CN113109559A - Immunochromatography gold-labeled test strip suitable for trifloxystrobin pesticide and rapid test method thereof - Google Patents
Immunochromatography gold-labeled test strip suitable for trifloxystrobin pesticide and rapid test method thereof Download PDFInfo
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- 239000005857 Trifloxystrobin Substances 0.000 title claims abstract description 106
- ONCZDRURRATYFI-TVJDWZFNSA-N trifloxystrobin Chemical compound CO\N=C(\C(=O)OC)C1=CC=CC=C1CO\N=C(/C)C1=CC=CC(C(F)(F)F)=C1 ONCZDRURRATYFI-TVJDWZFNSA-N 0.000 title claims abstract description 105
- 238000012360 testing method Methods 0.000 title claims abstract description 66
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 239000000575 pesticide Substances 0.000 title claims abstract description 41
- 238000010998 test method Methods 0.000 title claims abstract description 8
- 238000003317 immunochromatography Methods 0.000 title claims description 5
- 238000001514 detection method Methods 0.000 claims abstract description 82
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1826—Water organic contamination in water
- G01N2033/184—Water organic contamination in water herbicides, pesticides, fungicides, insecticides, or the like
Abstract
The invention discloses an immunochromatographic gold-labeled rapid test strip suitable for trifloxystrobin pesticide and a rapid test method thereof. The gold-labeled antibody combination pads are respectively coated with a colloidal gold-labeled trifloxystrobin specific antibody, and the nitrocellulose membrane is coated with a trifloxystrobin artificial antigen detection line and a goat anti-rabbit IgG quality control line for indirect competition. The invention also provides a corresponding rapid test method: homogenizing or cutting a strawberry sample, weighing 5g of a sample to be detected into 5mL of sample diluent, adding a matched grinding bead, fully shaking and uniformly mixing for 2min, standing for 3min after extraction, taking supernate, dropwise adding 2-3 drops of the supernate into a sample application area of a sample pad for chromatographic reaction, and directly judging the positive and negative results of trifloxystrobin pesticide in the strawberry sample according to the color development conditions of a detection line and a quality control line after 5-10 min.
Description
Technical Field
The invention designs a high-specificity rapid test strip aiming at trifloxystrobin pesticide based on competitive gold-labeled immunochromatography and the test strip, and the rapid test strip is suitable for rapid detection of the trifloxystrobin pesticide.
Background
The trifloxystrobin is a modern pesticide, belongs to a strobilurin fungicide, has a rapid and specific fungicidal function, and has a specific action mode. The trifloxystrobin has much lower toxicity to human beings and environment than the traditional antifungal pesticide, is safe and efficient, and is widely applied to various crops for resisting fungal diseases, such as grains, strawberries, oranges, grapes, tomatoes, cucumbers and the like.
In 2003, trifloxystrobin was approved by the european union as a low risk plant protection active. In europe and the united states, the Maximum Residual Limit (MRL) of trifloxystrobin ranges from 0.05 to 30 ppm. According to the 2010 european union food pesticide residue report (european food safety agency), trifloxystrobin bactericide is common in food sample analysis, with about 2% to 5% of samples having residues, which are generally equal to or lower than the maximum residual limit. Because the dosage of the compound fertilizer is increased year by year and certain safety risk exists, strict limit standards are established for the residue of the compound fertilizer in food at home and abroad, the export barrier of agricultural products in China is caused, and the export economic situation of China is influenced.
At present, the trifloxystrobin analysis method in food is mainly a chromatographic detection method, and comprises High Performance Liquid Chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS), gas chromatography (CC) and the like. The chromatographic detection method has high sensitivity and strong specificity, but the pretreatment of a detection sample is complicated, time-consuming, poor in timeliness and expensive in instrument, and professional detection technicians are required, so that the requirement of completing rapid detection of a sample in a short time cannot be met. In addition, immunochemical methods may be an attractive strategy for low cost, in situ and rapid application of samples with limited amounts of target analytes. At present, an enzyme-linked immunosorbent assay method aiming at oximido acetate bactericides has a potential market application prospect, but the detection needs multi-step reaction and cannot really realize on-site rapid screening. In contrast, the colloidal gold immunochromatographic assay has low requirements on instruments and equipment, low requirements on sample treatment and detection environment, no need of complex pretreatment on samples, simplicity, rapidness, portability, economy, high sensitivity and strong specificity, is easy to popularize and popularize at the basic level, can meet the requirements of rapid analysis and detection, and is particularly suitable for field screening and rapid analysis of a large number of samples.
The invention develops a colloidal gold immunochromatographic test strip which has high sensitivity and good specificity on trifloxystrobin and is used for rapidly detecting the trifloxystrobin.
The invention relates to one of four innovative engineering projects of scientific and technological cooperation of Ningbo city agricultural academy and China agricultural academy, which are approved and implemented by people's government in Ningbo city based on the country happy occasion, wherein the project number is 2019CXGC 007.
Disclosure of Invention
The invention aims to solve the primary technical problem of providing an immunochromatographic gold-labeled test strip which has the advantages of high sensitivity, simple and convenient operation, simple and visible result and the like, is suitable for quick detection of trifloxystrobin pesticide and is based on an indirect competitive immunochromatographic gold-labeled quick detection test strip.
The invention aims to solve another technical problem of providing an immunochromatographic gold-labeled test strip rapid test method which has the advantages of high sensitivity, simple and convenient operation, simple and visible results and the like, is suitable for rapid on-site test and is suitable for trifloxystrobin pesticide.
The technical scheme for solving the above-mentioned primary technical problems of the invention is as follows: an immunochromatographic gold-labeled test strip for trifloxystrobin pesticide comprises a PVC lining plate, a nitrocellulose membrane, a gold-labeled antibody binding pad, a sample pad (Oslon 8964 glass fiber paper) and a water absorption pad, wherein a pesticide specific antibody marked by colloidal gold is coated on the gold-labeled antibody binding pad, and a trifloxystrobin artificial antigen detection line and a goat anti-rabbit IgG secondary antibody quality control line are coated on the nitrocellulose membrane.
The test conditions of the indirect competition immunochromatographic gold-labeled test strip suitable for rapid detection of trifloxystrobin pesticide residue are as follows: the colloidal gold labeled pesticide specific antibody is a compound formed by combining 20-30 nm (preferably 25nm) nano gold particles and a pesticide specific antibody, wherein the combination concentration of the pesticide specific antibody and the colloidal gold is 40-60 mg/L (preferably 50mg/L), namely 50mg of the pesticide specific antibody is matched in every 1L of colloidal gold solution.
Remarks explanation: the mass concentration of the colloidal gold in the colloidal gold solution was 0.01%.
The test conditions of the indirect competition immunochromatographic gold-labeled test strip suitable for rapid detection of trifloxystrobin pesticide residue are as follows: the detection line coated artificial antigen for indirect competition is an artificial antigen coupling pesticide hapten to chicken Ovalbumin (OVA) respectively, and the concentration is 550-650 mg/L (preferably 600 mg/L); the coupling ratio of the pesticide hapten to the OVA is 8: 1-12: 1 (preferably 10: 1). The quality control line concentration of goat anti-rabbit 1gG (secondary antibody) is 900-1100 mg/L (preferably 1000 mg/L). The coating amount of the detection line and the control line is 0.05 mu L/mm.
The test conditions of the indirect competition immunochromatographic gold-labeled test strip suitable for rapid detection of trifloxystrobin pesticide residue are as follows: the diluent used by the detection line and the quality control line is 0.01M phosphate buffer solution with the mass concentration of 1-5% of sucrose and the pH value of 7.4.
According to the invention, 0.01M phosphate buffer solution with the mass concentration of 1-5% of sucrose and the pH value of 7.4 is used as diluent, so that the effect of protecting the coating antigen and the secondary antibody is achieved, and the activity of the antigen and the secondary antibody is prolonged.
The trifloxystrobin specific polyclonal antibody adopted by the invention is prepared by coupling BSA or OVA (the molar ratio is 15: 1-20: 1) with (E) -2- (methoxyimino) 2- (2- ((E)1- (3- (trifluoromethyl) phenyl) ethylideneaminooxy) methyl) phenyl) acetic acid (TFA) as a hapten, wherein the pH of the whole system for preparing the immunogen and the coating antigen is 7.3-7.5. Mixing the hapten with an immunologic adjuvant to prepare a trifloxystrobin complete antigen and immunize a white rabbit with a big ear, and preparing a polyclonal antibody capable of specifically reacting with the trifloxystrobin.
The invention adopts a sealing mode that the sample pad completely absorbs sealing liquid and then is dried. In the sample detection process, the chromatography of the sample to be detected drives the sealing liquid on the sample pad to the nitrocellulose membrane, so that the sealing effect is achieved. An indirectly competing trifloxystrobin antigen refers to a complete antigen complex coupling the trifloxystrobin hapten to chicken Ovalbumin (OVA). The rapid test strip can detect the trifloxystrobin pesticide residue in a sample.
The trifloxystrobin polyclonal antibody provided by the invention is prepared by the following basic steps:
(1) artificial hapten synthesis: (E) -2- (methoxyimino) 2- (2- ((E)1- (3- (trifluoromethyl) phenyl) ethyleneaminooxy) methyl) phenyl) acetic acid (TFA). 5g trifloxystrobin (12.24mmol), methanol (70mL) and 5M aqueous sodium hydroxide (12.5mL) were stirred under reflux. After hydrolysis was complete, the reaction mixture was neutralized with 5M aqueous HCl and methanol evaporated, as monitored by thin film chromatography (about 5 h). Water was added to the residue, and neutral impurities were removed by extraction with ethyl acetate. The aqueous layer was cooled to 0 deg.C, acidified to pH3 with 2M HCl solution, extracted with ethyl acetate, washed with brine, and finally anhydrous MgSO4And (5) drying. Evaporation gave 4.48g of a clear solution, which was recrystallized from n-hexane/acetone (95:5) to give 3.52g of the acid derivative as white crystals (72.9%).
(2) Synthesis of complete antigen and coatingen: 0.1mmol of hapten, 0.1mmol of NHS and 0.1mmol of 1 DCC were dissolved in 1.0mL of DMF, respectively. And (3) dropwise adding the DCC solution into the hapten and the NHS under magnetic stirring, stirring at room temperature for reacting for 1h, and stirring in a refrigerator at 4 ℃ overnight. The next day, the supernatant was centrifuged and added dropwise to 5.0mL of 0.001mmol BSA or OVA pH8.0 boric acid buffer solution under stirring for about half an hour, reacted at room temperature with magnetic stirring for 1 hour, reacted at 4 ℃ with stirring overnight, and taken out the next day. Transferring the solution into dialysis bag, dialyzing with PBS buffer solution of pH7.4 for 3 days, changing water every 4h at 4 deg.C, and freeze-drying for storage.
(3) On the basis, the prepared immunogen is used for immunizing a white rabbit of the big ear of Japan to prepare a high-sensitivity polyclonal antibody aiming at the trifloxystrobin:
1) preparation of trifloxystrobin antibody serum: preparing the trifloxystrobin complete antigen obtained in the step (2) into a suspension, and injecting animals to obtain trifloxystrobin antibody serum;
2) and (3) purifying the trifloxystrobin polyclonal antibody: and purifying the trifloxystrobin antibody serum by adopting an antigen affinity purification method to obtain the polyclonal antibody.
As a preferred technical scheme of the invention, in the step 1), the specific process of preparing the trifloxystrobin complete antigen into the suspension and injecting the suspension is as follows:
in the first immunization, 0.3mg of trifloxystrobin complete antigen is mixed with Freund's complete adjuvant to immunize animals, and in the second, third and fourth immunizations, 0.15mg of trifloxystrobin complete antigen is mixed with incomplete Freund's adjuvant to form water-in-oil emulsion, and the emulsion is sucked into a needle tube to be injected after being vibrated and uniformly mixed.
As a preferred technical scheme of the present invention, in the step 1), the specific process for preparing the trifloxystrobin antibody from the serum of the animal is as follows: animals are usually immunized by complete Freund's adjuvant on day 1, are immunized by incomplete Freund's adjuvant for the second, third and fourth times on days 12, 26 and 40, and are sacrificed on day 52 to obtain the anti-trifloxystrobin serum antibody.
The technical scheme for solving the other technical problem is as follows: a rapid detection method for trifloxystrobin residue in strawberry by using the test strip comprises the steps of taking a strawberry sample as a sample to be detected, chopping the sample, weighing 5.0g of the sample, putting the sample into 5mL of extracting solution (taking 5.0g of the sample as a reference), adding a matched grinding bead, extracting for 2min by shaking, standing for 3min, and then taking supernatant for detection. The sample extract was 0.01M phosphate buffer pH7.4 containing 20% ethanol.
Taking the supernatant, dropwise adding 2-3 drops into a sample application area (located in the middle of the sample pad) of each test strip sample pad for chromatographic reaction, and judging the negative and positive of the pesticide in the sample according to the color development conditions of the detection line and the quality control line after 5-10 minutes;
the red color of the quality control line indicates that the test strip is effective, and the test strip is ineffective if the test strip is not colored;
the quality control line shows red, and the detection line shows red, the detection line is judged to be negative, which indicates that the trifloxystrobin is not detected in the strawberry sample, or the concentration of the trifloxystrobin in the strawberry sample is lower than the corresponding sensitivity;
and the quality control line shows red color, and the detection line is judged to be positive if not, which indicates that the residual concentration of the trifloxystrobin pesticide in the strawberry sample is higher than the corresponding sensitivity.
Remarks explanation: the light red is incomplete color development and indicates partial positive, and the condition that the detection line is not colored is higher than the condition that the detection line displays light red, which indicates that the concentration of the trifloxystrobin pesticide residue in the strawberry sample is high.
The invention has the beneficial effects that: the colloidal gold labeled pesticide specific antibody is used as an immune gold labeled probe, so that the trifloxystrobin pesticide residue in the sample can be monitored, and the method is a rapid, simple and convenient pesticide residue screening means. The sensitivity of the prepared test strip to trifloxystrobin is 1 ppm. The test strip has the advantages of high sensitivity, simple and convenient operation, suitability for on-site rapid test, simple and visible result and the like, and is expected to be applied and expanded on a series of agricultural products in the future.
The invention utilizes the principle of the reaction of the pesticide specific antibody and the antigen, realizes the rapid detection of the trifloxystrobin pesticide, and has the advantages of good specificity, high sensitivity, accurate result, convenient use and the like. The test strip has good practicability, can be applied to on-site screening of the residual quantity of trifloxystrobin in agricultural products and standard-exceeding detection and diagnosis, has the sensitivity of 1ppm and the detection time of 5-10 minutes. The product technology has the advantages of convenience and rapidness in use, high sensitivity, accurate result and low cost, and has a good application prospect in quality safety supervision and management of agricultural product production.
In conclusion, the invention adopts the trifloxystrobin specific antibody and the antigen thereof to establish an indirect competitive immunochromatography technology, and the prepared gold-labeled test strip can detect the residue of the trifloxystrobin pesticide in the sample. Meanwhile, the product can realize the rapid screening of agricultural products (such as strawberries) during field picking, and if the pesticide exceeding the standard in the agricultural products (such as strawberries) is found, the pesticide residue of the agricultural products (such as strawberries) can be reduced by delaying the picking time and the like.
Drawings
FIG. 1 is a schematic structural diagram of trifloxystrobin hapten;
FIG. 2 is a curve of an indirect competitive ELISA method using trifloxystrobin polyclonal antibody;
FIG. 3 is a schematic diagram of the composition of a single-linked test strip for rapid detection of pesticide residues;
FIG. 4 is a schematic top view of FIG. 1;
FIG. 5 is a method for determining the result of detection;
in fig. 5, from left to right;
both line C and line T show red → negative;
line C shows red, line T shows light red → partial positive;
line C shows red, line T does not show color → positive;
line C does not appear color, line T shows red → fails;
the C line and the T line are not developed → failed;
FIG. 6 shows the results of the precision of trifloxystrobin assay in strawberry;
FIG. 7 is the results of the actual sample detection of trifloxystrobin in strawberry.
Detailed Description
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Example 1: preparation of immune and polyclonal antibodies
1. Synthesis of trifloxystrobin hapten
The specific steps are that 5g of trifloxystrobin (12.24mmol), methanol (70mL) and 5M aqueous solution of sodium hydroxide (12.5mL) are refluxed and stirred. After hydrolysis was complete, the reaction mixture was neutralized with 5M aqueous HCl and methanol evaporated, as monitored by thin film chromatography (about 5 h). Water was added to the residue, and neutral impurities were removed by extraction with ethyl acetate. The aqueous layer was cooled to 0 deg.C, acidified to pH3 with 2M HCl solution, extracted with ethyl acetate, washed with brine, and finally anhydrous MgSO4And (5) drying. Evaporating to obtain 4.48g of clear liquid, and recrystallizing with n-hexane/acetone (95:5) to obtain 3.52g of acid derivative, wherein the white crystal is trifloxystrobin hapten.
2. Synthesis of complete antigens
The specific steps are dissolving hapten 0.1mmol, NHS 0.1mmol and DCC 0.1mmol 1mmol in DMF 1.0mL respectively. And (3) dropwise adding the DCC solution into the hapten and the NHS under magnetic stirring, stirring at room temperature for reacting for 1h, and stirring in a refrigerator at 4 ℃ overnight. The next day, the supernatant was centrifuged and added dropwise to 5.0ml of 0.001mmol of BSA or OVA in pH8.0 boric acid buffer solution with stirring for about half an hour, the reaction was magnetically stirred at room temperature for 1 hour, stirred at 4 ℃ for overnight reaction, and taken out the next day. Transferring the solution into dialysis bag, dialyzing with PBS buffer solution of pH7.4 for 3 days, changing water every 4h at 4 deg.C, and freeze-drying for storage.
3. Preparation of polyclonal antibody serum
On day 1, a Japanese big ear white rabbit is immunized by adopting complete Freund's adjuvant and 0.3mg of trifloxystrobin complete antigen, on days 12, 26 and 40, the Japanese big ear white rabbit is immunized for the second, third and fourth times by adopting the complete Freund's adjuvant and 0.15mg of trifloxystrobin complete antigen respectively, and on day 52, the animal is killed to take blood to obtain the anti-trifloxystrobin serum antibody.
4. Polyclonal antibody serum purification
By adopting an antigen affinity purification method, 2ml of Sulfolink couping gel (Thermo Fisher) is put into a chromatographic column, 10mg of trifloxystrobin complete antigen solution is added, the chromatographic column is placed on a rotary incubator to rotate for 2-3 hours and then is placed on a chromatographic frame for 30 minutes, and a constant flow pump is connected. 40ml of phosphate buffer, 30ml of 100mM glycine buffer, and 30ml of phosphate buffer were pumped in this order to equilibrate the column to neutrality. Adding 4M sodium chloride solution into the antibody serum to be purified according to the volume ratio of 1:20, performing column chromatography twice, adding 40ml washing solution (320 mM sodium chloride is added into phosphate buffer solution) for washing once, adding 100mM glycine buffer solution for eluting the antibody, collecting 8 tubes, wherein each tube contains 16ml, adding into a dialysis bag, mixing uniformly, placing into the phosphate buffer solution mixed with 50% glycerol, dialyzing and concentrating overnight to obtain the concentrated polyclonal antibody.
5. Titer and titer determination of polyclonal antibodies
Coating a 96-well plate by using OVA coupled trifloxystrobin hapten as an antigen (200ng) of the polyclonal antibody obtained in the example 4, detecting the titer by an indirect ELISA method, respectively diluting the obtained polyclonal antibody by 1:1000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000 times, using serum before immunization as a control, performing ELISA detection on the polyclonal antibody under different dilutions, sequentially adding an antibody, a substrate and a color developing agent, detecting the absorbance value after incubation, and obtaining the result shown in figure 2, wherein the titer of the purified polyclonal antibody is 1: 256000.
6. polyclonal antibody specificity detection
WB detection is carried out by using trifloxystrobin complete antigen (see example 1 for a complete antigen synthesis method), purified polyclonal antibody is used as a primary antibody (1:2000), HRP coat Anti-Rabbit IgG (H + L) (abllone) is used as a secondary antibody (1:5000), and WB detection results are carried out by using Tanon 5200, and the antibody can specifically recognize the trifloxystrobin complete antigen.
Example 2: preparation of test paper strip
1. Preparation of colloidal gold
Colloidal gold particles were prepared using trisodium citrate (sodium citrate) modification. Before preparation, the vessel is cleaned and dried for later use. Preparing 100mL of 0.01% chloroauric acid by using newly prepared deionized water, placing the chloroauric acid in a conical flask, heating the chloroauric acid in a microwave oven until the chloroauric acid is boiled, accurately absorbing 2.5mL of 1% trisodium citrate under magnetic stirring, quickly adding the trisodium citrate into the conical flask, stirring the trisodium citrate uniformly, immediately placing the mixture into the microwave oven to be continuously boiled until the chloroauric acid is red, taking the mixture out, cooling the mixture to room temperature, recovering the volume of the chloroauric acid to the original volume by using the deionized water, placing the cooled chloroauric acid into a reagent bottle, and storing the reagent bottle. Scanning the obtained colloidal gold solution with the mass concentration of 0.01% in a visible light range by using an ultraviolet-visible spectrophotometer to obtain colloidal gold particles with the maximum absorption wavelength of 520nm and the light absorption value of 0.85; observing the colloidal gold particles by a transmission electron microscope, wherein the particles are uniform in size; the average particle size of the particles was 25nm as measured by a Malvern laser particle size Analyzer.
2. Preparation of gold-labeled antibody
Placing the antibody into a dialysis bag, dialyzing in deionized water or saline with extremely low concentration (0.005M NaCl, pH 7.0), and changing the dialysate for 3 times; centrifuging at 10000rpm and 4 deg.C for 60min to remove antibody precipitate, and collecting supernatant. Adjusting the pH value of the colloidal gold solution to 7.5-8.5. Taking 10mL of colloidal gold solution with the mass concentration of 0.01%, dropwise adding 1mL of 50mg/L trifloxystrobin specific antibody solution while stirring at room temperature, uniformly mixing, and standing for 30 min. Then, 2.5mL of 20mM boric acid buffer solution containing 5% bovine serum albumin and 0.1% polyethylene glycol was added, and the mixture was mixed uniformly and allowed to stand for 30 min. Centrifuging the gold-labeled antibody at 3000rpm and 4 ℃ for 10min, and removing the precipitate; centrifuging the supernatant at 12000rpm at 4 deg.C for 1 hr, and discarding the supernatant; the pellet was dissolved in 20mM Borate Buffer (BBS) in the original volume and centrifuged repeatedly 2-3 times; finally, the red precipitate was dissolved in 20mM boric acid buffer solution containing 1% BSA and 5% sucrose and made to volume of 1mL, and stored at 4 ℃ until use. Thus obtaining the purified trifloxystrobin gold-labeled primary antibody.
Remarks explanation: the above% concentrations are mass concentrations. For example, the above 20mM boric acid buffer solution containing 5% bovine serum albumin and 0.1% polyethylene glycol is prepared by: to 100mL of a 20mM boric acid buffer solution (pH 8.5) were added 5g of bovine serum albumin and 0.1g of polyethylene glycol.
3. Sample pad treatment
Prepare blocking solution, weigh 50mg Bovine Serum Albumin (BSA), 50mg polyvinylpyrrolidone (PVP), 1000mg sucrose, add to 20mL 0.01M Phosphate Buffered Saline (PBST) containing 0.05% Tween.
Remarks explanation: the above 0.01M Phosphate Buffered Saline (PBST) containing 0.05% Tween means that 0.05mL of Tween 20 was added to 100mL of 0.01M phosphate buffered saline (PBS, pH 7.4).
And fully and uniformly mixing the reagent and the solution by vortex until the reagent and the solution are completely dissolved, thus finishing the preparation of the sealing liquid. Immersing the sample pad into the confining liquid, taking out after the confining liquid is completely adsorbed (namely the residual amount of the confining liquid is not changed and is generally 20-30s), and drying in an oven at 37 ℃ for 3 hours; put into a sealed bag and stored in a glass desiccator.
Remarks explanation: the sample pad described above was also subsequently used as the starting material for the gold-labeled antibody conjugate pad.
4. Preparation of gold-labeled antibody conjugate pad
Uniformly coating the pesticide-specific gold-labeled antibody obtained in the step 2 on the sample pad dried in the step 3, wherein the coating amount is 0.5 mu L/mm2Drying in a 37 ℃ oven for 30min to obtain a gold-labeled antibody binding pad; put into a sealed bag and stored in a glass desiccator.
5. Coating of detection line and quality control line
Firstly, preparing a protective agent, wherein the protective agent is 0.01M PBS solution containing 1% of sucrose. Then, the prepared protective agent is used as a diluent to prepare 600mg/L trifloxystrobin hapten-OVA conjugate (detection line) and 1000mg/L goat anti-rabbit IgG (quality control line) respectively.
Remarks explanation: the coupling method of the trifloxystrobin hapten-OVA conjugate is an active ester method, wherein a pesticide hapten is coupled to Ovalbumin (OVA), and the coupling ratio of the hapten to the OVA is 10: 1. The goat anti-rabbit 1gG was purchased from Beijing Boolong immuno-technology, Inc. at an original concentration of 5 mg/mL.
Scribing with automatic scribing instrument, and coating detection line and control line on nitrocellulose membrane (scribing position shown in figure 1 and figure 2), wherein the coating amount of the detection line and the control line is 0.5 μ L/mm2. And after the scribing is finished, drying the nitrocellulose membrane in a 37 ℃ oven for 10min to obtain the detection line coated with the trifloxystrobin hapten-OVA and the nitrocellulose membrane coated with the secondary quality control line of goat anti-rabbit IgG.
6. Assembly of test strips
The nitrocellulose membrane coated with the detection line and the control line prepared in step 5 is firstly attached to the middle section of the PVC backing plate, and then a gold-labeled antibody binding pad (the longitudinal length is 2mm) is attached to the nitrocellulose membrane at a position such that about half of the gold-labeled antibody binding pad is positioned above the nitrocellulose membrane. Then, the blocking solution-treated sample pad was stuck on the top of the gold-labeled antibody conjugate pad at a position covering about half of the gold-labeled antibody conjugate pad. And finally, adhering the water absorption pad, wherein the adhering position is about 3mm for covering the nitrocellulose membrane. After the lining plate is assembled, the lining plate is horizontally placed into an automatic strip cutting machine, and the strip cutting machine is used for cutting the lining plate into test strips with the width of 3-4 mm.
Example 3: application of test strip on strawberries
1. Test strip sensitivity test
(1) Standard solution preparation
And preparing a standard stock solution of trifloxystrobin by using methanol, wherein the concentration of the standard stock solution is 1 mg/L. The strawberry blank sample matrix extracted with 20% ethanol in 0.01M PBS buffer was diluted to serial concentrations of standard working solutions: 100. mu.g/mL, 20. mu.g/mL, 4. mu.g/mL, 1. mu.g/mL, 0.2. mu.g/mL, 0.04. mu.g/mL.
(2) Sensitivity detection
7 treatment groups were set, one of which was a control of strawberry blank matrix (strawberry blank sample was treated with 20% ethanol in 0.01M PBS buffer), and the others were serial concentrations of standard working solution diluted with strawberry blank matrix. The method comprises the following steps of (1) detecting by using the same batch of residue quick test paper strips: and (3) dropwise adding 2-3 drops of strawberry blank matrix supernatant and diluted series of standard products into a sample application area for chromatography, and judging the negative and positive of the pesticide in the sample according to the color development conditions of the detection line and the quality control line after 5-10 minutes. And taking the lowest concentration at which the detection line does not develop color as the sensitivity of the test strip.
The detection result is shown in table 1, and under the detection condition of the standard working solution, the sensitivity of the trifloxystrobin is 1 mug/mL, namely the lowest detection limit judged by naked eyes is 1 mug/mL.
TABLE 1 results of sensitivity detection of trifloxystrobin standard working solution in strawberry
Note: the control line and the detection line are completely colored, and the result is judged to be negative; + indicates that the color development is not obvious, that is, the detection line is not completely developed, is light red, and is judged to be positive (does not reach the set lowest detection limit); and + represents that the control line is colored, the detection line is not colored, and the result is judged to be positive. A partial positive indication shows a pink color (light red) indicating that a lower concentration of pesticide than that which did not develop was detected. The result determination method is shown in fig. 5.
2. Trifloxystrobin standard solution addition test in strawberry blank sample
Weighing 4 parts of strawberry sample, respectively weighing 5g of strawberry sample, respectively adding 100 mu L of methanol and the trifloxystrobin standard sample with the lowest detection limit, standing for 10min, respectively adding 5mL of phosphate buffer solution containing 20% ethanol into a blank sample and a standard sample, fully oscillating and uniformly mixing, taking obtained supernatant as a sample matrix, usually, adding a part of matched grinding beads, fully oscillating and uniformly mixing for 2min, standing for 3min after extraction is finished, and detecting by using a residual rapid detection card of the same batch. The result shows that only the corresponding test strip is a positive result after 5 pesticides with the lowest detection limit are added to a single standard product.
TABLE 2 test results of standard working solution addition for strawberry trifloxystrobin quick test strip
Note: the control line and the detection line are completely colored, and the result is judged to be negative; and + represents that the control line is colored, the detection line is not colored, and the result is judged to be positive.
3. Rapid test method precision test of trifloxystrobin test strip in strawberry sample
Weighing 4 parts of strawberry sample, adding 5g of each strawberry sample, adding 100 mu L of methanol into one part of strawberry sample, adding 1ppm of trifloxystrobin pesticide standard substance into one part of strawberry sample, standing for 10min, respectively adding 5mL of phosphate buffer solution containing 20% ethanol into a blank sample and a standard sample, fully oscillating and uniformly mixing, taking obtained supernatant as a sample matrix, usually, adding one part of matched grinding beads, fully oscillating and uniformly mixing for 2min, standing for 3min after extraction is finished, and detecting by using a residual quick test card of the same batch. The result shows that after the trifloxystrobin pesticide mixed standard with the lowest detection limit is added, 3 test strips are positive results, and the specific result is shown in fig. 6.
TABLE 3 Rapid test method precision test results of trifloxystrobin test strip in strawberry samples
Note: + indicates that the complete coloration was judged negative: and + or + indicates incomplete color development and is judged to be positive (not reaching the set minimum detection limit): the non-color development was judged to be positive.
4. Strawberry sample detection and result judgment
The strawberry samples are detected by using the same batch of residual quick test paper strips, and the detection method is the same as the above. The results of the assay are shown in FIG. 7, which shows: the test paper strip of the strawberry sample with the detection result of the instrument exceeding the minimum detection limit of the test paper strip is positive in color development, and the test paper strip of the strawberry sample with the detection result of the instrument not exceeding the minimum detection limit of the test paper strip is negative or weakly positive in color development, so that the detection result of the test paper strip is consistent with the detection result of the instrument, and the accuracy and the practicability of the detection result of the test paper strip are verified.
Claims (8)
1. The utility model provides an immunochromatography gold-labeled test paper strip suitable for trifloxystrobin pesticide, it includes welt, nitrocellulose membrane, gold-labeled antibody combination pad, sample pad, absorbent pad, characterized by: the gold-labeled antibody combination pad is coated with a colloidal gold-labeled trifloxystrobin specific antibody, and the nitrocellulose membrane is respectively coated with a trifloxystrobin artificial antigen detection line and a goat anti-rabbit IgG quality control line for indirect competition.
2. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 1, which is characterized in that: the colloidal gold labeled trifloxystrobin specific antibody is a compound formed by combining 20-30 nm nano-gold particles and the trifloxystrobin specific antibody, and the combination concentration of the trifloxystrobin specific antibody and colloidal gold is 40-60 mg/L.
3. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 1, which is characterized in that: the trifloxystrobin artificial antigen coated by the detection line and used for indirect competition is an artificial antigen coupling a trifloxystrobin hapten to OVA (ovalbumin in chicken), and the concentration is 550-650 mg/L; the coupling ratio of the pesticide hapten to the OVA is 8: 1-12: 1.
4. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 1, which is characterized in that: the concentration of the quality control line of the goat anti-rabbit IgG is 900-1100 mg/L.
5. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 1, which is characterized in that: the diluent used by the detection line and the quality control line is 0.01M phosphate buffer solution with the mass concentration of 1-5% of sucrose and the pH value of 7.4.
6. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 1, which is characterized in that: the antibody with the trifloxystrobin specificity is prepared by coupling BSA or OVA with a molar ratio of 15: 1-20: 1 by taking (E) -2- (methoxyimino) 2- (2- ((E)1- (3- (trifluoromethyl) phenyl) ethylideneaminooxy) methyl) phenyl) acetic acid (TFA) as a hapten, preparing an immunogen and a whole system of a coating antigen, wherein the pH value of the whole system is 7.3-7.5, mixing the hapten with an immunologic adjuvant, preparing a trifloxystrobin complete antigen, immunizing a white rabbit with big ear, and preparing a polyclonal antibody capable of specifically reacting with the trifloxystrobin.
7. The immunochromatographic gold-labeled test strip suitable for the trifloxystrobin pesticide according to claim 6, which is characterized in that: the specific preparation basic steps of the trifloxystrobin specific antibody are as follows:
(1) artificial hapten synthesis: (E) -2- (methoxyimino) 2- (2- ((E)1- (3- (trifluoromethyl) phenyl) ethyleneaminooxy) methyl) phenyl) acetic acid (TFA); 5g of trifloxystrobin, 70mL of methanol and 12.5mL of 5M sodium hydroxide aqueous solution are refluxed and stirred; after hydrolysis was complete, the reaction mixture was neutralized with 5M aqueous HCl and methanol evaporated, monitored for about 5h according to thin film chromatography; adding water into the residue, and extracting with ethyl acetate to remove neutral impurities; the aqueous layer was cooled to 0 deg.C, acidified to pH3 with 2M HCl solution, extracted with ethyl acetate, washed with brine, and finally anhydrous MgSO4Drying; evaporating to obtain 4.48g of clear solution, and recrystallizing with n-hexane/acetone at a volume ratio of 95:5 to obtain 3.52g of acid derivative, which is 72.9% white crystal;
(2) synthesis of complete antigen and coatingen: dissolving hapten 0.1mmol, NHS 0.1mmol and DCC 0.1mmol 1mmol in DMF 1.0mL respectively; under magnetic stirring, dropwise adding a DCC solution into the hapten and NHS, stirring at room temperature for reaction for 1h, and stirring in a refrigerator at 4 ℃ overnight; centrifuging the mixture to obtain supernatant, adding the supernatant into 5.0mL of 0.001mmol BSA or OVA boric acid buffer solution with pH8.0 dropwise under stirring for about half an hour, magnetically stirring the mixture at room temperature for reaction for 1h, stirring the mixture at 4 ℃ for reaction overnight, and taking the mixture out the next day; transferring the solution into a dialysis bag, dialyzing with PBS buffer solution with pH of 7.4 for 3 days at 4 deg.C, changing water every 4h, and freeze-drying for storage;
(3) on the basis, the prepared immunogen is used for immunizing a white rabbit of the big ear of Japan to prepare a polyclonal antibody with high sensitivity aiming at the trifloxystrobin.
8. A rapid test method for trifloxystrobin in strawberries by using the test strip of any one of claims 1 to 7, which is characterized in that: homogenizing or cutting a strawberry sample, weighing 5g of the sample to be detected into 5mL of sample diluent, adding a matched grinding bead, fully shaking and uniformly mixing for 2min, standing for 3min after extraction, taking supernate, dropwise adding 2-3 drops of the supernate into a sample application area of a sample pad for chromatographic reaction, and directly judging the positive and negative results of trifloxystrobin pesticide in the strawberry sample according to the color development conditions of a detection line and a quality control line after 5-10 min; the quality control line shows red to indicate that the test strip is effective, and the test strip fails if the test strip is not developed; if the detection line shows red or light red, the detection line is judged to be negative or more positive, which indicates that the trifloxystrobin is not detected in the strawberry or the concentration of the trifloxystrobin in the strawberry is lower than the corresponding lowest detection limit; and if the detection line is not developed, judging the detection line to be positive, and indicating that the concentration of the trifloxystrobin in the strawberry is higher than the corresponding lowest detection limit.
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