CN113960307B - 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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Abstract
The invention provides a test strip for detecting isoprothiolane, 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 a 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 a isoprothiolane monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting isoprothiolane in a sample by using 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 limit by detection equipment, and can realize rapid detection and on-site monitoring of a large number of isoprothiolane samples.
Description
Technical Field
The invention relates to the 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) is also known as Isoprothiolane and Fuji I, and the chemical name of Isoprothiolane-2-subunit diisopropyl malonate is a high-efficiency systemic bactericide, has a narrow bactericidal spectrum, is a specific drug for preventing and treating rice blast, has excellent effect on neck blast, can be systemic and conducted through various parts of roots, stems and leaves of plants, and can reach heart leaves. It is also effective on rice seedling plague and micrococcus nuclear disease, can treat rice planthoppers and leafhoppers simultaneously when used in a large area, and has the effect of regulating plant growth.
However, with the large-scale application of isoprothiolane, the isoprothiolane may pollute the surrounding environment and may remain in agricultural products, thus posing a potential health threat to human beings, so the problem of residues in the production of fruits, vegetables and tobacco is receiving more and more attention. The maximum residue limit standard of the isoprothiolane is formulated for different crops in China, wherein the maximum residue limit of the watermelon is 0.1mg/kg, the maximum residue limit of the rice is 1mg/kg, and the maximum residue limit of the tobacco is 2mg/kg.
At present, the literature only reports the detection of the residual quantity of the bactericide in grains, fruits and vegetables, but no report on the measurement method of the residual quantity in tobacco leaves is seen. The detection method for the isoprothiolane residue at home and abroad mainly comprises gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), gas Chromatography (GC) and the like. The instrument method has the advantages of high detection sensitivity, strong specificity and the like, but the pretreatment of the detection sample is tedious and time-consuming, the sample also needs to be extracted and purified, and meanwhile, the instrument detection method needs expensive large-scale instruments and equipment, is provided with professional detection technicians to operate and manage, cannot perform on-site large-scale detection, has poor timeliness and is difficult to popularize. Therefore, developing a product and method that is not limited by the detection apparatus and that enables rapid detection of large volumes of samples is an urgent problem to be solved.
Disclosure of Invention
The invention aims to overcome the defects that the existing method for detecting the isoprothiolane has high equipment dependence and cannot realize the rapid detection of a large number of samples, and provides a test strip which has the advantages of simple operation, high sensitivity, high detection speed, low cost and no limitation of detection equipment, and a preparation method and application thereof, so as to realize the rapid detection and on-site monitoring of the isoprothiolane samples in a large number.
In order to achieve 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 antibody; the binding substance release pad is sprayed with a isoprothiolane monoclonal antibody-colloidal gold marker.
The isoprothiolane monoclonal antibody is prepared by taking a isoprothiolane hapten-carrier protein conjugate as an immunogen.
The isoprothiolane hapten-carrier protein conjugate is obtained by coupling isoprothiolane hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, the isoprothiolane hapten is obtained by condensation reaction of diisopropyl malonate serving as a raw material, carbon disulfide and 3, 4-dichlorobutyric acid in strong alkali, and the molecular structural formula is as follows:
the sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, and 1/3-1/2 of the conjugate releasing pad is covered under the sample absorbing pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing 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 absorbing pad is water absorbing paper; the reaction membrane may 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 binding substance 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 antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
Specifically, the method comprises the following steps:
1) Taking diisopropyl malonate as a raw material, and carrying out condensation reaction on the diisopropyl malonate, carbon disulfide and 3, 4-dichlorobutyric acid in strong alkali to prepare isoprothiolane hapten;
2) Coupling the isoprothiolane hapten with carrier protein to prepare a isoprothiolane hapten-carrier protein conjugate;
3) Immunizing a mouse by using a isoprothiolane hapten-carrier protein conjugate, and fusing and screening spleen cells 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-antibody;
5) Coating a isoprothiolane hapten-carrier protein conjugate and a 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 the reaction of trisodium citrate and chloroauric acid;
7) Adding the prepared isoprothiolane monoclonal antibody into the prepared colloidal gold to obtain a isoprothiolane monoclonal antibody-colloidal gold marker;
8) Spraying a Magnaporthe grisea monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 2 hours, taking out, and storing in a dry environment for later use;
9) Soaking the sample absorption pad in 0.02mol/L phosphate buffer solution containing 1% bovine serum albumin and having a pH of 7.2 for 2 hours, and drying at 37 ℃ for 2 hours for later use;
10 A sample absorbing pad, a conjugate releasing pad, a reaction membrane, and a water absorbing pad are sequentially stuck on the bottom plate, and 1/3 area of the conjugate releasing pad from the initial end is covered by the sample absorbing pad. Finally cutting into small strips with the width of 3.95mm, placing the small strips in a specially made plastic card-making shell, sealing the small strips by an aluminum foil bag, and preserving the small strips for 12 months at the temperature of 4-30 ℃. The 1/3 of the conjugate release pad is covered by the sample absorption pad, so that the observation time of the detection result can be prolonged, and the sample absorption pad can fully absorb the detection liquid and fully react with the gold-labeled antibody, thereby reducing errors.
The invention also provides a method for detecting isoprothiolane residues in a sample by using the test strip, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The invention relates to a fast test strip for isoprothiolane, which adopts a highly specific antibody antigen reaction and immunochromatography analysis technology, and fixes a isoprothiolane monoclonal antibody-colloidal gold marker on a conjugate release pad, wherein the 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 membrane detection line compete for combining with the isoprothiolane monoclonal antibody-colloidal gold marker, and whether the isoprothiolane residue is contained in the sample liquid to be detected is judged according to the red stripe depth of the detection line.
During detection, a sample is dripped into a sample absorption pad after treatment, when the concentration of the isoprothiolane in the sample is lower than the detection limit or is zero, the monoclonal antibody-colloidal gold marker is combined with the isoprothiolane hapten-carrier protein conjugate fixed on the reaction membrane 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 or 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-labeled substance can be completely combined with isoprothiolane, so that red bands are not generated or the color development is lighter than that of C line because the monoclonal antibody-colloidal gold-labeled substance can not be combined with the isoprothiolane hapten-carrier protein conjugate at the T line due to competition reaction. As shown in fig. 2.
Negative: and when the quality control line (C) shows red stripes, the detection line (T) also shows red stripes, and the color of the (T) line is close to or deeper than that of the (C), the judgment is negative.
Positive: and judging positive when the quality control line (C) shows red stripes and the detection line (T) does not develop color or the color of the detection line (T) is lighter than that of the detection line (C).
Invalidation: when the quality control line (C) does not display red stripes, whether the detection line (T) displays red stripes or not, the test strip is judged to be invalid.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, no limit by 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, quick, 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 membrane; 4. a water absorbing pad; 5. a detection line; 6. a quality control line; 7. a bottom plate;
FIG. 2 is a test strip detection result judgment chart;
FIG. 3 shows the synthesis of isoprothiolane hapten.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention. In addition, various changes or modifications may be made by those skilled in the art within the scope of the appended claims, and such changes or modifications should also fall within the scope of the invention.
Example 1 preparation of test strip for detecting Pyricularia oryzae
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a binding substance 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 antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
The following is a stepwise detailed description:
1. synthesis of Magnaporthe grisea hapten (synthetic route see figure 3)
Taking 1.88g of diisopropyl malonate, adding 20mL of carbon disulfide, stirring at room temperature, fully mixing, adding 13mL of 2mol/L KOH solution, stirring at room temperature for 1h, adding 1.56g of 3, 4-dichlorobutyric acid, adding 5mL of 2mol/L KOH solution, 3.8g of tetrabutylammonium bromide and 80mL of acetonitrile, heating and refluxing for 4h, stopping the reaction, rotary steaming, removing acetonitrile and 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, merging organic phases, washing, drying by anhydrous sodium sulfate, evaporating to obtain yellow oily matter, 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, thus obtaining 0.76g of isoprothiolane acetate 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 for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1mol/L CB9.1 mL for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the isoprothiolane-BSA conjugate, namely the immunogen.
3. Preparation of coating Material
Taking 7.7mg of isoprothiolane hapten, adding 1mL of dimethyl sulfoxide (DMSO), dissolving and clarifying, adding 5.1mg of NHS and 9.7mg of EDC, fully dissolving and mixing uniformly, and reacting for 2 hours at room temperature to obtain hapten activating solution A; taking 50mg of Ovalbumin (OVA), and adding 0.1mol/L CB9.1 mL for dissolution to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, stopping the reaction, dialyzing and purifying for 3 days by using 0.02mol/L PBS buffer solution, changing the solution 3 times a day, centrifuging and subpackaging to obtain the isoprothiolane-OVA conjugate, namely the coating source.
4. Preparation of Magnaporthe grisea monoclonal antibody
(1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.
(2) Cell fusion and cloning
Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting indirect competition ELISA, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation
The hybridoma cells were prepared into 1X 10 by using a frozen stock solution 6 Cell suspensions of individual/mL were stored in liquid nitrogen for long periods. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, wherein the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
6. Preparation of Magnaporthe grisea monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 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 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering to original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, has no sediment or floating matters, and has a wine red color when observed in sunlight.
(2) Preparation of Magnaporthe grisea 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 of adding 5-50 mug antibody into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30min; after 10min of standing, 10% BSA was added to give a final concentration of 1% in the colloidal gold solution, and the mixture was left for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.
Reconstitution buffer: 0.02mol/L phosphate buffer solution containing 0.1-0.5% of BSA, 2-4% of sucrose and pH 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in 0.02mol/L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, uniformly soaked for 2h, and dried at 37℃for further use. Uniformly spraying the prepared isoprothiolane monoclonal antibody-colloidal gold marker on a conjugate release pad by using a Bio dot film-drawing instrument, spraying 0.01mL of the isoprothiolane monoclonal antibody-colloidal gold marker on each 1cm of conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 2 hours, 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 pad was soaked in 0.02mol/L phosphate buffer containing 1% BSA at pH7.2 for 2h and dried at 37℃for 2 h.
9. Preparation of reaction film
The isoprothiolane hapten-OVA conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the isoprothiolane hapten-OVA conjugate to 1mg/mL by using 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coating the isoprothiolane hapten-OVA conjugate on a detection line (T line) on a nitrocellulose membrane by using a Bio dot-based membrane-drawing instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/mL with 0.01mol/L, pH 7.2.7.2 phosphate buffer, and coated on a quality control line (C line) on a nitrocellulose membrane in an amount of 1.0. Mu.L/cm using a Bio dot-based membrane cutter. And (5) drying the coated reaction film for 16 hours at 37 ℃ for standby.
10. Assembly of test strips
According to the section structure of the test strip shown in figure 1, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially stuck on a PVC bottom plate (7); the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film 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 perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3.95mm by a machine, putting the small strips into a specially made plastic card shell, sealing the small strips by an aluminum foil bag, and storing the small strips in an environment with the temperature of 4-30 ℃ for 12 months.
Example 2 detection of Pyricularia oryzae in samples
1. Sample pretreatment
Crushing or shearing the sample into fragments smaller than 1 cm; weighing 1.00 g+/-0.05 g of crushed sample into a polystyrene centrifuge tube; adding 5mL of methanol, and carrying out vortex oscillation for 1min; 100 mu L of supernatant is taken, 900 mu L of 0.02mol/L phosphate buffer solution is added, and the mixture is uniformly mixed and tested.
2. Detection by test strips
100 mu L of sample liquid to be detected is sucked by a micropipette and vertically dripped into a sample adding hole; the flow of the liquid was started to time, the reaction was continued for 10 minutes, and the result was judged.
3. Analyzing the detection result
Negative (-). The color development of the T line is deeper than or 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 figures 2a and 2b.
Positive (+): the T line developed less than the C line developed or the T line did not develop, indicating that the concentration of isoprothiolane in the sample was equal to or higher than the limit of detection, as shown in FIGS. 2C, 2d.
Invalidation: the absence of line C indicates an incorrect procedure or that the test strip has failed due to deterioration, as shown in fig. 2e, 2f.
Example 3 sample detection example
1. Limit of detection test
Taking blank tobacco leaves to be cured and primary cured tobacco leaf samples, respectively adding isoprothiolane to the blank tobacco leaves to be cured and primary cured tobacco leaf samples, wherein final concentrations are 1mg/kg, 2mg/kg and 4mg/kg, taking test strips for detection, and repeatedly measuring each sample for three times.
When the test paper strip is used for detecting the tobacco leaves to be cured after the sampling and the primary flue-cured tobacco leaf samples, when the isoprothiolane and the additive concentration thereof are 1mg/kg, the test paper strip shows that the color development of the T line is deeper than or consistent with that of the C line and 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 the test strip is positive, so that the test strip can meet the requirement of the maximum residual limit on the detection limit of the isoprothiolane in tobacco leaves, which is 2mg/kg.
2. False positive rate and false negative rate test
Taking blank tobacco leaves to be cured, primary flue-cured tobacco leaf samples and 20 parts of positive tobacco leaves to be cured and primary flue-cured tobacco leaf samples with final concentration of 2mg/kg, respectively detecting the blank tobacco leaves to be cured and primary flue-cured tobacco leaf samples by using 3 batch production test strips, and calculating the negative and positive rate of the test strips.
The results show that: when positive samples are detected by using test strips produced in 3 batches, the results are positive, the positive coincidence rate is 100%, and the false negative rate is 0; when a negative sample was detected, the result was all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting the isoprothiolane can be used for rapidly detecting the isoprothiolane residue in a tobacco leaf sample.
3. Specificity test
The common bactericides in the 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 test strip of the isoprothiolane is used for detection. 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 color development of the T line of the test strip is deeper than or consistent with that of the C line, and the test strip is negative. The test strip has no cross reaction to common bactericides in tobacco leaves such as chlorothalonil, carbendazim, oxadixyl, thiophanate-methyl, metalaxyl, myclobutanil, triazolone, triadimenol, propamocarb, tebuconazole, iprodione and the like.
4. Comparison test with instrumental method
Taking 20 samples of tobacco leaves to be cured and primary cured tobacco leaves after picking, and carrying out comparison detection on the samples with numbers 1-20 by using the test strip and an instrument detection method, wherein the instrument method is as follows: "GB/T20769-2008 determination of residual quantity of 450 pesticides and related chemicals in fruits and vegetables liquid chromatography-tandem mass spectrometry" (the detection limit of isoprothiolane is 0.46 mg/kg), the isoprothiolane content in tobacco leaves to be cured and flue-cured tobacco leaf samples after the test strip is picked is lower than 2mg/kg, and is regarded as undetected, and the result is expressed by "-" and the sample instrument method higher than 2mg/kg is expressed by actual result, and the test strip detection result is expressed by "+", and the results are shown in the following table.
Table 1 comparing the test strip of the cured tobacco leaf sample after picking with the detection result of the instrument method (mg/kg)
Table 2 test strip of flue-cured tobacco leaf sample and test result of instrument method comparison (mg/kg)
As can be seen from the above table, the test strip detection results are identical to the instrument detection results.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (5)
1. The test strip for detecting the 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 a 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 a isoprothiolane monoclonal antibody-colloidal gold marker; the isoprothiolane monoclonal antibody is prepared by taking isoprothiolane hapten-carrier protein conjugate as 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, thyroxine or human serum albumin; the synthesis method of the isoprothiolane hapten is characterized by comprising the following steps: taking 1.88g of diisopropyl malonate, adding 20mL of carbon disulfide, stirring at room temperature, fully mixing, adding 13mL of 2mol/L KOH solution, stirring at room temperature for 1h, adding 1.56g of 3, 4-dichlorobutyric acid, adding 5mL of 2mol/L KOH solution, 3.8g of tetrabutylammonium bromide and 80mL of acetonitrile, heating and refluxing for 4h, stopping the reaction, removing acetonitrile and carbon disulfide by rotary evaporation, adding 80mL of water, adding 6mol/LHCl to adjust the pH value to 6, adding 100mL multiplied by 3 of ethyl acetate, extracting for three times, merging organic phases, washing, drying and evaporating to dryness by anhydrous sodium sulfate to obtain yellow oily matter, 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 isoprothiolane acetate, wherein the molecular structural formula 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 test strip of any one of claims 1-2, wherein the conjugate release pad is 1/3 to 1/2 coated under the sample absorbent pad.
4. A method of preparing the test strip of any one of claims 1-3, comprising the steps of:
1) Preparing a binding substance 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 antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
5. A method of detecting isoprothiolane in a sample using the test strip of any one of claims 1 to 3, the method comprising the steps of:
1) Pretreating a sample;
2) Detecting with the test strip of any one of claims 1-3;
3) And analyzing the detection result.
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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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| 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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