CN113960307A - Test strip for detecting isoprothiolane and preparation method and application thereof - Google Patents
Test strip for detecting isoprothiolane and preparation method and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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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- 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
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Abstract
The invention provides a test strip for detecting isoprothiolane and a preparation method and application thereof, the test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with an isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with an isoprothiolane monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting isoprothiolane in a sample by applying the test strip. The test strip and the detection method provided by the invention have the advantages of simple operation, high sensitivity, high detection speed, low cost and no limitation of detection equipment, and can realize rapid detection and on-site monitoring on large batches of isoprothiolane samples.
Description
Technical Field
The invention relates to detection of isoprothiolane, in particular to a colloidal gold test strip for detecting isoprothiolane, which is particularly suitable for detecting isoprothiolane residues in tobacco leaves.
Background
Isoprothiolane (Isoprothiolane), also known as Isoprothiolane and Fushiyi, has a chemical name of 1, 3-dithiolane-2-isopropylidene malonate, is a high-efficiency systemic fungicide, has a narrow fungicide spectrum, is a specific drug for preventing and treating rice blast, particularly has an excellent effect on panicle neck blast, can be systemically transmitted through roots, stems and leaves of plants, and can reach the heart and leaves. It is also effective on rice seedling plague and sclerotinia scleotiorum, and can be used for treating rice planthopper and leafhopper in large area, and has regulating effect on plant growth.
However, with the application of the isoprothiolane in a large amount, the surrounding environment is possibly polluted, the isoprothiolane can be left in agricultural products and pose potential health threats to human beings, so the problem of residue in the production of fruits, vegetables and tobaccos is receiving more and more attention. China sets the maximum residue limit standard of isoprothiolane aiming at different crops, wherein the maximum residue limit of watermelons is 0.1mg/kg, the maximum residue limit of rice is 1mg/kg, and the maximum residue limit of tobacco is 2 mg/kg.
At present, the literature only shows the detection report of the residual quantity of the bactericides in grains, fruits and vegetables, but does not show the report of a measuring method of the residual quantity in tobacco leaves. The domestic and foreign detection methods for isoprothiolane residues mainly comprise gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), Gas Chromatography (GC) and the like. The instrument and the method have the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of a detection sample is complicated and time-consuming, the sample also needs to be extracted and purified, and meanwhile, the instrument and the detection method need expensive large-scale instruments and equipment and are equipped with professional detection technicians for operation and management, so that the field large-scale detection cannot be carried out, the timeliness is poor, and the popularization is difficult. Therefore, it is an urgent problem to develop a product and a method which are not limited by the detection equipment and can realize rapid detection of a large number of samples.
Disclosure of Invention
The invention aims to overcome the characteristics of high equipment dependence and incapability of realizing rapid detection on a large batch of samples in the conventional method for detecting isoprothiolane, and provides a test strip which is simple to operate, high in sensitivity, high in detection speed, low in cost and not limited by detection equipment, a preparation method and application thereof so as to realize rapid detection and on-site monitoring on a large batch of isoprothiolane samples.
In order to realize the purpose of the invention, the invention provides a test strip for detecting isoprothiolane, which comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; the conjugate release pad is sprayed with a isoprothiolane monoclonal antibody-colloidal gold marker.
The isoprothiolane monoclonal antibody is prepared by taking an isoprothiolane hapten-carrier protein conjugate as an immunogen.
The isoprothiolane hapten-carrier protein conjugate is obtained by coupling isoprothiolane hapten and carrier protein, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the isoprothiolane hapten is obtained by taking diisopropyl malonate as a raw material and carrying out condensation reaction with carbon disulfide and 3, 4-dichlorobutyric acid in strong alkali, 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.
The invention also provides a method for preparing the test strip, which comprises the following steps:
1) preparing a conjugate release pad sprayed with a isoprothiolane monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
Specifically, the steps include:
1) carrying out condensation reaction on diisopropyl malonate serving as a raw material, carbon disulfide and 3, 4-dichlorobutyric acid in strong alkali to prepare isoprothiolane hapten;
2) coupling the isoprothiolane hapten with carrier protein to prepare an isoprothiolane hapten-carrier protein conjugate;
3) immunizing a mouse by using the isoprothiolane hapten-carrier protein conjugate, and fusing and screening splenocytes of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting the isoprothiolane monoclonal antibody;
4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;
5) coating the isoprothiolane 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 isoprothiolane monoclonal antibody into the prepared colloidal gold to obtain an isoprothiolane monoclonal antibody-colloidal gold marker;
8) spraying the isoprothiolane monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 2h, taking out, and storing in a dry environment for later use;
9) soaking a sample absorption pad in 0.02mol/L phosphate buffer solution containing 1% bovine serum albumin and having pH of 7.2 for 2h, and drying at 37 ℃ for 2h for later use;
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. Finally cutting into small strips with the width of 3.95mm, putting the small strips into a special plastic card shell, sealing the card shell by an aluminum foil bag, and storing the card shell for 12 months at the temperature of 4-30 ℃. Covering 1/3 of the conjugate release pad with the sample absorbent pad can prolong the observation time of the test result, so that the sample absorbent pad can sufficiently absorb the test liquid and sufficiently react with the gold-labeled antibody, thereby reducing errors.
The invention also provides a method for detecting the isoprothiolane residue in a sample by applying the test strip, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using a test strip;
(3) and analyzing the detection result.
The invention relates to a test paper strip for quickly detecting isoprothiolane, which adopts a highly specific antibody-antigen reaction and immunochromatography analysis technology to fix isoprothiolane monoclonal antibody-colloidal gold marker on a conjugate release pad, wherein isoprothiolane in a sample is combined with the isoprothiolane monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the isoprothiolane-antibody-colloidal gold marker. The isoprothiolane in the sample and the isoprothiolane hapten-carrier protein conjugate on the reaction film detection line compete to combine with the isoprothiolane monoclonal antibody-colloidal gold marker, and whether the sample solution to be detected contains isoprothiolane residues or not is judged according to the depth of a red strip of the detection line.
During detection, a sample is treated and then dripped into a sample absorption pad, when the concentration of isoprothiolane in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with the isoprothiolane hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, a red strip is respectively generated 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 isoprothiolane in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold label will bind to all of the isoprothiolane, and thus no red band will appear or the color will be lighter than that of the C line at the T line because the competitive reaction will not bind to the isoprothiolane hapten-carrier protein conjugate. As shown in fig. 2.
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, no limitation of detection equipment, suitability for various units, simple storage and long quality guarantee period. The method for detecting the isoprothiolane residue by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
Fig. 1 is a schematic diagram of a cross-sectional structure of a test strip, in which: 1. a sample absorbing pad; 2. a conjugate release pad; 3. a reaction film; 4. a water absorbent pad; 5. detecting lines; 6. a quality control line; 7. a base plate;
FIG. 2 is a diagram showing the test result of the test strip;
FIG. 3 is a synthetic diagram of isoprothiolane hapten.
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. In addition, various changes or modifications of the present invention may be made by those skilled in the art within the scope defined by the appended claims, and these changes or modifications should also fall within the scope of the present invention.
Example 1 preparation of test strip for detecting isoprothiolane
The preparation method of the test strip mainly comprises the following steps:
1) preparing a conjugate release pad sprayed with a isoprothiolane monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
The following steps are detailed:
1. synthesis of isoprothiolane hapten (the synthetic route is shown in figure 3)
Taking 1.88g of diisopropyl malonate, adding 20mL of carbon disulfide, stirring at room temperature, fully and uniformly mixing, adding 13mL of 2mol/L KOH solution, stirring at room temperature for 1h, adding 1.56g of 3, 4-dichlorobutyric acid, supplementing 5mL of 2mol/L KOH solution, 3.8g of tetrabutylammonium bromide and 80mL of acetonitrile, heating and refluxing for 4h, stopping the reaction, carrying out rotary evaporation, removing the acetonitrile and the carbon disulfide, adding 80mL of water, adding 6mol/L HCl to adjust the pH value to 6, adding 100mL multiplied by 3 of ethyl acetate, extracting for three times, combining organic phases, washing with water, drying and evaporating anhydrous sodium sulfate to dryness to obtain yellow oily substances, loading on a silica gel column, eluting, separating and purifying by using a petroleum ether-ethyl acetate mixed solution with the volume ratio of 3:1 to obtain 0.76g of acetic acid-isoprothiolane hapten.
2. Preparation of immunogens
Taking 12.9mg of isoprothiolane hapten, adding 1mL of tetrahydrofuran for dissolving and clarifying, adding 8.5mg of N-hydroxysuccinimide (NHS) and 14.8mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain a hapten activation solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1mol/L CB9.18mL for dissolving to obtain a solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging and packaging to obtain isoprothiolane-BSA conjugate, namely the immunogen.
3. Preparation of coating antigen
Taking 7.7mg of isoprothiolane hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving and clarifying, adding 5.1mg of NHS and 9.7mg of EDC, fully dissolving and uniformly mixing, and reacting at room temperature for 2 hours to obtain hapten activating solution A; dissolving Ovalbumin (OVA)50mg in 0.1mol/L CB9.17mL to obtain solution B; dropwise adding the solution A into the solution B, reacting at room temperature for 4h, stopping the reaction, dialyzing and purifying with 0.02mol/L PBS buffer solution for 3 days, changing the solution 3 times per day, centrifuging and packaging to obtain isoprothiolane-OVA conjugate, namely the coating antigen.
4. Preparation of isoprothiolane monoclonal antibody
(1) Animal immunization
Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.
(2) Cell fusion and cloning
Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.
(3) Cell cryopreservation and recovery
Making hybridoma cell into 1 × 10 with frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
(4) Preparation and purification of monoclonal antibodies
An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse anti-antibody
The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.
6. Preparation of isoprothiolane 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 1.5mL of 1% trisodium citrate under continuous stirring at high temperature, stirring and heating at constant speed 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 isoprothiolane 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 isoprothiolane monoclonal antibody into the colloidal gold solution according to the standard that 5-50 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: 0.02mol/L phosphate buffer solution with BSA of 0.1-0.5 percent, sucrose of 2-4 percent and pH of 7.2.
7. Preparation of conjugate Release pad
The conjugate release pad was soaked in 0.02mol/L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, soaked for 2h, and dried at 37 deg.C for use. And (3) uniformly spraying the prepared isoprothiolane monoclonal antibody-colloidal gold marker on a conjugate release pad by using a Bio dot film scratching instrument, spraying 0.01mL isoprothiolane monoclonal antibody-colloidal gold marker on every 1cm of conjugate release pad, placing the conjugate release pad in an environment (humidity is less than 20%) at 37 ℃ for 2h, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.
8. Preparation of sample absorbent pad
The sample absorbing pad is soaked in 0.02mol/L phosphate buffer solution containing 1% BSA and having pH of 7.2 for 2h, and dried at 37 ℃ for 2h for later use.
9. Preparation of the reaction film
Coating the isoprothiolane hapten-OVA 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 isoprothiolane hapten-OVA conjugate to 1mg/mL with 0.01mol/L, pH 7.2.2 phosphate buffer, and coating the conjugate on a detection line (T line) on a nitrocellulose membrane by using a Bio dot film cutting 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 7.2.2 phosphate buffer and coated on a control line on nitrocellulose membrane (line C) with a Bio dot striping machine in an amount of 1.0. mu.L/cm. And (3) drying the coated reaction membrane at 37 ℃ for 16h for later use.
10. Assembly of test strips
According to the section structure of the test strip shown in the attached figure 1, 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 3.95mm by a machine, placing the small strips in a specially-made plastic card shell, sealing the card shell by an aluminum foil bag, and storing the card shell in an environment at the temperature of 4-30 ℃ for 12 months.
Example 2 detection of isoprothiolane in samples
1. Sample pretreatment
Crushing or shearing the sample into pieces smaller than 1 cm; weighing 1.00g +/-0.05 g of crushed sample into a polystyrene centrifuge tube; adding 5mL of methanol, and performing vortex oscillation for 1 min; and adding 900 mu L of 0.02mol/L phosphate buffer solution into 100 mu L of supernatant, and uniformly mixing to be tested.
2. Detection with test strips
Sucking 100 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 is consistent with that of the C line, which indicates that the concentration of isoprothiolane in the sample is lower than the detection limit, as shown in FIGS. 2a and 2 b.
Positive (+): the color development of the T line is lighter than that of the C line or the T line is not developed, which indicates that the concentration of isoprothiolane in the sample is equal to or higher than the detection limit, as shown in FIGS. 2C and 2 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. 2e and 2 f.
Example 3 sample testing example
1. Limit of detection test
Taking blank tobacco leaf samples to be roasted and primary roasted after picking, respectively adding isoprothiolane into the blank tobacco leaf samples until the final concentrations are 1mg/kg, 2mg/kg and 4mg/kg, taking test paper strips for detection, and repeatedly measuring each sample for three times.
When the test paper strip is used for detecting samples of the tobacco leaves to be roasted and the flue-cured tobacco leaves after being picked, when the isoprothiolane is not contained and the adding concentration of the isoprothiolane is 1mg/kg, the test paper strip shows that the color development of a T line is darker than that of a C line or is consistent with that of the C line, and the test paper strip is negative; when the adding concentration of the isoprothiolane is 2mg/kg and 4mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed and is positive, which shows that the detection limit of the test strip on the isoprothiolane in the tobacco leaves is 2mg/kg, and the requirement of the maximum residual limit can be met.
2. Test for false positive and false negative rates
Taking 20 parts of blank tobacco leaves to be baked and primary baked tobacco leaves after being picked and positive tobacco leaves to be baked and primary baked tobacco leaves after being picked and added with isoprothiolane to the final concentration of 2mg/kg, respectively detecting by using test strips produced in 3 batches, and calculating the negative and positive rates of the samples.
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 isoprothiolane can be used for quickly detecting isoprothiolane residues in tobacco samples.
3. Specificity test
Common bactericides in tobacco leaves such as chlorothalonil, carbendazim, oxadixyl, thiophanate-methyl, metalaxyl, myclobutanil, triadimefon, triadimenol, propamocarb, tebuconazole, iprodione and the like are diluted to 500mg/L by phosphate buffer solution with pH of 7.2 and 0.2mol/L, and the detection is carried out by a isoprothiolane test strip. The result shows that when the test strip is used for detecting 500mg/L chlorothalonil, carbendazim, oxadixyl, thiophanate-methyl, metalaxyl, myclobutanil, triadimefon, triadimenol, propamocarb, tebuconazole and iprodione, the T-line color development of the test strip is darker than or consistent with the C-line color development, and the test strip is negative. The test strip shows that the test strip has no cross reaction to the commonly used bactericides in tobacco leaves such as chlorothalonil, carbendazim, oxadixyl, thiophanate-methyl, metalaxyl, myclobutanil, triadimefon, triadimenol, propamocarb, tebuconazole, iprodione and the like.
4. Comparison test with instrumental methods
Taking 20 parts of the collected tobacco leaves to be roasted and the tobacco leaves to be primarily roasted respectively, numbering 1-20, using the test strip to carry out contrast detection with an instrument detection method, wherein the instrument method refers to: GB/T20769-.
TABLE 1 comparison of test paper strip of tobacco leaf sample to be roasted after harvest with the test result of instrumental method (mg/kg)
TABLE 2 comparison of test paper strips of flue-cured tobacco leaf samples with the results of the instrumental methods (mg/kg)
As can be seen from the above table, the test result of the test strip is identical to the test result of the instrument.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A test strip for detecting isoprothiolane comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with an isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and the conjugate release pad is sprayed with an isoprothiolane monoclonal antibody-colloidal gold marker; the isoprothiolane monoclonal antibody is prepared by taking an isoprothiolane hapten-carrier protein conjugate as an immunogen; the isoprothiolane hapten-carrier protein conjugate is obtained by coupling isoprothiolane 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 isoprothiolane hapten is obtained by taking diisopropyl malonate as a raw material and carrying out condensation reaction with carbon disulfide and 3, 4-dichlorobutyric acid in strong alkali, and the molecular structural formula of the isoprothiolane hapten-carrier protein conjugate is as follows:
2. the test 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 preparing the test strip of any one of claims 1-3, comprising the steps of:
1) preparing a conjugate release pad sprayed with a isoprothiolane monoclonal antibody-colloidal gold marker;
2) preparing a reaction membrane with a detection line coated with a isoprothiolane hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.
5. A method for detecting isoprothiolane in a sample using the test strip of any one of claims 1 to 3, 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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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000086654A (en) * | 1998-09-11 | 2000-03-28 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of isoprothiolane, antibody and assay |
| CN102146072A (en) * | 2011-03-10 | 2011-08-10 | 湖南化工职业技术学院 | Method for preparing isoprothiolane |
| CN104391068A (en) * | 2014-11-05 | 2015-03-04 | 中国烟草总公司四川省公司 | Method for determining residual quantity of conventional bactericides in tobacco |
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| JP2000086654A (en) * | 1998-09-11 | 2000-03-28 | Kankyo Meneki Gijutsu Kenkyusho:Kk | Hapten compound of isoprothiolane, antibody and assay |
| CN102146072A (en) * | 2011-03-10 | 2011-08-10 | 湖南化工职业技术学院 | Method for preparing isoprothiolane |
| CN104391068A (en) * | 2014-11-05 | 2015-03-04 | 中国烟草总公司四川省公司 | Method for determining residual quantity of conventional bactericides in tobacco |
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| 尤青山;: "气相色谱法测定大米中稻瘟灵残留量", 黑龙江农业科学, no. 05, pages 105 - 107 * |
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