阅读说明：本技术 一种腈菌唑胶体金检测试剂盒及其应用 (Myclobutanil colloidal gold detection kit and application thereof ) 是由 蒲小容 徐毓琴 周立冬 罗贵昆 于 2020-05-20 设计创作，主要内容包括：本发明公开了一种腈菌唑胶体金检测试剂盒及其应用,所述检测试剂盒包括试剂盒盒体,在所述盒体内有一次性吸管、样本提取液及检测试纸卡；其中,所述检测试纸卡包括底板、吸收垫、反应膜、吸水垫；所述检测试纸卡反应膜上包被有腈菌唑半抗原-HSA偶联物构成的检测线和包被有抗抗体构成的质控线；所述吸收垫内包被有腈菌唑特异性抗体-胶体金标记物。本发明腈菌唑胶体金检测试剂盒是利用胶体金免疫层析技术对样本中腈菌唑药物残留进行检测,与现有技术相比,具有成本低、重复性好,检测快速,检测灵敏度高等优点,是特别适用于粮食、烟草中腈菌唑残留检测的试剂盒。(The invention discloses a myclobutanil colloidal gold detection kit and application thereof, wherein the detection kit comprises a kit body, and a disposable straw, a sample extracting solution and a detection test paper card are arranged in the kit body; the detection test paper card comprises a bottom plate, an absorption pad, a reaction membrane and a water absorption pad; the detection line formed by myclobutanil hapten-HSA conjugate and the quality control line formed by anti-antibody are coated on the reaction film of the detection test paper card; the absorption pad is internally coated with a myclobutanil specific antibody-colloidal gold marker. The invention discloses a myclobutanil colloidal gold detection kit, which is used for detecting myclobutanil drug residue in a sample by using a colloidal gold immunochromatography technology.)

1. A myclobutanil colloidal gold detection kit comprises a kit body, wherein a disposable straw, a sample extracting solution and a detection test paper card are arranged in the kit body, and the detection test paper card is characterized by comprising a bottom plate, an absorption pad, a reaction membrane and a water absorption pad; the reaction membrane is coated with a detection line consisting of a myclobutanil hapten-HSA conjugate and a quality control line consisting of an anti-antibody; the absorption pad is coated with a myclobutanil specific antibody-colloidal gold marker.

2. The colloidal gold myclobutanil assay kit of claim 1, wherein: the grain size of the colloidal gold is 30 nm.

3. The colloidal gold myclobutanil assay kit of claim 1, wherein: the myclobutanil hapten-HSA conjugate is obtained by coupling myclobutanil hapten and HSA.

4. The colloidal gold myclobutanil assay kit of claim 1, wherein: the myclobutanil hapten is prepared by the following steps: weighing 0.86 g of myclobutanil in a100 mL round-bottom flask, adding 20 mL of propionic acid for dissolving, adding 0.5 g of succinic anhydride and 0.5 g of anhydrous aluminum trichloride, carrying out reflux reaction at 80 ℃ for 10 hours, and cooling to room temperature after the reaction is finished; washing with acetone for 2 times, and evaporating the reaction solution to dryness at 100 deg.C to obtain oily substance; silica gel column, ethyl acetate: petroleum ether = 2: 1, eluting, separating and purifying to obtain a myclobutanil hapten solution, and evaporating to dryness at 100 ℃ to obtain a solid myclobutanil hapten.

5. The colloidal gold myclobutanil assay kit of claim 1, wherein: the test paper card is also provided with a plastic shell, a sample adding hole is formed in the plastic shell above the absorption pad, and an observation window is formed in the plastic shell above the detection line and the quality control line.

6. The colloidal gold myclobutanil assay kit of claim 1, wherein: the sample extracting solution is 0.05 mol/LMES buffer solution containing 70% ethanol and pH 6.0.

7. Use of the colloidal gold myclobutanil test kit according to any one of claims 1-6 for testing myclobutanil drug residue in foodstuffs and tobacco.

Technical Field

The invention relates to a colloidal gold immunochromatographic assay rapid detection technology, in particular to a myclobutanil colloidal gold detection kit and application thereof.

Background

Myclobutanil is a triazole fungicide, mainly plays a role in inhibiting the biosynthesis of ergosterol of pathogenic bacteria, has an effect of inhibiting the biosynthesis of ergosterol, has the characteristics of strong systemic property, high drug effect, safety to crops and long lasting period, and is commonly used for preventing and treating common diseases in the production process of vegetables, fruits and tobaccos, such as the powdery mildew, rust disease and scab of apples and pears, the powdery mildew of cucumbers and the powdery mildew of tobaccos, and the loose smut, the hard smut, the net smut, the wheat stalk blight, net blotch and barley stripe disease of wheat. Myclobutanil can generate certain pesticide residue after being used in crops for a long time, and if people take the myclobutanil for a long time, the myclobutanil threatens the health of human bodies, mainly damages nervous systems, reduces the immune function of the human bodies, induces tumors and the like, so the problem of the residual myclobutanil must be considered.

At present, instrument detection methods are widely applied to detection of myclobutanil drugs, and although the instrument detection methods have high detection sensitivity and accurate detection results, the instrument detection methods have the characteristics of high cost, low detection speed and strict requirements on samples, equipment and detection personnel, and are not favorable for rapid detection. The immunological detection method has the advantages of low relative detection cost, quick detection and low requirement on detection personnel, and can be suitable for the field quick detection of a large number of samples, and particularly, the colloidal gold immunochromatography detection method is a more ideal immunological detection method in recent years. However, the existing immunological detection method still has the defects of low detection sensitivity, poor repeatability, inaccurate detection result and long detection time.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a myclobutanil colloidal gold detection kit.

The detection line formed by the myclobutanil hapten-HSA conjugate on the reaction membrane and the quality control line formed by the coated anti-antibody are coated, and the absorption pad is coated with a myclobutanil specific antibody-colloidal gold marker.

Preferably, the particle size of the colloidal gold is 30 nm, the 30 nm colloidal gold has strong binding capacity with a myclobutanil antibody, and a firm binding substance can be formed, so that myclobutanil drugs in a sample can be better detected, and the sensitivity of the myclobutanil colloidal gold detection kit to myclobutanil drug detection is effectively improved. Preferably, the preparation process of the 30 nm colloidal gold particles comprises the following steps: heating and boiling 100 mL of 0.01% chloroauric acid, adding 1.6 mL of 1% trisodium citrate, boiling for 10 min to obtain transparent orange red, and naturally cooling to obtain 30 nm colloidal gold with uniform particle size and no aggregation.

The myclobutanil specific antibody-colloidal gold marker is coated on the absorption pad, and the traditional sample pad is combined with the gold-labeled conjugate release pad, so that the production process of the test paper is simplified, the production cost is reduced, the difference between detection batches is reduced, the repeatability of detection is improved, and the detection time is shorter.

Preferably, the myclobutanil hapten-HSA conjugate is obtained by coupling myclobutanil hapten and HSA,

the myclobutanil hapten is prepared by the following steps: weighing 0.86 g of myclobutanil in a100 mL round-bottom flask, adding 20 mL of propionic acid for dissolving, adding 0.5 g of succinic anhydride and 0.5 g of anhydrous aluminum trichloride, carrying out reflux reaction at 80 ℃ for 10 hours, and cooling to room temperature after the reaction is finished; washing with acetone for 2 times, and evaporating the reaction solution to dryness at 100 deg.C to obtain oily substance; silica gel column, ethyl acetate: petroleum ether = 2: 1, eluting, separating and purifying to obtain a myclobutanil hapten solution, and evaporating to dryness at 100 ℃ to obtain a solid myclobutanil hapten.

Preferably, the test paper card is further provided with a plastic outer box, a sample adding hole is formed in the plastic outer box and corresponds to the upper portion of the absorption pad, and an observation window is formed in the plastic outer box and corresponds to the upper portions of the detection line and the quality control line.

Preferably, the sample extract is 70% ethanol, pH6.0, 0.05 mol/LMES buffer.

The invention also provides application of the myclobutanil colloidal gold kit in detection of myclobutanil drug residues in food and tobacco, and the detection sensitivity of the kit is up to 0.01 mu g/g.

The invention discloses a myclobutanil colloidal gold detection kit, which is a product for detecting myclobutanil in a sample by using a colloidal gold competitive immunochromatography. The hapten and the antigen prepared by the invention are matched with the detection system of the kit, the detection sensitivity of the kit to the myclobutanil medicament in the sample is very high, and compared with the prior art, the colloidal gold myclobutanil detection kit product of the invention also has the advantages of simplified production process, small batch difference of detection results, low cost, short time required for detection and the like, and is very suitable for detecting the residual myclobutanil medicament in the sample.

Drawings

FIG. 1 is a schematic view of the structure of a test paper card in a myclobutanil colloidal gold detection kit.

FIG. 2 is a schematic view showing the analysis of the test result of the test paper card of the present invention.

FIG. 3 shows the results of the repeatability tests between test paper batches of the present invention.

Detailed Description

The invention is further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the present invention. The reagents of the present invention are conventional reagents unless otherwise specified. Unless otherwise specified, the percentages by weight are by volume.

Example one

The rapid detection kit for myclobutanil colloidal gold comprises a kit body, wherein a disposable straw, a sample extracting solution and a detection test paper card are arranged in the kit body. The detection test paper card comprises a PVC bottom plate 6, an absorption pad 1, a reaction membrane 3 and a water absorption pad 2 as shown in figure 1; in order to avoid pollution to the test paper card in the detection process of taking and placing the test paper card, the test paper card is also provided with a plastic outer box, a sample adding hole is formed in the plastic outer box above the corresponding absorption pad, and an observation window is formed in the plastic outer box above the corresponding detection line and the quality control line. The reaction membrane is also coated with a detection line 4 consisting of a myclobutanil hapten-HSA conjugate and a quality control line 5 consisting of an anti-antibody; the absorption pad is coated with a myclobutanil specific antibody-colloidal gold marker, wherein the particle size of the colloidal gold particles is preferably 30 nm.

Preparation of main materials:

preparation of first, detection line coating substance myclobutanil hapten-HSA conjugate

Preparation of myclobutanil hapten: weighing 0.86 g of myclobutanil into a100 mL round-bottom flask, adding 20 mL of propionic acid for dissolving, adding 0.5 g of succinic anhydride and 0.5 g of anhydrous aluminum trichloride, carrying out reflux reaction at 80 ℃ for 10 h, and cooling to room temperature after the reaction is finished. Washing with acetone for 2 times, and evaporating the reaction solution to dryness at 100 deg.C to obtain oily substance. Silica gel column, ethyl acetate: petroleum ether = 2: 1, (v/v, 2/1) eluting, separating and purifying to obtain a myclobutanil hapten solution, and evaporating to dryness at 100 ℃ to obtain solid myclobutanil hapten.

Preparation of myclobutanil hapten-HSA conjugate: (1) weighing 60 mg of myclobutanil hapten, and dissolving in 1 mLDMF; (2) weighing NHS and EDC, and dissolving 80 mg of NHS and EDC in 1 mL of deionized water respectively; (3) slowly adding the mixed solution obtained in the step (2) into the step (1), and stirring for 1 h at room temperature; (4) weighing HSA100 mg and dissolving in 4 mL deionized water; (5) adding the reaction solution obtained in the step (3) into the step (4) in an equivalent amount under stirring, reacting for 30 min, adjusting the pH value to 8-9 by using 1mol/L sodium hydroxide solution, and stirring for 24 h at room temperature; (6) dialyzing with 0.02 mol/L PBS buffer solution (pH7.2), changing the buffer solution every day, and dialyzing for 3 days. Reaction of

The synthetic route is as follows:

preparation of monoclonal antibody-colloidal gold marker of myclobutanil and myclobutanil

1. The preparation method of the myclobutanil hapten-KLH conjugate as the immunogen is the same as the synthesis step of the myclobutanil hapten-HSA conjugate, and the difference is that the carrier protein is hemocyanin.

2. Preparation of myclobutanil monoclonal antibody:

2.1 animal immunization

Injecting the obtained myclobutanil hapten-KLH conjugate as immunogen into a Balb/c mouse, adding Freund's complete adjuvant for the first time, wherein the immunization dose is 50 mug/mouse; adding Freund's incomplete adjuvant for the second immunization after 15 days, wherein the immunization dose is 50 mug/piece, and the immunization dose is 50 mug/piece for the third immunization after 15 days; 3 weeks later, the immunization was boosted at an immunization dose of 200 μ g/mouse, and antiserum was generated.

2.2 cell fusion and cloning

Taking splenocytes of the immune Balb/c mice, and carrying out reaction according to the weight ratio of 10: 1 ratio was fused with SP2/0 myeloma cells, and positive wells were screened by measuring cell supernatants using an indirect competitive ELISA. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain of stable secretion of monoclonal antibody-monoclonal hybridoma cell strain of myclobutanil.

2.3 cell cryopreservation and Resuscitation

Making hybridoma cell into 1 × 10 with frozen stock solution⁹Cell suspension per ml, preserved for long periods 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. The frozen stock solution is 30% calf serum, 60%1640 culture solution and 10% DMSO.

2.4 preparation and purification of monoclonal antibodies

Injecting sterilized paraffin oil 0.5 mL/mouse into Balb/c mouse abdominal cavity, injecting myclobutanil monoclonal hybridoma cell strain 1X 10 into abdominal cavity 7 days later⁶Ascites were collected 7 days later. Purifying by octanoic acid-saturated ammonium sulfate method, and storing the purified ascites at-20 deg.C. Before use, the solution is dialyzed for 48 hours at the temperature of 4 ℃ through 0.01mol/L NaCl, and the solution is changed every 6 hours.

3. Preparation of myclobutanil monoclonal antibody-colloidal gold marker

In this example, 30 nm colloidal gold is adopted, and the preparation method of 30 nm colloidal gold is as follows: heating and boiling 100 mL of 0.01% chloroauric acid, adding 1.6 mL of 1% trisodium citrate, boiling for 10 min until transparent orange red appears, and naturally cooling to obtain the product. The 30 nm colloidal gold particle size and the myclobutanil monoclonal antibody of the embodiment have strong binding capacity, so that the sensitivity of the myclobutanil colloidal gold detection kit to myclobutanil drug detection is effectively improved, and the detection sensitivity is greatly improved.

Myclobutanil monoclonal antibody-colloidal gold marker: taking 100 mL of the colloidal gold solution, adding 1% potassium carbonate to adjust the pH value of the colloidal gold to 7.2 under magnetic stirring, adding 10-20 mu L of myclobutanil monoclonal antibody into each mL of the colloidal gold solution, stirring for 10 min, standing for 5 min, adding 5% BSA (bovine serum albumin) until the volume concentration of the BSA is 1%, and standing for 20 min; centrifuging at 10000rpm and 4 ℃ for 15 min, discarding the supernatant, and resuspending the precipitate into 1/10 of the original volume with PBS buffer solution containing 1% BSA and 0.002% sodium azide, and standing at 4 ℃ for later use.

In this embodiment, a myclobutanil monoclonal antibody is used, and a myclobutanil polyclonal antibody is also suitable.

Third, anti-antibody preparation

The goat anti-mouse anti-antibody is prepared by immunizing a pathogen-free goat by using a murine antibody as an immunogen, and is used for coating a quality control line. When the specific antibody is myclobutanil polyclonal antibody, the quality control line can coat goat anti-rabbit anti-antibody, and the goat anti-rabbit anti-antibody is prepared by immunizing a pathogen-free goat by taking a rabbit derived antibody as immunogen.

And fourthly, the reaction membrane can be a cellulose acetate membrane or a cellulose nitrate membrane, preferably a cellulose nitrate membrane, is soaked for 30 min by using sucrose with the pH value of 7.2 and the concentration of 5 percent and 0.01mol/LPBS before being coated on the detection line and the quality control line, and is dried for 2 h at the temperature of 37 ℃. The detection line myclobutanil hapten-HSA coating concentration is 10 mg/mL, and the sheep anti-mouse anti-antibody coating concentration is 1 mg/mL. After the reaction membrane is prepared, the reaction membrane is placed in PBS buffer solution containing 1% bovine serum albumin, 1% NaCl, the pH value of 7.0 and the concentration of 0.01mol/L for sealing for 30 min, and then the reaction membrane is dried for 2 h at 37 ℃ for standby. The nitrocellulose membrane can increase the hydrophilic rate, reduce the signal intensity and reduce the false positive after being treated.

The absorption pad is a glass fiber membrane, and is soaked in PBS buffer solution containing 20% Tween-20 at pH7.0 for 30 min before coating the myclobutanil specific antibody-colloidal gold marker, and dried at 37 deg.C for 2 h. Coating the myclobutanil specific antibody-colloidal gold label according to the combination of l mL of myclobutanil specific antibody-colloidal gold label per 5 square centimeters, and then drying in vacuum for later use.

The absorbent pad is absorbent paper.

Example two

The colloidal gold immunochromatography is an immune gold-labeling technology which applies a colloidal gold labeling technology, takes colloidal gold as a tracer and is applied to antigen-antibody reaction. The myclobutanil colloidal gold detection kit adopts a competitive colloidal gold immunochromatography method, a myclobutanil hapten-HSA conjugate is pre-coated on a nitrocellulose membrane quality control line, myclobutanil in a sample and a coupled antigen pre-coated on the nitrocellulose membrane compete for a myclobutanil specific antibody marked by colloidal gold in the chromatography process, and whether myclobutanil medicine exists in the sample is judged through whether a detection line develops color or not.

1. The operation steps of the myclobutanil colloidal gold kit of the invention

Sucking a sample to be detected by using a disposable suction tube, and vertically dropping 2-3 drops of the sample to be detected into a sample adding hole; timing is started when the liquid flows, and the result is judged after the reaction is carried out for 3-5 min.

2. Analysis of the results, see FIG. 2

Negative: the detection line (T) and the quality control line (C) are both red, which indicates that the concentration of the myclobutanil drug in the sample is lower than the detection limit.

Positive: the detection line (T) has no color or is lighter than the quality control line (C), and the quality control line (C) has color, which indicates that the concentration of the myclobutanil drug in the sample is equal to or higher than the detection limit.

And (4) invalidation: the absence of a control line (C) indicates an incorrect procedure or the test strip has deteriorated and failed. It should be re-detected.

3. Sample pretreatment

The wheat and tobacco sample processing method comprises the following steps: cutting tobacco leaves into 1 cm with scissors, and directly taking wheat; weighing 1.0 g of sample to be detected into a 50 mL polystyrene centrifuge tube; adding 2 mL of sample extracting solution, fully mixing the samples for 5 min by using an oscillator, and standing for 2 min at room temperature; the supernatant was aspirated with a disposable pipette for analysis. The sample extract is 0.05 mol/LMES buffer solution containing 70% ethanol at pH6.0, and the percentage is volume percentage.

4. Sensitivity of detection

Myclobutanil standard was diluted to different concentrations as follows: 0.005 mug/g, 0.01 mug/g, 0.02 mug/g and 0.04 mug/g, and the myclobutanil test paper card is taken for detection, and each sample is repeatedly measured for 3 times.

When the myclobutanil standard solution of 0.005 mug/g is measured, two red lines visible to the naked eye are displayed on the test paper card and are negative; when the myclobutanil standard solution is measured to be 0.01 mug/g, 0.02 mug/g and 0.04 mug/g, the test paper card quality control line is colored, the detection line is not colored, and the test paper card quality control line is positive. The detection sensitivity of the product of the invention to myclobutanil is 0.01 mug/g.

Limit of sample detection

Taking blank wheat and tobacco samples, respectively adding myclobutanil to the blank wheat and tobacco samples until the final concentration is 0, 0.005, 0.01, 0.02 and 0.04 mu g/g, taking the myclobutanil colloidal gold detection kit for detection, and repeatedly measuring each sample for 5 times.

When the concentration of myclobutanil added in wheat and tobacco is 0 and 0.005 mu g/g, two macroscopic red lines are displayed on the test strip and are negative; when the concentration of myclobutanil is 0.01, 0.02 and 0.04 mug/g, the quality control line of the test strip shows red, and the detection line shows no color and is positive; the detection limit of the product of the invention to myclobutanil medicines in wheat and tobacco is 0.01 mug/g.

5. Repeatability test

Taking a myclobutanil standard solution of 0.02 mug/g, and measuring by using myclobutanil test paper cards produced in 5 different batches, wherein the measurement result is shown in figure 3. The myclobutanil colloidal gold detection kit has the advantages of uniform detection color development, small batch difference and good repeatability.

6. Specificity test

Specificity refers to the ability of an antibody to bind to structurally different antigenic determinants. The sensitivity of the myclobutanil detection product to myclobutanil detection is 0.01 mug/g. The detection result of the detection product is negative when the concentration of the drug standard substance is 50 mug/g, so that the product does not have cross reaction to the drugs and can specifically detect the myclobutanil drug.

6. Negative and positive rechecking rate

Taking 20 parts of wheat positive samples with known addition of 0.05 mu g/g of myclobutanil and 20 parts of wheat negative samples with the content of myclobutanil being 0 mu g/g determined by an instrument, and detecting by using the myclobutanil colloidal gold detection kit. When 20 positive samples are detected, and the false negative rate is 0; when 20 negative samples were detected, all the results were negative, and the false positive rate was 0.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.