CN111289752A - Test strip and method for detecting dicofol - Google Patents
Test strip and method for detecting dicofol Download PDFInfo
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- CN111289752A CN111289752A CN202010146384.3A CN202010146384A CN111289752A CN 111289752 A CN111289752 A CN 111289752A CN 202010146384 A CN202010146384 A CN 202010146384A CN 111289752 A CN111289752 A CN 111289752A
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- dicofol
- pad
- test strip
- conjugate
- hapten
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- UOAMTSKGCBMZTC-UHFFFAOYSA-N dicofol Chemical compound C=1C=C(Cl)C=CC=1C(C(Cl)(Cl)Cl)(O)C1=CC=C(Cl)C=C1 UOAMTSKGCBMZTC-UHFFFAOYSA-N 0.000 title claims abstract description 90
- 238000012360 testing method Methods 0.000 title claims abstract description 55
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Images
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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
- G01N33/58—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
- 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
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a test strip and a method for detecting dicofol. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction film, a water absorption pad and a bottom plate, wherein the reaction film is provided with a detection line coated with dicofol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with a dicofol monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting dicofol in a fruit sample by applying the test strip. The test strip provided by the invention has the advantages of simple operation, high sensitivity, high detection speed, low cost, suitability for screening large-batch samples and the like, and can meet the requirements of food supervision departments in China on-site monitoring and detection.
Description
Technical Field
The invention relates to a test strip and a method for detecting dicofol, in particular to a colloidal gold test strip for detecting dicofol, which is particularly suitable for detecting residual dicofol in fruits.
Background
Dicofol is an organic chlorine acaricide, can be used for preventing and treating acarid on crops such as cotton, fruit trees, vegetables, etc., has strong contact poisoning and stomach poisoning effects on adult acarid, nymph and ovum, and has long effective period and quick-acting period. The research shows that the medicine has certain toxicity to human and livestock, the main toxic symptoms are headache, dizziness, hyperhidrosis, chest distress, mydriasis, blurred vision, nausea, vomit and diarrhea, and contact dermatitis can be caused by local contact. The dicofol is forbidden in China, and the national standard GB2763-2019 maximum limit of pesticide residue in food safety national standard food also provides that the residue limit of the dicofol in fruits is 1 mg/kg.
The reported methods for detecting dicofol mainly comprise gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry and other instrument methods. The methods are operated under laboratory conditions, the sample pretreatment is complicated and time-consuming, expensive instruments and equipment are required, the detection cost is high, the time consumption is long, the operation is complex, the method has great limitation in the practical application process, and the requirement for rapidly detecting a large number of samples and field samples is difficult to meet. Therefore, the colloidal gold test strip which is simple and quick and is suitable for residual dicofol in fruits is developed, a large number of samples can be screened and monitored on site, and detection work of food supervision departments and the like in China can be better met.
Disclosure of Invention
The invention aims to provide a colloidal gold test strip capable of detecting residual dicofol in fruits, and provides a detection method which is efficient, accurate, simple and convenient and is suitable for field monitoring and large-scale sample screening.
The test strip for detecting dicofol provided by the invention comprises a sample absorption pad, a conjugate release pad, a reaction film, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with dicofol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody; the conjugate release pad is sprayed with a dicofol monoclonal antibody-colloidal gold marker.
The dicofol monoclonal antibody is prepared by taking a dicofol hapten-carrier protein conjugate as an immunogen.
The dicofol hapten-carrier protein conjugate is obtained by coupling dicofol hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the dicofol hapten is obtained by reacting o-chlorophenylacetic acid, chlorobenzene and chloral under the catalysis of sulfuric acid, and the molecular structural formula is as follows:
the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.
The bottom plate can be a PVC bottom plate or other hard non-absorbent materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorption pad is absorbent paper; the reaction membrane can be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the test strip, which comprises the steps of:
1) preparing a conjugate release pad sprayed with dicofol monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with dicofol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
Specifically, the steps include:
1) o-chlorophenylacetic acid, chlorobenzene and chloral react under the catalysis of sulfuric acid to prepare dicofol hapten;
2) the dicofol hapten and carrier protein are coupled to prepare a dicofol hapten-carrier protein conjugate;
3) immunizing a mouse by using the dicofol hapten-carrier protein conjugate, and fusing and screening spleen cells and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the dicofol monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) coating the dicofol hapten-carrier protein conjugate and the goat anti-mouse anti-antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;
6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;
7) adding the prepared dicofol monoclonal antibody into the prepared colloidal gold to obtain a dicofol monoclonal antibody-colloidal gold marker;
8) spraying dicofol monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 deg.C for 1 hr, taking out, and storing in dry environment;
9) soaking the sample absorption pad in 0.5% bovine serum albumin-containing phosphate buffer solution with pH of 7.2 and 0.1mol/L for 2h, and drying at 37 deg.C for 2 h;
10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. And finally cutting into small strips with the width of 3mm, adding a plastic box, carrying out vacuum packaging, and storing for 12 months at the temperature of 4-30 ℃.
The invention also aims to provide a method for detecting residual dicofol in fruits by using the test strip, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using a test strip;
(3) and analyzing the detection result.
The dicofol rapid detection test strip adopts a highly specific antibody antigen reaction and immunochromatographic analysis technology, a dicofol monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and dicofol in a sample is combined with the dicofol monoclonal antibody-colloidal gold marker on the conjugate release pad in a flowing process to form a drug-antibody-colloidal gold marker. The medicament in the sample and the dicofol hapten-carrier protein conjugate on the reaction film detection line compete to combine with the dicofol monoclonal antibody-colloidal gold marker, and whether the dicofol residue is contained in the sample liquid to be detected is judged according to the depth of a red strip of the detection line.
During detection, a sample is treated and then dropped into a test strip card hole, when the concentration of the dicofol in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker can be combined with the dicofol hapten-carrier protein conjugate fixed on a reaction film in the chromatography process, a red strip appears on a detection line (T) and a quality control line (C), and the color development of the T line is deeper than that of the C line or is consistent with that of the C line; if the concentration of dicofol in the sample is equal to or higher than the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the dicofol, so that no red band or less coloration will appear at the T-line due to the competitive reaction with the dicofol hapten-carrier protein conjugate. As shown in fig. 3.
Negative: when the quality control line (C) shows a red strip, the detection line (T) also shows a red strip, and the color of the line (T) is close to or deeper than that of the line (C), the line (C) is judged to be negative.
Positive: and when the quality control line (C) shows a red strip, the detection line (T) does not show color or the color of the line (T) is lighter than that of the line (C), the test line is judged to be positive.
And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period. The method for detecting residual dicofol by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a synthesis diagram of dicofol hapten.
FIG. 2 is a schematic cross-sectional view of a test strip.
FIG. 3 is a diagram showing the test result of the test strip.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 preparation of test strip for detecting dicofol
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with dicofol monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with dicofol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
The following steps are detailed:
1. synthesis of dicofol hapten (the synthetic route is shown in figure 1)
Dissolving 1.7g of o-chlorophenylacetic acid in 100mL of carbon tetrachloride, adding 1.12g of chlorobenzene, fully stirring, adding 1.47g of trichloroacetaldehyde, cooling to 10-15 ℃, dropwise adding 10mL of 10% sulfuric acid solution, continuously stirring for reacting for 4 hours, adding 100mL of 0-5 ℃ cold water after the reaction is finished, transferring to a separating funnel, vibrating, standing for layering, extracting the water phase with trichloromethane for three times, 80mL each time, and combining the organic phases; and (3) washing the carbon tetrachloride with water again, combining the carbon tetrachloride and the chloroform extract, evaporating the mixture to dryness to obtain red oily matter, purifying the red oily matter by using a silica gel column, and eluting and separating the red oily matter by using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 6:1 to obtain the dicofol hapten.
2. Preparation of immunogens
Taking 14mg of dicofol hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving, adding 8.2mg of N-hydroxysuccinimide (NHS) and 15.7mg of carbodiimide (EDC), and reacting at room temperature for 3h to obtain a hapten solution A; taking 50mg of Bovine Serum Albumin (BSA), and adding 0.05mol/L PB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times every day to obtain the dicofol hapten-BSA conjugate which is the immunogen, and subpackaging and storing at-20 ℃.
3. Preparation of coating antigen
Taking 8.1mg of dicofol hapten, adding 1mL of 1, 4-dioxane for dissolution, adding 6.1mg of NHS and 9.7mg of EDC, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving Ovalbumin (OVA)50mg in PB buffer solution 0.05mol/L to obtain solution B; dripping the A solution into the B solution, reacting at 4 deg.C for 12h, dialyzing and purifying with 0.02mol/L PBS for 3 days, changing the solution 3 times per day to obtain dicofol hapten-OVA conjugate, i.e. coating antigen, packaging, and storing at-20 deg.C.
4. Preparation of dicofol monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Using hybridoma cellsMaking frozen stock solution into 1 × 106Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
6. Preparation of dicofol monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5mL of 1% trisodium citrate under continuous stirring at high temperature, stirring at constant speed, heating until the solution is bright red, cooling to room temperature, recovering the volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, is transparent and bright, has no sediment or floating objects, and is wine red when observed in sunlight.
(2) Preparation of dicofol monoclonal antibody-colloidal gold marker
Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution, adding the dicofol monoclonal antibody into the colloidal gold solution according to the standard that 20-50 mu g of antibody is added into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30 min; after standing for 10min, 10% BSA was added to make the final concentration of the solution in colloidal gold 1%, and the solution was allowed to stand for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.
Redissolving buffer solution: 0.1 to 0.3 percent of BSA, 0.05 to 0.2 percent of Tween-80 and 0.02mol/L of phosphate buffer solution with pH of 7.2.
7. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.5% BSA, pH 7.2, 0.5mol/L phosphate buffer, soaked for 1h, and baked at 37 deg.C for 3 h. And uniformly spraying the prepared dicofol monoclonal antibody-colloidal gold marker on a conjugate release pad by using an Isoflow film spraying instrument, spraying 0.01mL of dicofol monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing in an environment at 37 ℃ (humidity is less than 20%) for 60min, taking out, and placing in a dry environment (humidity is less than 20%) for storage.
8. Preparation of sample absorbent pad
The sample absorption pad is soaked in 0.5 percent bovine serum albumin-containing phosphate buffer solution with the pH value of 7.2 and the mol/L of 0.1 for 2 hours, and is dried for 2 hours at the temperature of 37 ℃ for standby.
9. Preparation of the reaction film
Coating the dicofol hapten-ovalbumin conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.
Coating process: diluting dicofol hapten-ovalbumin conjugate to 1mg/mL by using phosphate buffer solution, and coating the dicofol hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01mol/L, pH 7.4.4 phosphate buffer and coated on a quality control line (line C) on a nitrocellulose membrane in an amount of 1.0. mu.L/cm using an Isoflow dot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
10. Assembly of test strips
According to the section structure of the test strip shown in the attached figure 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially adhered to a PVC bottom plate (7); the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC base plate; the reaction membrane is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; cutting the test strip into small strips with the width of 3mm by a machine, putting the small strips into a special plastic card, and storing the small strips in an environment with the temperature of 4-30 ℃ for 12 months.
Example 2 detection of dicofol in fruit
1. Sample pretreatment
Before detection, the sample is deslimed and then cut into fragments smaller than 1cm or small finely-divided dices; weighing (2.00 +/-0.05) g of chopped sample into a 15mL centrifuge tube, adding 4mL of phosphate buffer solution, covering a cover, manually shaking for 30s, standing for 1min, and taking supernatant as sample solution to be detected.
2. Detection with test strips
Sucking 70 mu L of sample liquid to be detected by a micropipette and vertically dripping the sample liquid into the sample adding hole; the liquid flow was started, the reaction was carried out for 10min, and the results were judged.
3. Analyzing the results of the detection
Negative (-): the color development of the T line is darker than that of the C line or consistent with that of the C line, which indicates that the concentration of the dicofol in the sample is lower than the detection limit, as shown in FIGS. 3a and 3 b.
Positive (+): the T line is lighter than the C line or the T line is not colored, which indicates that the concentration of the dicofol in the sample is equal to or higher than the detection limit, as shown in FIGS. 3C and 3 d.
And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIGS. 3e and 3 f.
Example 3 sample testing example
1. Limit of detection test
Taking blank apple and pear samples, respectively adding dicofol into the samples until the final concentrations are 0.5mg/kg, 1.0mg/kg and 2.0mg/kg, taking test strips for detection, and repeatedly measuring each sample for three times.
When the test strip is used for detecting apple and pear samples, when no dicofol exists and the addition concentration of the dicofol is 0.5mg/kg, the test strip shows that the color development of a T line is deeper than that of a C line or is consistent with that of the C line and is negative; when the concentration of the dicofol is 1.0mg/kg and 2.0mg/kg, the test strip shows that the T line color development is lighter than the C line color development or the T line color is not developed, and the test strip is positive, which shows that the detection limit of the test strip on the dicofol in the fruits is 1.0 mg/kg.
2. Test for false positive and false negative rates
Taking blank apple and pear samples and 20 parts of positive apple and pear samples added with dicofol to the final concentration of 1.0mg/kg respectively, detecting by using test strips produced in 3 batches respectively, and calculating the negative and positive rates.
The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The test strip for detecting the dicofol can be used for rapidly detecting the residual dicofol in the fruit sample.
3. Specificity test
When the test strip is used for detecting other organic chlorine pesticides such as 10mg/kg dichlorodiphenyl trichloroethane, hexachloro cyclohexane, dicofol, quintozene and the like, the test strip shows that the color development of a T line is darker than or consistent with that of a C line, and the test strip is negative, so that the test strip has no cross reaction to the drugs.
Claims (5)
1. A test strip for detecting dicofol comprises a sample absorption pad, a conjugate release pad, a reaction film, a water absorption pad and a bottom plate, wherein the reaction film is provided with a detection line coated with a dicofol hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and a dicofol monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; the dicofol monoclonal antibody is prepared by taking a dicofol hapten-carrier protein conjugate as an immunogen; the dicofol hapten-carrier protein conjugate is obtained by coupling dicofol hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, and is characterized in that the dicofol hapten is obtained by reacting o-chlorophenylacetic acid, chlorobenzene and chloral under the catalysis of sulfuric acid, and the molecular structural formula is as follows:
2. the strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane, and the absorbent pad are sequentially attached to the base plate.
3. The strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.
4. A method of making the test strip of any one of claims 1-3, comprising the steps of:
1) preparing a conjugate release pad sprayed with dicofol monoclonal antibody-colloidal gold marker;
2) preparing a reaction film with a detection line coated with dicofol hapten-carrier protein conjugate and a quality control line coated with goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
5. A method for detecting residual dicofol in a fruit sample, comprising the steps of:
1) pretreating a sample;
2) performing a test using the test strip of any one of claims 1-3;
3) and analyzing the detection result.
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Cited By (1)
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| CN113637642A (en) * | 2021-09-16 | 2021-11-12 | 江南大学 | Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain |
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