line.

During detection, a sample is treated and then dropped into a test strip card hole, when the concentration of the difenoconazole in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with the difenoconazole hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, 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 is consistent with that of the C line; if the concentration of difenoconazole in the sample is equal to or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the difenoconazole and no red band will appear or the color will be lighter than the C-line at the T-line because the competition reaction will not bind to the difenoconazole hapten-carrier protein conjugate. As shown in fig. 3.

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

Positive: and when the quality control line (C) shows a red strip, the detection line (T) does not show color or the color of the line (T) is lighter than that of the line (C), the test line is judged to be positive.

And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.

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 quality guarantee period. The method for detecting the difenoconazole residue by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

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

FIG. 2 is a schematic cross-sectional view of a test strip.

FIG. 3 is a diagram showing the test result of the test strip.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present 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 membrane 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;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

The following steps are detailed:

1. synthesis of difenoconazole hapten (the synthetic route is shown in figure 1)

1) Dissolving 4.2g of difenoconazole impurity 1 in 100mL of 1, 2-dichloroethane, 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, dropwise adding the other half after the color becomes light, and continuously reacting for 2 hours; after the reaction is finished, adding 100mL of saturated saline solution, fully shaking, standing, separating out the water phase, adding 100mL of water again, shaking, standing, separating out the water phase, concentrating and evaporating the organic phase to dryness to obtain an intermediate 1;

2) dissolving all the 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 an 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 value to 7, extracting with 300mL of ethyl acetate, oscillating, standing, and separating a water phase; adding 100mL of saturated saline solution, washing, standing, separating out a water phase, concentrating an organic phase, applying to a silica gel column, and eluting and separating by using a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1 to obtain the difenoconazole hapten.

2. Preparation of immunogens

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

3. Preparation of coating antigen

Taking 12mg of difenoconazole hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving, adding 11mg of 1-Hydroxybenzotriazole (HOBT) and 11mg of EDC, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving Ovalbumin (OVA)50mg in PB buffer solution 0.05mol/L to obtain solution B; dripping the A solution into the B solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times every day to obtain the difenoconazole hapten-OVA conjugate which is the coating antigen, subpackaging and storing at-20 ℃.

4. Preparation of difenoconazole monoclonal antibody

(1) Animal immunization

Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.

(2) Cell fusion and cloning

Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.

(3) Cell cryopreservation and recovery

Making hybridoma cell into 1 × 10 with frozen stock solution⁶Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.

(4) Preparation and purification of monoclonal antibodies

An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.

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

5. Preparation of goat anti-mouse anti-antibody

The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.

6. Preparation of difenoconazole monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid to 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 2.5mL 1% trisodium citrate under continuous stirring at high temperature, stirring at constant speed, heating until the solution is bright red, cooling to room temperature, recovering to original volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, is transparent and bright, has no sediment or floating objects, and is wine red 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 that 20-50 mu g of antibody is added into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30 min; after standing for 10min, 10% BSA was added to make the final concentration of the solution in colloidal gold 1%, and the solution was allowed to stand for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.

Redissolving buffer solution: 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 of 7.2.

7. Preparation of conjugate Release pad

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

8. Preparation of sample absorbent pad

The sample absorption pad is soaked in 0.5 percent bovine serum albumin-containing phosphate buffer solution with the pH value of 7.2 and the mol/L of 0.1 for 2 hours, and is dried for 2 hours at the temperature of 37 ℃ for standby.

9. Preparation of the reaction film

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

Coating process: 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 Isoflow point membrane instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01mol/L, pH 7.4.4 phosphate buffer and coated on a quality control line (line C) on a nitrocellulose membrane in an amount of 1.0. mu.L/cm using an Isoflow dot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.

10. Assembly of test strips

According to the section structure of the test strip shown in the attached figure 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially adhered to a PVC bottom plate (7); the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC base plate; the reaction membrane 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 vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; cutting the test strip into small strips with the width of 3mm by a machine, putting the small strips into a special plastic card, and storing the small strips in an environment with the temperature of 4-30 ℃ for 12 months.

Example 2 detection of Difenoconazole in fruits and vegetables

1. Sample pretreatment

Taking a fresh sample, wiping off soil, and cutting the fresh sample into fragments with the square length of less than 1 cm; weighing (2.00 +/-0.05) g of 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 sample liquid to be detected.

2. Detection with test strips

Sucking 70 mu L of sample liquid to be detected by a micropipette and vertically dripping the sample liquid into the sample adding hole; the liquid flow was started, the reaction was carried out for 10min, and the results were judged.

3. Analyzing the results of the detection

Negative (-): the color development of the T line is darker than that of the C line 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 3 b.

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

And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIGS. 3e and 3 f.

Example 3 sample testing example

1. Limit of detection test

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

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

2. Test for false positive and false negative rates

Taking blank onion, tomato, kidney bean, grape, pear and apple samples and 20 parts of positive onion, tomato, kidney bean, grape, pear and apple samples which are added with difenoconazole to the final concentration of 0.5mg/kg respectively, detecting by using test strips produced in 3 batches respectively, and calculating the negative and positive rates.

The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The test strip for detecting difenoconazole can be used for rapidly detecting the difenoconazole residue in the fruit and vegetable sample.

3. Specificity test

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

Claims (5)

1. A 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, thyroid protein or human serum albumin, and is characterized in that the difenoconazole hapten is obtained by taking difenoconazole impurity 1 as an initial raw material, performing bromination reaction to obtain an intermediate bromide, and performing nucleophilic substitution reaction with aminopropionic acid, wherein the molecular structural formula of the difenoconazole hapten-carrier protein conjugate is as follows:

2. the 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 strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.

4. A method of making 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 membrane 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;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

5. A method for detecting difenoconazole residue in a fruit and vegetable sample comprises the following steps:

1) pretreating a sample;

2) performing a test using the test strip of any one of claims 1-3;

3) and analyzing the detection result.

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