CN113702631A - Triplex immunochromatography detection kit and detection method for procymidone, carbofuran and carbendazim - Google Patents
Triplex immunochromatography detection kit and detection method for procymidone, carbofuran and carbendazim Download PDFInfo
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- CN113702631A CN113702631A CN202110970387.3A CN202110970387A CN113702631A CN 113702631 A CN113702631 A CN 113702631A CN 202110970387 A CN202110970387 A CN 202110970387A CN 113702631 A CN113702631 A CN 113702631A
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- procymidone
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- 238000001514 detection method Methods 0.000 title claims abstract description 131
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 239000006013 carbendazim Substances 0.000 title claims abstract description 74
- QXJKBPAVAHBARF-BETUJISGSA-N procymidone Chemical compound O=C([C@]1(C)C[C@@]1(C1=O)C)N1C1=CC(Cl)=CC(Cl)=C1 QXJKBPAVAHBARF-BETUJISGSA-N 0.000 title claims abstract description 74
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 23
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 title claims abstract 15
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- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 14
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- -1 3, 5-dichlorophenyl Chemical group 0.000 description 1
- ZOCSXAVNDGMNBV-UHFFFAOYSA-N 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile Chemical compound NC1=C(S(=O)C(F)(F)F)C(C#N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl ZOCSXAVNDGMNBV-UHFFFAOYSA-N 0.000 description 1
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- CFNHVUGPXZUTRR-UHFFFAOYSA-N n'-propylethane-1,2-diamine Chemical compound CCCNCCN CFNHVUGPXZUTRR-UHFFFAOYSA-N 0.000 description 1
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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- Immunology (AREA)
- Engineering & Computer Science (AREA)
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- Chemical & Material Sciences (AREA)
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- Microbiology (AREA)
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- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention provides a triple immunochromatography detection kit and a detection method for procymidone, carbofuran and carbendazim. The kit comprises a sample extraction and purification tube, a reaction substrate with reaction holes and a procymidone, carbofuran and carbendazim triple immunochromatography detection test strip. The three-in-one colloidal gold immunochromatographic assay kit provided by the invention can be used for rapidly and accurately carrying out simultaneous extraction, purification and detection on the procymidone, carbofuran and carbendazim remained in vegetables on site and in multiple flux, greatly simplifying the pretreatment process, improving the detection efficiency and providing powerful technical support for food safety production, basic supervision, food inspection, large-scale activity guarantee and the like.
Description
Technical Field
The invention relates to the technical field of pesticide residue detection, and particularly relates to a procymidone, carbofuran and carbendazim triple immunochromatography detection kit and a detection method.
Background
Procymidone (Procymidone), the chemical name of which is N- (3, 5-dichlorophenyl) -1, 2-dimethylcyclopropane-1, 2-diformylimide, is a low-toxicity bactericide which can inhibit the synthesis of triglyceride in thalli. Carbofuran (Carbofuran), chemical name 2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl methyl carbamate, is a carbamate insecticide and nematicide. Carbendazim (Carbendazin), the chemical name of which is 2- (methoxycarbamoyl) benzimidazole, is a high-efficiency and low-toxicity fungicide and can be used for preventing and treating various plant diseases. Procymidone, carbofuran and carbendazim are commonly used pesticides in plant cultivation. At present, the detection rates of the pesticide residues of procymidone, carbofuran and carbendazim in vegetables are high, the reject ratio is also high, and the existing detection method is relatively complicated.
According to the national standard, the method for detecting procymidone is a gas chromatography-mass spectrometry combined method, the method for detecting carbofuran is a liquid chromatography-post-column derivatization method, and the currently effective method for detecting carbendazim is a liquid chromatography-tandem mass spectrometry. Although the method has good stability, high accuracy and strong repeatability, the method needs expensive instruments and equipment, professional operators and complex pretreatment processes, is not environment-friendly and cannot meet the requirements of timely detection, on-site office work and on-site law enforcement. And the pretreatment method and the used instruments are different, so the aim of simultaneously detecting several pesticide residues is difficult to realize.
In the rapid detection method, a surface-enhanced Raman spectroscopy method and an immunochromatographic test paper rapid detection method are more applied. However, many detection objects do not have raman signals, the detection object of the immunochromatographic test paper is single, and the pretreatment methods of different detection objects are different, which is not beneficial to the simultaneous detection of several pesticide residues.
Disclosure of Invention
The invention aims to provide a triple immunochromatography detection kit and a detection method for procymidone, carbofuran and carbendazim.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a procymidone, carbofuran and carbendazim triple immunochromatography detection test strip which comprises a sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the nitrocellulose membrane is sequentially provided with three detection lines and a quality control line in the direction from the sample pad to the water absorption pad, the three detection lines are respectively coated with a procymidone hapten-carrier protein conjugate, a carbofuran hapten-carrier protein conjugate and a carbendazim hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse polyclonal antibody.
The sample pad, the nitrocellulose membrane and the absorbent pad are alternately laminated and stuck on the bottom plate at intervals of 2-3mm in sequence.
The concentration of the Pythium hapten-carrier protein conjugate coated on the detection line is 0.10-0.20mg/mL, and preferably 0.15 mg/mL.
The concentration of carbofuran hapten-carrier protein conjugate coated on the detection line is 0.25-0.8mg/mL, and preferably 0.6 mg/mL.
The concentration of the carbendazim hapten-carrier protein conjugate coated on the detection line is 0.25-0.6mg/mL, and preferably 0.5 mg/mL.
The concentration of the goat anti-mouse polyclonal antibody coated on the quality control line is 0.25 mg/mL.
The structures of the procymidone hapten, the carbofuran hapten and the carbendazim hapten are respectively shown in formulas 1) to 3):
Preferably, the carrier protein is bovine serum albumin.
The distances between the detection lines on the nitrocellulose membrane and between the detection line close to the end of the absorbent pad and the quality control line are 4-5mm, preferably 4.3 mm. In the invention, if the distance between two adjacent detection lines is too close, the color development can be interfered; if the distance is too far, the chromatography time is too long and more detection solution is required.
The width of the test strip is 3-5mm, preferably 3.8 mm. The test strip is too narrow to judge the result, and too wide to save cost, because more antigens, antibodies and detection solutions are needed.
The material of the sample pad can be a nitrocellulose membrane. The material of the bottom plate can be PVC.
FIG. 1 is a schematic structural diagram of the immunochromatographic assay test strip of the present invention.
In a second aspect, the invention provides a triple immunochromatography detection kit for procymidone, carbofuran and carbendazim, which comprises a sample extraction purification tube (containing a sample extraction purification solution), a reaction substrate with reaction holes and the triple immunochromatography detection test strip. The sample extraction purification tube, the reaction substrate and the immunochromatography detection test strip are sealed and then are put into a kit for storage at 4 ℃.
The preparation method of the sample extraction and purification tube comprises the following steps: 200mg of PSA (N-propylethylenediamine solid phase adsorbent), 200mg of C18Placing (octadecyl bonded silica gel) and 30mg GCB (graphitized carbon black) in a 5mL centrifuge tube, adding 2mL of 0.05mol/L PBS (pH7.4), and mixing uniformly to obtain the sample extraction purification tube filled with the sample extraction purification liquid.
The reaction hole is coated with a colloidal gold-labeled procymidone antibody, a colloidal gold-labeled carbofuran antibody and a colloidal gold-labeled carbendazim antibody. The bottom area of the reaction hole is 0.32cm2The volume of the reaction well is 365 mu L, and a 96-well plate can be directly used as a reaction well so as to prepare the kit in large batch.
The preparation method of the reaction hole comprises the following steps: adding 2 mu L of colloidal gold-labeled procymidone antibody solution, 3 mu L of colloidal gold-labeled carbofuran antibody solution and 2 mu L of colloidal gold-labeled carbendazim antibody solution into the reaction hole, diluting to 60 mu L with a freeze-drying protective solution, and carrying out vacuum freeze-drying.
Preferably, the freeze-drying protection solution is: 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% tween 20 and 0.03% ProClin 300, in water.
Preferably, the preparation method of the colloidal gold-labeled procymidone antibody solution comprises the following steps: 1mL of the colloidal gold solution was added to 0.2mol/L K2CO3Mixing the solution 5-10 μ L (preferably 7 μ L), adding procymidone antibody 6 μ g, mixing, and standing at 25 deg.C for 10 min; then adding 50 mu L of 10% BSA blocking solution (mass percent), mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution.
Preferably, the preparation method of the colloidal gold-labeled carbofuran antibody solution comprises: 1mL of the colloidal gold solution was added to 0.2mol/L K2CO3Mixing the solution 6-12 μ L (preferably 7 μ L), adding carbofuran antibody 5 μ g, mixing, and standing at 25 deg.C for 10 min; then adding 50 mu L of 10% BSA blocking solution (mass percent), mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution.
Preferably, the method for preparing the colloidal gold-labeled carbendazim antibody solution comprises: 1mL of colloidal gold solution was added to the reaction well, and 0.2mol/L K was added2CO3Mixing the solution 3-7 μ L (preferably 4 μ L), adding carbendazim antibody 6 μ g, mixing, and standing at 25 deg.C for 10 min; then adding 50 mu L of 10% BSA blocking solution (mass percent), mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution.
The preparation method of the colloidal gold solution comprises the following steps: first, 2g of chloroauric acid (HAuCl)4) Dissolving the powder in 100mL of ultrapure water to obtain a chloroauric acid solution with the concentration of 2%; and heating 1L of ultrapure water to boil, adding 10mL of trisodium citrate solution with the mass concentration of 1%, continuing to heat for 8 minutes after boiling, adding 10mL of 2% chloroauric acid solution, heating for 10 minutes after the solution turns to wine red, stopping heating, naturally cooling, and fixing the volume to 1L to obtain the colloidal gold solution.
The reconstituted solution may be 0.05mol/L PBS containing 2% trehalose and 1% BSA, pH 7.4.
The average grain diameter of the colloidal gold solution is 40 nm.
The colloidal gold labeled procymidone antibody can be used for labeling 5-10 mu g (preferably 6 mu g) of procymidone antibody by adjusting the pH of 1mL of colloidal gold by 7 mu L of 0.2mol/L potassium carbonate solution.
The colloidal gold-labeled carbofuran antibody can be used for labeling 5-8 mu g (preferably 5 mu g) of carbofuran antibody by adjusting the pH of 1mL of colloidal gold with 7 mu L of 0.2mol/L potassium carbonate solution.
The pH of 1mL of colloidal gold is adjusted by 4 mu L of 0.2mol/L potassium carbonate solution, and the colloidal gold-labeled carbendazim antibody can be used for labeling 5-12 mu g (preferably 6 mu g) of carbendazim antibody.
In the preparation method of the reaction hole, the vacuum freeze-drying conditions are as follows: -50 ℃ for 4h, -25 ℃ for 4h, -15 ℃ for 4h, 5 ℃ for 2h, 25 ℃ for 10 h.
In a third aspect, the invention provides the use of the kit in procymidone, carbofuran and carbendazim detection (including non-disease diagnosis and treatment purposes). The procymidone, carbofuran and carbendazim triple colloidal gold immunochromatography kit can simultaneously extract, purify and detect residual procymidone, carbofuran and carbendazim in vegetables.
In a fourth aspect, the invention provides a method for simultaneously extracting, purifying and detecting procymidone, carbofuran and carbendazim residues in vegetables and fruits, which comprises the following steps:
(1) pretreatment of the sample: cutting vegetable samples into pieces, homogenizing, then placing 2g of the vegetable samples into an extraction purification tube, uniformly mixing, centrifuging, and collecting supernatant as a liquid to be detected;
(2) and (3) detection of the sample: adding the solution to be detected into the reaction hole, sucking and uniformly mixing, and inserting the sample pad end of the immunochromatography detection test strip into the reaction hole for detection;
(3) and analyzing the detection result.
The sample extraction purification tube, the reaction hole and the immunochromatography detection test strip are from the immunochromatography detection kit.
The specific detection method comprises the following steps:
(1) pretreatment of the sample: cutting vegetable sample, homogenizing, weighing 2g, adding into extraction purification tube, vortexing for 1min to mix well, centrifuging at 4000rpm for 2min, and collecting supernatant as to-be-detected liquid. Taking another vegetable sample, adding a certain amount of the standard solution of procymidone, carbofuran and carbendazim to ensure that the content of the procymidone is 0.2ppm, the content of the carbofuran is 0.02ppm and the content of the carbendazim is 2ppm, and then operating according to a pretreatment method to obtain a standard-added liquid to be detected.
(2) And (3) detection of the sample: 100 mu L of actual sample solution to be detected and standard sample solution to be detected are respectively taken by a pipette and are respectively placed in the reaction holes, and the solution is pumped up and down and mixed evenly. And after standing and reacting for 2min, inserting the sample pad end of the test strip into the reaction hole, timing for 5min, taking out the test strip, slightly scraping the sample pad to terminate the chromatography process, and judging the result.
(3) The results are shown in FIG. 2:
and (4) invalidation: the line C does not develop color, indicating that the test strip is invalid or improperly operated, and the newly unpacked test strip is used for retesting.
Negative: and C line color development, wherein the three detection lines are all color development, and the color is darker than or equal to the C line, which indicates that the concentration of the detection object in the sample is lower than the detection limit.
Positive: and C line is developed, the detection line is not developed or the color is lighter than the C line, namely the concentration of the detection object is more than or equal to the detection limit, and then the quantitative detection is carried out by using a standard method.
In the present invention, the procymidone hapten-carrier protein conjugate, carbofuran hapten-carrier protein conjugate, carbendazim hapten-carrier protein conjugate, and the procymidone antibody, carbofuran antibody and carbendazim antibody can be prepared by a method known in the art or can be obtained by purchase.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the three kinds of pesticide residues which are more used in vegetables and have more serious overproof conditions can be simultaneously extracted, purified and detected by the procymidone, carbofuran and carbendazim triple colloidal gold immunochromatography kit, and the detection object is clear and has strong pertinence.
And (II) the sample extraction and purification tube, the test strip and the kit are simple in preparation method and good in stability, and are suitable for mass production.
And (III) the detection method is simple, convenient and efficient, the total time consumption is about ten minutes from the pretreatment of the sample to the completion of the reaction and the judgment of the result, and the method is very suitable for large-scale on-site screening.
And (IV) the test strip has high sensitivity, low detection limit, good specificity and good stability, and is convenient for basic level inspectors to use.
Drawings
FIG. 1 is a schematic structural diagram of the immunochromatographic assay test strip of the present invention. Wherein, the sample comprises 1-sample pad, 2-T1 line, 3-T2 line, 4-T3 line, 5-C line, 6-nitrocellulose membrane, 7-water absorption pad and 8-bottom plate.
FIG. 2 is a schematic diagram illustrating the detection result determination of the test strip of the present invention.
Detailed Description
The invention provides a procymidone, carbofuran and carbendazim three-in-one colloidal gold immunochromatographic test strip, which is shown in figure 1: comprises a bottom plate (8), a sample pad (1), a nitrocellulose membrane (6) and a water absorption pad (7); the cellulose nitrate membrane (6) is sequentially coated with a first detection line (2) of a procymidone hapten-carrier protein conjugate, a second detection line (3) of a carbofuran hapten-carrier protein conjugate, a third detection line (4) of a carbendazim hapten-carrier protein conjugate and a quality control line (5) combined with a goat anti-mouse polyclonal antibody.
The invention provides a quick detection reagent box which consists of the test strip, a reaction hole containing a gold-labeled antibody and a sample extraction and purification tube. The reaction holes contain procymidone, carbofuran and carbendazim antibodies marked by colloidal gold. The bottom area of the reaction hole is 0.32cm2The volume of the reaction well was 365. mu.L, and a 96-well plate was used as a reaction well for mass production of the kit.
In the present invention, the average particle size of the colloidal gold solution used is about 40 nm. The procymidone hapten-carrier protein conjugate and an antibody thereof, the carbofuran hapten-carrier protein conjugate and an antibody thereof, the carbendazim hapten-carrier protein conjugate and an antibody thereof can be prepared by adopting a method known in the field or can be obtained by purchasing.
The colloidal gold immunochromatographic test strip and the quick detection kit provided by the invention can realize quick detection of procymidone, carbofuran and carbendazim at the same time, and have the advantages of accurate result and simple operation. The method comprises the following specific steps:
1. colloidal gold-labeled procymidone antibody, carbofuran antibody and carbendazim antibody were prepared and lyophilized in reaction wells.
2. Preparing a first detection line coated with a procymidone hapten-carrier protein conjugate, a second detection line coated with a carbofuran hapten-carrier protein conjugate, a third detection line coated with a carbendazim hapten-carrier protein conjugate and a nitrocellulose membrane combined with a quality control line of a goat anti-mouse polyclonal antibody.
3. And (3) assembling the nitrocellulose membrane prepared in the step (2) with a sample pad, a water absorption pad and a bottom plate to form the test strip.
4. 200mg of PSA, 200mg of C18 and 30mg of GCB are put into a 5mL centrifuge tube, and then 2mL of 0.05mol/L PBS 7.4 (namely PBS with pH 7.4) is added and mixed uniformly, thus obtaining the sample extraction purification tube filled with the sample extraction purification liquid.
5. And (3) filling the reaction holes in the step (1), the test paper strips in the step (3) and the sample extraction and purification tube in the step (4) into a kit, and storing the kit in a cool and dry place at 4-25 ℃ for later use.
6. And (4) pretreating a sample to be detected by using the sample extraction and purification tube in the step (4) to prepare a detection solution, transferring the detection solution into the reaction hole, and detecting by using the prepared test strip.
Preferably, the colloidal gold labeled with the procymidone antibody in the step 1 is used for adjusting the pH of 1mL of the colloidal gold by 0.2mol/L potassium carbonate solution, and the dosage is 5-10 μ L, and is preferably 7 μ L.
Preferably, the colloidal gold is labeled with the procymidone antibody in the step 1, and 1mL of the colloidal gold can label 5-10 μ g of the procymidone antibody, preferably 6 μ g.
Preferably, the carbofuran antibody is labeled with the colloidal gold in step 1, and the pH of 1mL of the colloidal gold is adjusted by 0.2mol/L potassium carbonate solution, and the dosage is 6-12 μ L, preferably 7 μ L.
Preferably, the colloidal gold is labeled with the carbofuran antibody in the step 1, and 1mL of the colloidal gold can be labeled with 5-8 μ g of the carbofuran antibody, preferably 5 μ g.
Preferably, the colloidal gold labeled carbendazim antibody in step 1 is used to adjust the pH of 1mL of colloidal gold with 0.2mol/L potassium carbonate solution in an amount of 3-7. mu.L, preferably 4. mu.L.
Preferably, the colloidal gold is labeled with carbendazim antibody in step 1, and 1mL of colloidal gold can be labeled with 5-12 μ g of carbendazim antibody, preferably 6 μ g.
The preparation method of the colloidal gold solution comprises the following steps: first, 2g of chloroauric acid (HAuCl)4) Dissolving the powder in 100mL of ultrapure water to obtain a chloroauric acid solution with the concentration of 2%; and heating 1L of ultrapure water to boil, adding 10mL of trisodium citrate solution with the mass concentration of 1%, continuing to heat for 8 minutes after boiling, adding 10mL of 2% chloroauric acid solution, heating for 10 minutes after the solution turns to wine red, stopping heating, naturally cooling, and fixing the volume to 1L to obtain the colloidal gold solution.
Preferably, 2 μ L, 3 μ L and 2 μ L of the colloidal gold-labeled procymidone antibody, the colloidal gold-labeled carbofuran antibody and the colloidal gold-labeled carbendazim antibody in step 1 are respectively placed in the reaction well, diluted to 60 μ L by using a freeze-drying protective solution, and freeze-dried.
Preferably, the concentration of the procymidone hapten-carrier protein conjugate bound on the first detection line in the step 2 is 0.10-0.20mg/mL, and preferably 0.15 mg/mL.
Preferably, the concentration of the carbofuran hapten-carrier protein conjugate bound on the second detection line in step 2 is 0.25-0.8mg/mL, preferably 0.6 mg/mL.
Preferably, the concentration of the carbendazim hapten-carrier protein conjugate bound on the third detection line in step 2 is 0.25-0.6mg/mL, preferably 0.5 mg/mL.
Preferably, the concentration of the goat anti-mouse polyclonal antibody bound on the quality control line in the step 2 is 0.25 mg/mL.
Preferably, the distance between the first detection line and the second detection line, the distance between the second detection line and the third detection line, and the distance between the third detection line and the quality control line in step 2 are all 4-5mm, preferably 4.3 mm. In the invention, if the distance between two adjacent detection lines is too close, the color development can be interfered; if the distance is too far, the chromatography time is too long and more detection solution is required.
Preferably, the width of the test strip in step 3 is 3-5mm, preferably 3.8mm, too narrow is not favorable for judging the result, and too wide needs more antigens, antibodies and detection solution, which is not favorable for saving the cost.
The procymidone, carbofuran and carbendazim triple colloidal gold immunochromatography kit can simultaneously extract, purify and detect procymidone, carbofuran and carbendazim in vegetables, has the advantages of simple and convenient operation, high result accuracy, low detection limit, good specificity and stable performance, can meet the requirement of on-site rapid determination, and is particularly suitable for large-scale screening.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
The procymidone antibodies used in the examples below were purchased from guangzhou excellent multi-biotechnology limited. The carbofuran antibody and the carbendazim antibody are purchased from the institute of food safety and science of south China university. The method for synthesizing the procymidone hapten-carrier protein conjugate reference document 1 (Zhouqiang. anti-chlorothalonil and procymidone monoclonal antibodies and characteristic analysis thereof [ D ]. Huazhong agriculture university), the carbofuran hapten-carrier protein conjugate reference document 2 (sun phoenix, Kanligong, and the like. carbofuran artificial antigen synthesis and immune application thereof [ J ]. northwest agriculture bulletin 2018), and the method for synthesizing the carbendazim hapten-carrier protein conjugate reference document 3 (Jianwenhui, Wuxiasheng, and the like. A carbendazim enzyme-linked immunosorbent rapid detection method [ J ]. food and machinery.2021).
Example 1 construction of Triplex immunochromatography assay kit for Pythium, carbofuran and carbendazim
The preparation method of the procymidone, carbofuran and carbendazim triple immunochromatography detection kit provided by the embodiment is as follows:
1. preparation of colloidal gold labeled procymidone antibody
1mL of the colloidal gold solution was added to 7. mu. L K2CO3The solution (0.2mol/L) is mixed evenly, then 6 mu g of the procymidone antibody is added and mixed evenly, and the mixture is kept stand for 10min at 25 ℃. Adding 50 μ L BSA blocking solution (mass fraction of 10%), mixing, and standing at 25 deg.C for 15 min. Centrifugation was carried out at 11000rpm for 15min at 4 ℃ and the supernatant carefully removed, followed by addition of 200. mu.L of a reconstituted solution (0.05mol/L PBS 7.4 containing 2% trehalose and 1% BSA).
2. Preparation of colloidal gold labeled carbofuran antibody
1mL of the colloidal gold solution was added to 7. mu. L K2CO3The solution (0.2mol/L) is mixed evenly, then 5 mu g carbofuran antibody is added and mixed evenly, and the mixture is kept stand for 10min at 25 ℃. Adding 50 μ L BSA blocking solution (mass fraction of 10%), mixing, and standing at 25 deg.C for 15 min. Centrifugation was carried out at 11000rpm for 15min at 4 ℃ and the supernatant carefully removed, followed by addition of 200. mu.L of a reconstituted solution (0.05mol/L PBS 7.4 containing 2% trehalose and 1% BSA).
3. Preparation of colloidal gold labeled carbendazim antibody
1mL of the colloidal gold solution was added to the reaction well, and 4. mu. L K was added2CO3Mixing the solution (0.2mol/L), adding 6 μ g carbendazim antibody, mixing, and standing at 25 deg.C for 10 min. Adding 50 μ L BSA blocking solution (mass fraction of 10%), mixing, and standing at 25 deg.C for 15 min. Centrifugation was carried out at 11000rpm for 15min at 4 ℃ and the supernatant carefully removed, followed by addition of 200. mu.L of a reconstituted solution (0.05mol/L PBS 7.4 containing 2% trehalose and 1% BSA).
4. Preparation of reaction well of kit
The colloidal gold-labeled procymidone antibody, the colloidal gold-labeled carbofuran antibody and the colloidal gold-labeled carbendazim antibody prepared in the above steps are respectively placed in reaction wells in a volume of 2. mu.L, 3. mu.L and 2. mu.L, and are diluted to 60. mu.L with a freeze-dried protective solution (containing 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% Tween 20 and 0.03% ProClin 300). Transferring into vacuum freeze drying machine, and freeze drying at-50 deg.C for 4 hr, at-25 deg.C for 4 hr, at-15 deg.C for 4 hr, at 5 deg.C for 2 hr, and at 25 deg.C for 10 hr.
5. Preparation of sample extraction purification tube
200mg of PSA, 200mg of C18 and 30mg of GCB are put into a 5mL centrifuge tube, and then 2mL of 0.05mol/LPBS 7.4 is added and mixed evenly, thus obtaining the sample extraction purification tube filled with the sample extraction purification liquid.
6. Kit assembly
The method comprises the steps of coating procymidone hapten-carrier protein conjugate, carbofuran hapten-carrier protein conjugate, carbendazim hapten-carrier protein conjugate and goat anti-mouse polyclonal antibody on a nitrocellulose membrane, wherein the first detection line is a T1 line (2), the second detection line is a T2 line (3), the third detection line is a T3 line (4) and the quality control line is a C line (5). And (3) assembling the nitrocellulose membrane (6), the sample pad (1), the water absorption pad (7) and the bottom plate (8) and cutting into the test strip. The bottom plate is made of PVC. The specific method comprises the following steps:
1) coating of procymidone hapten-carrier protein conjugate, carbofuran hapten-carrier protein conjugate, carbendazim hapten-carrier protein conjugate and goat anti-mouse polyclonal antibody
According to the method, a Saidolite CN95 film is selected as a coating basement film, the Pythium semiantigen-carrier protein conjugate is diluted to 0.15mg/mL by 0.02mol/L PBS and pH7.4 (containing 0.625% NaCl), the carbofuran semiantigen-carrier protein conjugate is diluted to 0.6mg/mL, the carbendazim semiantigen-carrier protein conjugate is diluted to 0.5mg/mL, and the goat anti-mouse polyclonal antibody is diluted to 0.25mg/mL by 0.02mol/L PBS and pH7.4 (containing 2% sucrose). The film scratching condition during coating was 0.8. mu.L/cm, and the distance between each detection line was 4.3 mm. And after coating, drying in an oven at 60 ℃ for 2-4 h.
2) Preparation of test paper strip
And (3) adhering the dried nitrocellulose membrane (6) to the middle position of the bottom plate (8), then respectively adhering the sample pad (1) and the absorbent paper (7) to the bottom plates at the two ends of the nitrocellulose membrane, and slightly pressing the nitrocellulose membrane to ensure the smooth chromatography of the detection solution and the gold-labeled antibody. Then cutting the whole plate into test strips with the thickness of 3.8mm by a slitter, sealing and packaging the test strips at 4 ℃, and unsealing the test strips when the test strips are used.
The quick detection reagent box provided by the invention consists of the test strip, a reaction hole containing a gold-labeled antibody and a sample extraction and purification tube. The reaction holes contain procymidone, carbofuran and carbendazim antibodies marked by colloidal gold. The bottom area of the reaction hole is 0.32cm2The volume of the reaction well is 365 mu L, and a 96-well plate can be directly used as a reaction well so as to prepare the kit in large batch.
The kit is stored at 4-25 ℃ under the condition of cool and dry, and the effective period is 12 months.
EXAMPLE 2 testing of actual samples
The kit prepared in example 1 was used for actual sample detection, and the specific steps were as follows:
(1) pretreatment of the sample: cutting vegetable sample, homogenizing, weighing 2g, adding into sample extraction purification tube, vortexing for 1min to mix well, centrifuging at 4000rpm for 2min, and collecting supernatant as to-be-detected liquid. Taking another vegetable sample, adding a certain amount of the standard solution of procymidone, carbofuran and carbendazim to ensure that the procymidone content is 0.2mg/kg, the carbofuran content is 0.02mg/kg and the carbendazim content is 2mg/kg, and then operating according to a pretreatment method to obtain the standard-added liquid to be detected.
(2) And (3) detection of the sample: 100 mu L of actual sample solution to be detected and standard sample solution to be detected are respectively taken by a pipette and are respectively placed in the reaction holes, and the solution is pumped up and down and mixed evenly. After standing for 2min, inserting the sample pad end of the test strip into the hole, timing for 5min, taking out the test strip, gently scraping the sample pad to terminate the chromatography process, and determining the result.
(3) The results are shown in FIG. 2:
and (4) invalidation: the line C does not develop color, indicating that the test strip is invalid or improperly operated, and the newly unpacked test strip is used for retesting.
Negative: c line color development, 3 detection lines color development, and the color is darker than or equal to the C line, which indicates that the concentration of the detection object in the sample is lower than the detection limit.
Positive: and C line color development, and detection line vanishing or color lighter than C line indicates that the concentration of the detection object is more than or equal to the detection limit, and then the quantitative detection is carried out by using a standard method.
EXAMPLE 3 detection Limit of the kit
This example is intended to test the detection limits of the procymidone, carbofuran and carbendazim triple immunochromatographic assay kit.
1. Detection limit of procymidone: adding 10mg/L of different volumes of the procymidone standard solution into the leeks to ensure that the adding standard concentration is as follows: 0.1mg/kg, 0.2mg/kg, 0.4mg/kg, 0.8mg/kg, and the detection was carried out in the same manner as in example 2. The result shows that when the standard concentration is 0.1mg/kg, the color development of the T1 line is similar to that of the C line, the result is negative, and when the concentration is 0.2mg/kg, the color of the T1 line is obviously lighter than that of the C line, and the result is positive, so the detection limit of the kit on procymidone is 0.2 mg/kg.
2. Detection limit of carbofuran: adding carbofuran standard solutions with different volumes of 10mg/L into the Chinese chives to ensure that the standard adding concentrations are as follows: 5. mu.g/kg, 0.01mg/kg, 0.02mg/kg, 0.04mg/kg, and the detection was carried out in the same manner as in example 2. The result shows that when the standard concentration is 5 mug/kg, the color development of the T2 line is similar to the color of the C line, the result is negative, and when the concentration is 0.01mg/kg, the color of the T2 line is obviously lighter than that of the C line, and the result is positive, so the detection limit of the kit on carbofuran is 0.01 mg/kg.
3. Detection limit of carbendazim: adding 10mg/L carbendazim standard solutions with different volumes into the Chinese chives to ensure that the adding standard concentration is as follows: 5. mu.g/kg, 0.01mg/kg, 0.02mg/kg, 0.04mg/kg, and the detection was carried out in the same manner as in example 2. The result shows that when the standard concentration is 5 mug/kg, the color development of the T2 line is similar to the color of the C line, the result is negative, and when the concentration is 0.01mg/kg, the color of the T2 line is obviously lighter than that of the C line, and the result is positive, so the detection limit of the kit on carbofuran is 0.01 mg/kg.
EXAMPLE 4 investigation of specificity of the kit
In this example, the specificity of the triple immunochromatography detection kit for procymidone, carbofuran and carbendazim was tested by the following method:
respectively preparing common pesticides such as fipronil, chlorpyrifos, acetamiprid, fenpropathrin, imidacloprid and the like into standard solutions of 1mg/kg, transferring 100 mu L of the standard solutions into reaction holes, and pumping up and down to mix uniformly. And (3) after standing and reacting for 2min, inserting the sample pad end of the test strip into the reaction hole, timing for 5min, taking out the test strip, slightly scraping the sample pad, and judging the result. The results show that the C line, the T1 line, the T2 line and the T3 line are obvious in color development, and therefore the pesticides do not have cross reaction with antibodies, and the specificity of the test strip is good.
Example 5 comparison of kit Performance
The kit of the invention is compared with a commercially available kit or a kit in a published patent, and the specific steps are as follows:
1. the detection limits are compared in table 1:
TABLE 1
As can be seen from Table 1, the detection limit of the kit of the invention is lower than or equal to that of other commercially available kits, and the kit of the invention can simultaneously extract, purify and detect three types of pesticide residues, while the products on the market are single detection type kits. The detection performance of the kit on procymidone, carbofuran and carbendazim is better, more importantly, the simultaneous extraction, purification and detection of three different types of pesticide residues are realized, the market blank is filled, the resources are effectively saved, and the kit is more suitable for large-scale field screening.
The commercially available kits were purchased from Sumei Sci Biotech, Inc., and the product number of the commercially available kit 1 was MK107A1, the product number of the commercially available kit 2 was MK108A1, and the product number of the commercially available kit 3 was MK106C 1.
2. The pretreatment reagent comparison is shown in table 2:
TABLE 2
As is clear from Table 2, the pretreatment reagents were slightly different for different test subjects. The kit of the invention contains 200mg of PSA and 200mg of C18And 2mL of 0.05mol/L PBS 7.4 of 30mg GCB is used as an extraction purification solution, 3 different pesticide residues can be simultaneously extracted and purified into a solution to be detected, and the purification reagent can effectively adsorb pigments, so that the interference for judging the color of the test strip is reduced, the sensitivity of the detection method is improved, and the detection limit is reduced. The method can simultaneously extract 3 detection objects and carry out purification detection by one-time treatment, greatly saves manpower and material resources, improves the detection efficiency, and can provide powerful technical support for food safety basic supervision, food inspection, large-scale activity guarantee and the like.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The test strip for the triple immunochromatography detection of procymidone, carbofuran and carbendazim is characterized by comprising a sample pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the nitrocellulose membrane is sequentially provided with three detection lines and a quality control line in the direction from the sample pad to the water absorption pad, the three detection lines are respectively coated with a procymidone hapten-carrier protein conjugate, a carbofuran hapten-carrier protein conjugate and a carbendazim hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse polyclonal antibody;
the sample pad, the nitrocellulose membrane and the absorbent pad are alternately laminated and stuck on the bottom plate at intervals of 2-3mm in sequence.
2. The test strip of claim 1, wherein the concentration of the procymidone hapten-carrier protein conjugate coated on the test line is 0.10-0.20mg/mL, preferably 0.15 mg/mL; the concentration of the carbofuran hapten-carrier protein conjugate coated on the detection line is 0.25-0.8mg/mL, preferably 0.6 mg/mL; the concentration of the carbendazim hapten-carrier protein conjugate coated on the detection line is 0.25-0.6mg/mL, and preferably 0.5 mg/mL; the concentration of the goat anti-mouse polyclonal antibody coated on the quality control line is 0.25 mg/mL;
the structures of the procymidone hapten, the carbofuran hapten and the carbendazim hapten are respectively shown in formulas 1) to 3):
3. the test strip of claim 1, wherein the distances between the detection lines on the nitrocellulose membrane and between the detection line near the absorbent pad end and the quality control line are both 4-5mm, preferably 4.3 mm; the width of the test strip is 3-5mm, preferably 3.8 mm.
4. The test strip of any one of claims 1-3, wherein the sample pad is made of nitrocellulose membrane.
5. The triple immunochromatography detection kit for procymidone, carbofuran and carbendazim is characterized by comprising a sample extraction and purification tube, a reaction substrate with reaction holes and the test strip of any one of claims 1 to 4.
6. The kit of claim 5, wherein the sample extraction purge tube is prepared by a method comprising: 200mg of PSA, 200mg of C18And 30mg of GCB are placed in a 5mL centrifuge tube, and then 2mL of 0.05mol/L PBS with pH7.4 is added and mixed uniformly, thus obtaining the sample extraction purification tube filled with the sample extraction purification liquid.
7. The kit according to claim 5, wherein the reaction well is coated with a colloidal gold-labeled procymidone antibody, a colloidal gold-labeled carbofuran antibody and a colloidal gold-labeled carbendazim antibody;
the preparation method of the reaction hole comprises the following steps: adding 2 mu L of colloidal gold-labeled procymidone antibody solution, 3 mu L of colloidal gold-labeled carbofuran antibody solution and 2 mu L of colloidal gold-labeled carbendazim antibody solution into a reaction hole, diluting to 60 mu L by using a freeze-drying protective solution, and carrying out vacuum freeze-drying;
the preparation method of the colloidal gold solution comprises the following steps: dissolving 2g of chloroauric acid powder in 100mL of ultrapure water to obtain a chloroauric acid solution with the concentration of 2%; heating 1L of ultrapure water to boil, adding 10mL of trisodium citrate solution with the mass concentration of 1%, continuing to heat for 8 minutes after boiling, adding 10mL of 2% chloroauric acid solution, heating for 10 minutes after the solution turns to wine red, stopping heating, naturally cooling, and fixing the volume to 1L to obtain the colloidal gold solution;
the preparation method of the colloidal gold labeled procymidone antibody solution comprises the following steps: 1mL of the colloidal gold solution was added to 0.2mol/L K2CO35-10 mu L of the solution, uniformly mixing, adding 6 mu g of procymidone antibody, uniformly mixing, and standing for 10min at 25 ℃; then adding 50 mu L of 10% BSA blocking solution, mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution to obtain the final product;
the preparation method of the colloidal gold labeled carbofuran antibody solution comprises the following steps: 1mL of the colloidal gold solution was added to 0.2mol/L K2CO3Mixing 6-12 μ L of the solution, adding 5 μ g of carbofuran antibody, mixing, and standing at 25 deg.C for 10 min; then adding 50 mu L of 10% BSA blocking solution, mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution to obtain the final product;
the preparation method of the colloidal gold labeled carbendazim antibody solution comprises the following steps: 1mL of colloidal gold solution was added to the reaction well, and 0.2mol/L K was added2CO3Mixing the solution 3-7 μ L, adding carbendazim antibody 6 μ g, mixing, and cooling to 25 deg.CStanding for 10 min; then adding 50 mu L of 10% BSA blocking solution, mixing uniformly, and standing for 15min at 25 ℃; centrifuging at 4 deg.C and 11000rpm for 15min, removing supernatant, and adding 200 μ L of the complex solution to obtain the final product;
the compound solution is as follows: 0.05mol/L PBS containing 2% trehalose and 1% BSA, pH7.4;
the freeze-drying protective solution comprises: 2% BSA, 5% trehalose, 1% PEG 20000, 0.5% tween 20 and 0.03% ProClin 300, in water.
8. The kit according to claim 7, wherein the vacuum freeze-drying conditions of the reaction wells are: -50 ℃ for 4h, -25 ℃ for 4h, -15 ℃ for 4h, 5 ℃ for 2h, 25 ℃ for 10 h.
9. Use of a kit according to any one of claims 5 to 8 in procymidone, carbofuran and carbendazim assays; the use is for non-disease diagnostic and therapeutic purposes.
10. A method for simultaneously extracting, purifying and detecting procymidone, carbofuran and carbendazim residues in vegetables is characterized by comprising the following steps:
(1) pretreatment of the sample: cutting vegetable samples, homogenizing, taking 2g of vegetable samples in a sample extraction and purification tube, mixing uniformly, centrifuging, and collecting supernatant as a liquid to be detected;
(2) and (3) detection of the sample: adding the solution to be detected into the reaction hole, sucking and uniformly mixing, and inserting the sample pad end of the immunochromatography detection test strip into the reaction hole for detection;
(3) analyzing the detection result;
the sample extraction purification tube, the reaction hole and the immunochromatography detection test strip are from the kit of any one of claims 5 to 8.
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