CN109061145B - Test strip for detecting flumetralin, preparation method and application thereof - Google Patents

Abstract

A test paper strip for detecting flumetralin and a preparation method and application thereof, the test paper strip 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 flumetralin 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 flumetralin monoclonal antibody-colloidal gold marker, the flumetralin hapten is 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid generated by the reaction of 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid, and then reacting with iodoethane. The invention also provides a preparation method of the flumetralin test strip and a method for detecting the flumetralin residue in tobacco by using the test strip. The advantages are that: the method has the advantages of simple operation, high sensitivity, high detection speed and low cost, and is suitable for screening and field monitoring of a large number of samples.

Description

Test strip for detecting flumetralin, preparation method and application thereof

Technical Field

The invention belongs to the field of colloidal gold test strips, and particularly relates to a test strip for detecting flumetralin, a preparation method and application thereof, which are particularly suitable for detecting the residual flumetralin in tobacco.

Background

Flumetralin is a dinitroaniline plant growth regulator which is contact and local systemic and is used for inhibiting lateral buds of tobacco, and is an excellent tobacco bud inhibitor. Was successfully developed in 1977 by Ciba-Geigy, Switzerland. In 1990, Prime (Prime) is officially registered in China as a trade name with a registration number of PDll 6-90. It is a new type of highly effective sprout inhibitor which is popular internationally, and is suitable for flue-cured tobacco, open fire flue-cured tobacco, Maryland tobacco, sun-cured tobacco and cigar. The national Zhejiang chemical research institute is researched and developed at the earliest time and is successfully developed in 1998, and temporary registration of pesticides is obtained in 1999. The pesticide is applied once within 24 hours after the tobacco is artificially topped, and bud picking is not needed in the whole growing season. 60-70 mL of 25% flumetralin emulsifiable concentrate is used per mu, the effect is quick, the absorption is quick, the effect can be achieved as long as no rain exists after the pesticide is applied for 2 hours, and the pesticide application is convenient in rainy seasons. The contact of the medicament with the fully extended leaves does not cause phytotoxicity and does not contain harmful residues. The use of the flumetralin can save a great amount of artificial bud picking, lead the natural maturity to be consistent, increase the yield and improve the upper-middle proportion of the tobacco leaves and the internal quality of the tobacco leaves. In addition, the use of flumetralin can also reduce contact infection of mosaic disease in the field. Compared with other bud inhibitors, the flumetralin has high drug effect. The international cooperation center for tobacco science research (CORESTA) stipulates that the directive residual limit of the flunomide in the tobacco is 5 mg/kg, and the maximum residual limit of the flunomide in the food is not established in China.

At present, the detection method of the flumetralin is mainly an instrument detection method, such as gas chromatography, gas chromatography-mass spectrometry, gas chromatography tandem mass spectrometry and the like. However, these analysis methods require expensive large-scale instruments and equipment and professional detection personnel, and are complex in pretreatment process, complex in operation, high in detection cost and slow in analysis speed, so that the requirements of on-site monitoring and rapid screening of pesticide residues in a large number of samples are difficult to meet. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples. Until now, no test strip for flumetralin has appeared on the market.

Disclosure of Invention

The invention aims to provide the test strip for detecting the residual flumetralin, which has the advantages of high sensitivity, simple operation, low cost and short detection time.

The test strip for detecting the flumetralin 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 flumetralin 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 flumetralin monoclonal antibody-colloidal gold marker, the flumetralin monoclonal antibody is prepared by taking the flumetralin hapten-carrier protein conjugate as an immunogen, the flumetralin hapten-carrier protein conjugate is obtained by coupling flumetralin hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, and the flumetralin hapten is 3- (2-chloro-1, 3-dinitro-5-trifluoromethylbenzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid react to generate 3- (2-chloro-6-fluorophenyl) -3-fluorophenyl) - (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, and then reacting with iodoethane to obtain the compound with a molecular structural formula:

。

the preparation method of the fluvalinate hapten comprises the following steps:

1) taking 1.00 g of 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene, and adding 20 mL of absolute ethyl alcohol to dissolve the 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene to obtain solution A; dissolving 0.88 g of 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid in 10 mL of absolute ethanol, adding 1 mL of aqueous solution containing 0.37 g of sodium bicarbonate to obtain solution B, dropwise adding the solution A into the solution B, and reacting at room temperature for 3 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, carrying out rotary evaporation, removing ethanol, adding 80 mL of water for dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for shaking and layering, washing an organic phase by using water, carrying out rotary evaporation, applying to a silica gel column, and carrying out elution separation on n-hexane and ethyl acetate according to the volume ratio of 10:1 to obtain 1.53 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid;

2) taking 1.50 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, dissolving the intermediate in 50 mL of acetonitrile, adding 0.20 g of potassium hydroxide, adding 0.57 g of iodoethane, reacting for 4 h at 50 ℃, detecting, stopping the reaction when the raw materials completely react, removing the acetonitrile by rotary evaporation, adding 100 mL of water, dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate, extracting, washing and drying an organic phase, evaporating to dryness to obtain a yellow oily substance, and recrystallizing with a dichloromethane solution by using n-hexane and a dichloromethane solution in a volume ratio of 1:1 to obtain 1.51 g of a flumetralin hapten product.

The flumetralin monoclonal antibody is prepared by taking a flumetralin hapten-carrier protein conjugate as an immunogen and is obtained by secreting a flumetralin monoclonal antibody hybridoma cell strain; the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.

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 flumetralin monoclonal antibody-colloidal gold marker;

2) preparing a reaction membrane with a detection line coated with a flumetralin 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.

More specifically, the process for preparing the test strip is as follows:

1) hapten preparation: 2-chloro-1, 3-dinitro-5-trifluoromethyl benzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid are subjected to a series of reactions to obtain a flumetralin hapten product;

2) coupling the flumetralin hapten with carrier protein to obtain a flumetralin hapten-carrier protein conjugate;

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

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

5) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;

6) adding the flumetralin monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain a flumetralin monoclonal antibody-colloidal gold marker;

7) spraying the flumetralin 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;

8) coating the fluvalinate hapten-carrier protein 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;

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

10) and sequentially adhering a sample absorption pad, a conjugate release pad, a reaction film and a water absorption pad on the bottom plate, covering the conjugate release pad with the sample absorption pad, finally cutting into small strips with the width of 3 mm, 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 the residual flumetralin in the tobacco 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 invention relates to a rapid detection test strip for flumetralin, which adopts a highly specific antibody-antigen reaction and immunochromatography analysis technology to fix a flumetralin monoclonal antibody-colloidal gold marker on a conjugate release pad, wherein flumetralin in a sample is combined with the flumetralin monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the flumetralin-antibody-colloidal gold marker. And (3) the flumetralin in the sample and the flumetralin hapten-carrier protein conjugate on the reaction membrane detection line compete to combine with the flumetralin monoclonal antibody-colloidal gold marker, and whether the liquid sample to be detected contains the flumetralin residue or not is judged according to the condition that the red strip of the detection line has nothing or dark color.

The judgment of the detection result is shown in FIG. 2.

Negative: the color development ratio of the detection line (T) to the quality control line (C) is dark or consistent, which indicates that the concentration of the flumetralin in the sample is lower than the detection limit, and the result is judged to be negative.

Positive: the color development of the detection line (T) is lighter than that of the quality control line (C) or the detection line (T) does not develop color, which indicates that the concentration of the flumetralin in the sample is equal to or higher than the detection limit, and the sample 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.

At present, no test strip for detecting flumetralin in tobacco exists, and the invention fills the gap. The hapten has proper terminal active groups, the length of a modification site and a spacer arm is properly selected, and the molecular structure of the flumetralin can be simulated to the greatest extent. Meanwhile, the test strip has the advantages of low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period. In conclusion, the method for detecting the residual flumetralin 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 schematic diagram of a cross-sectional structure of a test strip, in which: 1. a sample absorbing pad; 2. a conjugate release pad; 3. a reaction film; 4. a water absorbent pad; 5. detecting lines; 6. a quality control line; 7. a base plate.

FIG. 2 is a diagram showing the test result of the test strip.

FIG. 3 shows the synthesis of flumetralin hapten (the figure is taken as the abstract figure).

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 Flumetraline test strip

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

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

2) preparing a reaction membrane with a detection line coated with a flumetralin 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 PVC base plate to form the test strip.

The following steps are detailed:

1. preparation of flumetralin hapten

Taking 1.00 g of 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene, and adding 20 mL of absolute ethyl alcohol to dissolve the 2-chloro-1, 3-dinitro-5-trifluoromethylbenzene to obtain solution A; dissolving 0.88 g of 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid in 10 mL of absolute ethanol, adding 1 mL of aqueous solution containing 0.37 g of sodium bicarbonate to obtain solution B, dropwise adding the solution A into the solution B, and reacting at room temperature for 3 h; TLC detection, the raw materials are basically completely reacted; stopping the reaction, carrying out rotary evaporation, removing ethanol, adding 80 mL of water for dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate for shaking and layering, washing an organic phase by using water, carrying out rotary evaporation, applying to a silica gel column, eluting and separating normal hexane and ethyl acetate according to the volume ratio of 10:1 to obtain 1.53 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid with the yield of 92.17%;

taking 1.50 g of an intermediate 3- (2-chloro-6-fluorophenyl) -3- (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, dissolving the intermediate in 50 mL of acetonitrile, adding 0.20 g of potassium hydroxide, adding 0.57 g of iodoethane, reacting for 4 hours at 50 ℃, detecting, stopping the reaction when the raw materials completely react, removing the acetonitrile by rotary evaporation, adding 100 mL of water, dissolving, adjusting the pH value to 6 by using 1 mol/L hydrochloric acid, adding 80 mL of ethyl acetate, extracting, washing and drying an organic phase, evaporating to dryness to obtain a yellow oily substance, and recrystallizing a dichloromethane solution by using n-hexane and a dichloromethane solution in a volume ratio of 1:1 to obtain 1.51 g of a flumetralin hapten product with the yield of 94.97%.

Nuclear magnetic identification of 1H NMR (CDCl)₃300 MHZ) δ:11.00 (1H, s), 7.141 (1H, dd, J =8.271, J =1.347), 7.373 (2H, dd, J =8.373, J =8.271), 4.23 (2H, dd, J =8.373, J =1.347), 2.880 (2H, d, J =6.843), 3.631 (2H, q, J =7.108), 1.238 (3H, t, J =7.108), 8.75 (2H, d, J =0.000), chemical shift δ =11.0 is the carboxyhydrogenresonance absorption peak on the spacer arm, δ =2.88 is the resonance absorption peak of methylenehydrogenon the spacer arm, the presence of these peaks, proving successful spacer arm coupling.

2. Preparation of immunogens

Dissolving 18 mg of flumetralin hapten in 0.3 mL of Dimethylformamide (DMF), clarifying, adding 8.6 mg of carbodiimide (EDC), stirring, clarifying, adding 5.2 mg of N-hydroxysuccinimide (NHS), stirring at room temperature, and activating for 3h to obtain solution A; taking 50 mg of Bovine Serum Albumin (BSA), adding 8mL of 0.05 mol/L phosphate buffer solution (PB) with the pH value of 7.2 to dissolve to obtain solution B, slowly dripping the solution A into the solution B, and stirring at room temperature for reaction for 5 hours. Stopping reaction, dialyzing with 0.02M Phosphate Buffer Solution (PBS) for 3 days, changing the solution three times per day to obtain flumetralin-BSA immunogen, subpackaging, and storing at-20 ℃ for later use.

3. Preparation of coating antigen

Dissolving 8 mg of flumetralin hapten in 0.2 mL of DMF, clarifying, adding 4.13 mg of Dicyclohexylcarbodiimide (DCC) and 2.3 mg of NHS, stirring at room temperature for 2h, filtering, and removing precipitates to obtain a hapten activation solution A; dissolving Ovalbumin (OVA) 50 mg in 8mL of 0.05 mol/L PB buffer solution with pH value of 7.2 to obtain solution B, slowly dropping solution A into solution B, and stirring at room temperature for reaction for 5 h. Stopping reaction, dialyzing with 0.02M PBS buffer solution for 3 days, changing the solution three times per day to obtain flumetralin-OVA coating antigen, subpackaging, and storing at-20 ℃ for later use.

4. Preparation of Flumetralin 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 an indirect competitive ELISA method, 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) Determination of the potency of monoclonal antibodies

The titer of the antibody is 1 (100000-400000) by using an indirect competition ELISA method.

Indirect competitive ELISA method: coating an enzyme label plate with a flumetralin hapten-OVA conjugate, adding a flumetralin standard solution, a flumetralin monoclonal antibody solution and a horse radish peroxidase labeled goat anti-mouse anti-antibody solution, reacting at 25 ℃ for 30 min, pouring out liquid in a hole, washing for 3-5 times with a washing solution, and patting dry with absorbent paper; adding a substrate color developing solution, reacting for 15 min at 25 ℃, and adding a stop solution to stop the reaction; the microplate reader was set to measure the absorbance value per well at a wavelength of 450 nm.

6) Determination of monoclonal antibody specificity

Antibody specificity refers to the comparison of its ability to bind to a specific antigen with the ability to bind to such antigen analogs, often using cross-reactivity as an evaluation criterion. The smaller the cross-reactivity, the higher the specificity of the antibody.

In the experiment, dinitroaniline herbicides (flumetralin, butralin, pendimethalin and trifluralin) are serially diluted, respectively subjected to indirect competitive ELISA with monoclonal antibodies, a standard curve is prepared, and IC is obtained through analysis₅₀Then, the cross-reactivity was calculated as follows:

the results show that the cross-reactivity rate of each analog is: 100 percent of flumetralin, less than 1 percent of butralin, less than 1 percent of pendimethalin and less than 1 percent of trifluralin. The antibody of the invention has no cross reaction to other dinitroaniline herbicides such as butralin, pendimethalin, trifluralin and the like, and only has specific binding to flumetralin.

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 flumetralin 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 100 mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5 mL of 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 the volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating material.

2) Preparation of flumetralin monoclonal antibody-colloidal gold marker

Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.0 by using 0.2 mol/L potassium carbonate solution, adding 20-50 mu g of flumetralin monoclonal antibody into the colloidal gold solution according to the standard of adding each milliliter of colloidal gold solution, continuously stirring and uniformly mixing for 30 min, adding 10% BSA (bovine serum albumin) to enable the final concentration of the BSA in the colloidal gold solution to be 1% (volume fraction), and standing for 10 min. Centrifuging at 12000 r/min at 4 deg.C for 40 min, 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: 0.02 mol/L phosphate buffer solution containing casein 0.02% -0.1% (mass fraction), tween-800.05% -0.2% (mass fraction) and pH value of 7.2.

7. Preparation of conjugate Release pad

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

8. Preparation of the reaction film

Coating the fluvalinate 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 fluvalinate hapten-ovalbumin conjugate to 10 mg/ml by using a phosphate buffer solution, and coating the conjugate on a detection line (T line) on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 0.8 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01 mol/L, pH value of 7.4 phosphate buffer and coated on a quality control line (C line) 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.

9. Preparation of sample absorbent pad

The sample absorption pad is placed in 0.5 percent bovine serum albumin (volume fraction), pH value is 7.2, and 0.1 mol/L phosphate buffer solution to be soaked for 2h, and the sample absorption pad is baked for 2h at 37 ℃ for standby.

10. Assembly of test strips

Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; 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 soleplate, and the tail end of the water absorption pad is aligned with the tail end of the PVC soleplate; the reaction membrane is provided with a detection line and a quality control line, 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; the test paper strip is cut into small strips with the width of 3 mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃.

EXAMPLE 2 detection of Flumetraline residue in samples

1. Pretreatment of samples

1.0 + -0.05 g of the pulverized tobacco leaf sample is weighed into a 50 mL centrifuge tube, 10 mL of 50% methanol aqueous solution is added, and the pulverized tobacco leaf sample is sufficiently pulverized by a homogenizer. The fragmented sample was transferred to a syringe and filtered using a filter. 100 μ L of the sample solution was diluted with 400 μ L of deionized water.

2. Detection with test strip

Taking 70 mu L (2-3 drops of a dropper) of the diluted sample liquid by using a pipette, and vertically dropping the sample liquid into the sample adding hole; timing when the liquid flows, reacting for 10 min, and interpreting the result.

3. Analyzing the results of the detection

Negative (-): the color development of the T line is darker than that of the C line or is consistent with that of the C line, indicating that the concentration of the flumetralin in the sample is lower than the detection limit, as shown in FIG. 2 (a).

Positive (+): the color development of the T line was lighter than that of the C line or the T line was not developed, indicating that the concentration of flumetralin in the sample was equal to or higher than the detection limit, as shown in FIG. 2 (b).

And (4) invalidation: the absence of a line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIG. 2 (C). In this case, the instructions should be read carefully again and retested with a new test strip.

Example 3 sample testing example

1. Limit of detection test

Taking a blank tobacco sample, respectively adding fluvalinamide to the final concentration of 2.5, 5 and 10 mg/kg, taking a test strip for detection, and repeatedly measuring each sample for three times.

When the test strip is used for detecting a tobacco sample, the detection limit is judged according to the display of the test strip, which shows that the detection limit of the test strip on the flumetralin in the tobacco is 5 mg/kg.

2. Test for false positive and false negative rates

Respectively taking 20 parts of tobacco positive samples with the known flumetralin content being more than the detection limit and 20 parts of negative samples with the known flumetralin content being less than the detection limit, detecting by using three batches of test strips, and calculating the negative and positive rates of the tobacco positive samples and the tobacco negative samples. The results are shown in the following table.

TABLE 1 false Positive and false negative test results

The results show that: when 3 test strips produced in batches are used for detecting positive tobacco samples respectively, the results are all positive, the coincidence rate of the positive samples is 100 percent, and the false negative rate is 0; when 20 negative tobacco samples are respectively 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 the flumetralin can be used for rapidly detecting the flumetralin residue in the tobacco.

3. Specificity test

The test paper of flumetralin is used for detecting 500 mu g/L dinitroaniline herbicides such as pendimethalin, butralin, trifluralin and the like. The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test paper strip has no cross reaction to dinitroaniline herbicides such as pendimethalin, butralin, trifluralin and the like with the concentration of 500 mu g/L, and has good specificity.

Claims (7)

1. The utility model provides a test paper strip of detection flumetralin, includes sample absorption pad, conjugate release pad, reaction film, water absorption pad and bottom plate, its characterized in that: the reaction membrane is provided with a detection line coated with a flumetralin 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 flumetralin monoclonal antibody-colloidal gold marker, the flumetralin monoclonal antibody is prepared by taking the flumetralin hapten-carrier protein conjugate as an immunogen, the flumetralin hapten-carrier protein conjugate is obtained by coupling flumetralin hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, and the flumetralin hapten is 3- (2-chloro-1, 3-dinitro-5-trifluoromethylbenzene and 3-amino-3- (2-chloro-6-fluorophenyl) -propionic acid react to generate 3- (2-chloro-6-fluorophenyl) -3-fluorophenyl) - (2, 6-dinitro-4-trifluoromethyl-phenylamino) -propionic acid, and then reacting with iodoethane to obtain the compound with a molecular structural formula:

。

2. the test strip of claim 1, wherein: the preparation reaction process of the fluvalinate hapten is as follows:

。

3. the test strip of claim 1, wherein: the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially adhered to the bottom plate.

4. The test strip of claim 1 or 3, wherein: the conjugate release pads 1/3-1/2 are covered under the sample absorbing pad.

5. The test strip of claim 1, wherein: the goat anti-mouse anti-antibody is obtained by immunizing a goat with a murine antibody.

6. A method for preparing the test strip of any one of claims 1-5, wherein: the method comprises the following steps:

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

2) preparing a reaction membrane with a detection line coated with a flumetralin 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.

7. A method for detecting flumetralin in tobacco by using the test strip of any one of claims 1 to 5, which is characterized in that: the method comprises the following steps:

1) sample pretreatment;

2) detecting with the test strip;

3) and analyzing the detection result.

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