CN111007247A

CN111007247A - Colloidal gold immunochromatographic test strip for synchronously detecting ribes diacetate sickle-knife fungus enol, deoxynivalenol and T-2 toxin

Info

Publication number: CN111007247A
Application number: CN201911122589.1A
Authority: CN
Inventors: 张兆威; 唐晓倩; 王督; 张奇; 李培武
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-04-14
Anticipated expiration: 2039-11-15
Also published as: CN111007247B

Abstract

The invention relates to a colloidal gold immunochromatographic test strip for synchronously detecting sickle knife enol, deoxynivalenol and T-2 toxin of ribes diacetate. The detection pad takes a nitrocellulose membrane as a base pad, quality control lines and detection lines are transversely arranged on the nitrocellulose membrane, the detection lines are positioned below the quality control lines, the number of the detection lines is three, the detection lines are distributed at intervals, each toxin protein conjugate is respectively coated on each detection line, and the quality control lines are coated with rabbit anti-mouse polyclonal antibodies; the gold label pad is transversely sprayed with each antibody marked by colloidal gold; the anti-ribes diacetate fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881. The method can realize synchronous and rapid detection of three fungaltoxins, namely sickle enol diacetate, deoxynivalenol and T-2 toxin, on one test strip.

Description

Colloidal gold immunochromatographic test strip for synchronously detecting ribes diacetate sickle-knife fungus enol, deoxynivalenol and T-2 toxin

Technical Field

The invention provides a colloidal gold immunochromatographic test strip for synchronously detecting sickle knife fungus enol (S-shaped toxin), deoxynivalenol (vomitoxin) and T-2 toxin of ribes diacetate and a preparation method thereof.

Background

Mycotoxin is a metabolite produced by fungi growing in food or feed, and pollution caused by mycotoxin can be produced in the processes of grain and feed collection, storage and transportation. Ribes diacetate fusarenol, deoxynivalenol and T-2 toxin are common mycotoxins in grain feeds. Sickle knife fungus enol acetate is also called as snake-shaped toxin, belongs to trichothecene toxins, is mainly produced by sickle knife fungus and equisetum hiemalis, deoxynivalenol and T-2 toxin are trichothecene toxins produced by some fusarium, and three fungal toxins are widely produced in grains including wheat, corn, barley, oat, rye, rice, millet and the like, and grains and feeds such as peanut, bean and the like. The three toxins have great harm to people and animals, and after people and animals ingest food polluted by the toxin B.diacetic acid sickle knife fungus enol, deoxynivalenol and T-2, acute poisoning symptoms such as anorexia, vomiting, diarrhea, fever, unstable standing, slow reaction and the like can be caused, and the hematopoietic system is damaged to cause death in severe cases. In view of the harmful effects of mycotoxins and their widespread occurrence in food and feed, the contents are strictly limited in countries throughout the world. Therefore, the method is an important link for understanding and mastering food and feed safety and health information and enhancing food safety consumption in order to strengthen detection, particularly rapid detection, of the mycotoxins in the food and the feed.

The existing detection method for mycotoxin mainly comprises a thin layer chromatography method, a precise instrument analysis method and an immunological analysis method. When the mycotoxin is detected by the thin layer chromatography, special instruments and equipment are not needed, the mycotoxin can be detected in a common laboratory, but the detection reagent is large in dosage, complicated to operate, serious in interference of other components, poor in accuracy, incapable of accurately quantifying, large in pollution harm to experimenters and the surrounding environment, and not suitable for rapid detection on site. The precision instrument analysis method comprises a method of combining high performance liquid chromatography, mass spectrometry and tandem mass spectrometry, has high sensitivity and high accuracy, but has expensive instruments, requires high purification degree of detected samples, has complex sample pretreatment process, consumes long time, has high requirements on experimental environment and detection personnel, is difficult to realize rapid detection, and has high detection cost. The immunoassay method overcomes the defects of a thin layer chromatography method and an instrumental analysis method, and has been rapidly developed in recent years due to the advantages of strong specificity, high sensitivity, simple sample pretreatment, low cost, small pollution harm to laboratory personnel and the surrounding environment, suitability for field batch detection and the like. The detection result of the immunochromatographic test strip based on the specific binding reaction of the colloidal gold labeled antibody and the antigen is visible to naked eyes, large-scale instruments and equipment are not needed, the detection cost is low, and the analysis time is short, so that qualitative, online and rapid detection of trace residues such as various mycotoxins can be realized. In a decentralized planting mode of small farmers in China, the incidence rate of mycotoxins in agricultural products is high, and the possibility that the same agricultural product is polluted by various mycotoxins is high, so that a detection technology capable of quickly and synchronously detecting various mycotoxins is urgently needed to realize synchronous, quick and accurate monitoring of various mycotoxins in grains and feeds.

Disclosure of Invention

The invention aims to solve the problem of providing a colloidal gold immunochromatographic test strip for synchronously detecting mixed pollution of sickle knife enol, deoxynivalenol and T-2 toxin of ribes diacetate and a preparation method thereof. The method can be used for synchronously detecting the content of three fungaltoxins, namely, riberenol diacetate, deoxynivalenol and T-2 toxin in a sample, and has the characteristics of simple and rapid operation and high sensitivity.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a colloidal gold immunochromatographic test strip for synchronously detecting ribes oxydans, deoxynivalenol and T-2 toxins comprises a base plate, wherein a water absorption pad, a detection pad, a gold mark pad and a sample pad are sequentially pasted on one surface of the base plate from top to bottom, the adjacent pads are connected in an overlapped mode at the joint, the detection pad takes a nitrocellulose membrane as a base pad, quality control lines and detection lines are transversely arranged on the nitrocellulose membrane, the detection lines are located below the quality control lines, the number of the detection lines is three, the detection lines are distributed at intervals, and the three detection lines are respectively coated with a ribes oxydans-bovine serum albumin conjugate (DAS-BSA), a deoxynivalenol-bovine serum albumin conjugate (DON-BSA) and a T-2 toxin-bovine serum albumin conjugate (T-2-BSA), the quality control line is coated with a rabbit anti-mouse polyclonal antibody; the gold label pad is transversely sprayed with a colloidal gold-labeled anti-sickle cell enol diacetate monoclonal antibody, a colloidal gold-labeled anti-deoxynivalenol monoclonal antibody and a colloidal gold-labeled anti-T-2 toxin monoclonal antibody; the anti-ribes diacetate fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881.

According to the scheme, the water absorption pad is 16-18 mm long and 3-4 mm wide, and the detection pad is 18-30 mm long and 3-4 mm wide; the gold mark pad is 10-12 mm long and 3-4 mm wide; the sample pad is 12-15 mm long, and 3-4 mm wide, and the overlap length of each adjacent pad is 1-3 mm.

According to the scheme, the water absorption pad is absorbent paper.

According to the scheme, the distance between every two adjacent detection lines on the detection pad is 2-3mm, the distance between the detection line close to the quality control line and the upper edge of the nitrocellulose membrane is 15-20 mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7 mm.

According to the scheme, the coating amount of the sickle knife bacterium enol diacetate-bovine serum albumin conjugate (DAS-BSA) required per centimeter on the detection line of the sickle knife bacterium enol diacetate-bovine serum albumin conjugate (DAS-BSA) coated by the detection pad is 100-300 ng; the coating amount of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA) required by each centimeter on a detection line coated with the deoxynivalenol-bovine serum albumin conjugate is 100-300 ng; the coating amount of the T-2 toxin-bovine serum albumin conjugate (T-2-BSA) required by each centimeter on a detection line coated with the T-2 toxin-bovine serum albumin conjugate (T-2-BSA) is 100-300 ng; the coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter on the quality control line is 50-200 ng.

According to the scheme, the particle size of colloidal gold used in the gold label pad is 15-20 nm; the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by the spraying length per centimeter on the gold label pad is 100-200 ng, the dosage of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody is 100-200 ng, and the dosage of the colloidal gold labeled anti-T-2 toxin monoclonal antibody is 200-400 ng.

According to the scheme, the IC50 of the anti-deoxynivalenol monoclonal antibody is preferably less than or equal to 15 ppb.

According to the above protocol, the anti-T-2 toxin monoclonal antibody has an IC50 of 2ppb or less.

The preparation method of the immunochromatographic test strip for synchronously detecting the sickle knife enol, the deoxynivalenol and the T-2 toxin of the ribes diacetate comprises the following steps:

(1) preparation of absorbent pad

Cutting the absorbent paper to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

b, preparing a coating solution of 0.25-0.5 mg/mL by using a coating buffer solution respectively for a fusarenol diacetate-bovine serum albumin conjugate (DAS-BSA), a deoxynivalenol-bovine serum albumin conjugate (DON-BSA) and a T-2 toxin-bovine serum albumin conjugate (T-2-BSA), coating the coating solution on a nitrocellulose membrane respectively by using a dot spraying mode to obtain three detection lines, and drying the detection lines for 30-60 minutes at 37-40 ℃; the coating amount of the sickle knife enol diacetate-bovine serum albumin conjugate (DAS-BSA) required per centimeter on the detection line for coating the sickle knife enol diacetate-bovine serum albumin conjugate (DAS-BSA) is 100-300 ng; the coating amount of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA) required by each centimeter on a detection line coated with the deoxynivalenol-bovine serum albumin conjugate is 100-300 ng; the coating amount of the T-2 toxin-bovine serum albumin conjugate (T-2-BSA) required on the detection line coated with the T-2 toxin-bovine serum albumin conjugate is 100-300 ng, the distance between every two adjacent detection lines is 2-3mm, and the distance between the detection line close to the quality control line and the upper edge of the nitrocellulose membrane is 15-20 mm;

coating of quality control line:

preparing a coating buffer solution for a rabbit anti-mouse polyclonal antibody into a coating solution of 0.2-0.4 mg/mL, transversely coating the coating solution on a nitrocellulose membrane at a position 5-7 mm away from a detection line close to a quality control line by a dot spraying mode to obtain the quality control line, wherein the coating amount of the rabbit anti-mouse polyclonal antibody required on each centimeter of the quality control line is 50-200 ng, and drying for 1-2 hours at 37-40 ℃;

(3) preparation of sample pad

Soaking the glass fiber membrane in a sealing liquid, taking out, drying for 6-10 hours at 37-40 ℃ to obtain a sample pad, and then placing the sample pad in a dryer for storage at room temperature;

(4) preparation of gold label pad

Soaking a glass fiber membrane in a sealing solution, taking out, drying for 6-10 hours at 37-40 ℃, and spraying a mixed solution of colloidal gold-labeled anti-sickle enol diacetate monoclonal antibody solution, colloidal gold-labeled anti-deoxynivalenol monoclonal antibody solution and colloidal gold-labeled anti-T-2 toxin monoclonal antibody solution onto the dried glass fiber membrane in a spot spraying manner, wherein: the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by each centimeter of spraying length is 100-200 ng, the dosage of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody required by each centimeter is 100-200 ng, the dosage of the T-2 toxin monoclonal antibody required by each centimeter is 200-400 ng, and then the coating is subjected to vacuum freeze drying for 2-4 hours and is stored in a dryer at room temperature; the preservation number of the anti-ribes diacetate fusarenol monoclonal antibody is CCTCC NO, and the anti-ribes diacetate fusarenol monoclonal antibody is produced by secretion of a hybridoma cell strain DAS5G11E7 with the preservation number of C201881;

(5) assembly of test strips

Paste the pad that absorbs water, detect pad, gold mark pad and sample pad in proper order from the top down at the one side of cardboard, adjacent each pad is in junction overlap connection, and overlap length is 1 ~ 3mm, obtains the colloidal gold immunity chromatography test paper strip of synchronous detection with the mixed pollution of ribes diacetic acid sickle-knife enol, deoxynivalenol, T-2 toxin.

According to the scheme, every 10mL of the coating buffer solution contains: bovine serum albumin 0.1-0.2 g, sodium chloride 0.08g, disodium hydrogen phosphate dodecahydrate 0.029g, potassium chloride 0.002g and potassium dihydrogen phosphate 0.002 g;

according to the scheme, the blocking solution in the step (3) and the step (4) contains the following components in each 100 mL: 1-2 g of ovalbumin, 2-5 g of sucrose, 0.02-0.05 g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate.

According to the scheme, the colloidal gold labeled anti-ribes diacetate fusarenol monoclonal antibody solution is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercially available colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value by using 0.4mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 2mL of 0.1mg/mL aqueous solution of anti-sickle enol diacetate under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing preservation solution to obtain 5.0mL concentrate, and placing in 4 deg.C refrigerator;

the colloidal gold labeled anti-deoxynivalenol monoclonal antibody is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercial colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value with 0.4mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 1.5mL of 0.1mg/mL of anti-deoxynivalenol monoclonal antibody aqueous solution under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing preservation solution to obtain 5.0mL concentrate, and placing in 4 deg.C refrigerator;

the colloidal gold labeled anti-T-2 toxin monoclonal antibody solution is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercial colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value with 0.425mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 2.5mL of 0.1mg/mL of anti-T-2 toxin monoclonal antibody aqueous solution under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing preservation solution to obtain 5.0mL concentrate, and placing in 4 deg.C refrigerator;

the 0.1mol/L potassium carbonate aqueous solution is as follows: dissolving 13.8g of potassium carbonate in pure water to reach the constant volume of 1000mL, and filtering with a 0.22-micron filter membrane to obtain the potassium carbonate solution; the marked washing and preserving fluid is as follows: 2.0g of polyethylene glycol-20000, 0.2g of sodium azide, 0.1235g of boric acid and pure water to 1000mL, and filtering the mixture through a 0.22-micron filter membrane.

The application of the immunochromatographic test strip for synchronously detecting mixed pollution of sickle knife enol, deoxynivalenol and T-2 toxin of ribes diacetate comprises the following steps: weighing a levigated sample to be detected, adding a methanol aqueous solution with volume concentration of 60-80%, uniformly mixing, carrying out ultrasonic extraction for 5-10 minutes in a water bath at 50-60 ℃, standing for 5-10 minutes, diluting a supernatant, namely an extracting solution, with water to enable the final volume concentration of methanol in the diluting solution to be 20-30% to obtain a sample solution to be detected, dropwise adding 80-150 microliter of the sample solution to be detected as a detection solution onto a sample pad of an immunochromatographic test strip for synchronously detecting mixed pollution of sethoxydim diacetate, deoxynivalenol and T-2 toxin, taking another methanol aqueous solution with the same volume of methanol concentration as a negative control solution, dropwise adding another sample pad of the immunochromatographic test strip for synchronously detecting mixed pollution of sethoxydim diacetate, deoxynivalenol and T-2 toxin, the test strip is used as a control test strip, and the detection test strip and the control test strip are subjected to color development control after 15-20 minutes:

when the color of a detection line coated with a conjugate (DAS-BSA) of sickle knife enol-bovine serum albumin diacetate on a detection test strip is close to that of a corresponding detection line on a control test strip, the content of the sickle knife enol diacetate in a sample solution to be detected is lower than 5 ng/mL; when the color of the detection line is lighter than that of the corresponding detection line, the content of the sickle enol diacetate in the sample solution to be detected is equal to or higher than 5ng/mL and lower than 100 ng/mL; when the color is not developed, the content of the ribes diacetate sickle knife fungus enol in the sample solution to be detected is equal to or higher than 100 ng/mL;

when the color of a detection line coated with a conjugate (DON-BSA) of deoxynivalenol-bovine serum albumin on the detection test strip is close to that of a corresponding detection line on a control test strip, the content of the deoxynivalenol in a sample solution to be detected is lower than 10 ng/mL; when the color of the detection line is lighter than that of the corresponding detection line, the content of deoxynivalenol in the sample solution to be detected is equal to or higher than 10ng/mL and lower than 200 ng/mL; when the color is not developed, the content of the deoxynivalenol in the sample solution to be detected is equal to or higher than 200 ng/mL;

when the color of a detection line coated with a T-2 toxin-bovine serum albumin conjugate (T-2-BSA) on the detection test strip is close to that of a corresponding detection line on the control test strip, the content of the T-2 toxin in a sample solution to be detected is lower than 5 ng/mL; when the color is lighter than that of the corresponding detection line, the content of the T-2 toxin in the sample solution to be detected is equal to or higher than 5ng/mL and lower than 80 ng/mL; when the color is not developed, the content of the T-2 toxin in the sample solution to be detected is equal to or higher than 80 ng/mL;

when the quality control line does not develop color, the test strip is judged to be invalid no matter whether the detection line of the test strip develops color or not;

finally, the contents of the ribes diacetate fusarenol, the deoxynivalenol and the T-2 toxin in the sample to be detected are obtained through conversion.

The operating principle of the immunochromatographic test strip in the synchronous detection of mixed pollution of sickle enol diacetate, deoxynivalenol and T-2 toxin is as follows: when a sample solution to be detected is added on the sample pad at the lower end of the test strip, the sample solution to be detected moves towards the water absorption pad through capillary action along the test strip, and when the sample solution to be detected moves to the gold mark pad, the colloidal gold-marked anti-sickle enol diacetate monoclonal antibody, the colloidal gold-marked anti-deoxynivalenol monoclonal antibody and the colloidal gold-marked anti-T-2 toxin monoclonal antibody are dissolved. When the sample contains the ribes oxalacetate sickle knife enol, the ribes oxalacetate sickle knife enol is combined with the colloidal gold-labeled anti-ribes oxalacetate sickle enol monoclonal antibody on the gold-labeled pad and swims upwards together, and when the sample reaches a detection line I which fixes the antigens of the ribes oxalacetate sickle enol-bovine serum albumin conjugate, the antigens compete with the ribes oxalacetate sickle enol to be combined with limited antigen combining sites on the colloidal gold-labeled anti-ribes oxalacetate sickle enol monoclonal antibody, the higher the content of the ribes oxalacetate sickle enol in the sample is, the fewer the colloidal gold-labeled anti-ribes oxalate monoclonal antibody which can be combined with the antigens on the detection line is, and the lighter the color of a color zone formed on the detection line is; when the sample contains deoxynivalenol, the deoxynivalenol is combined with the anti-deoxynivalenol monoclonal antibody marked by colloidal gold on the gold-marked pad and swims upwards together, and when the deoxynivalenol reaches a detection line II fixed with the antigen of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA), the antigen is in competition with the deoxynivalenol and is combined with the limited antigen combination sites on the anti-deoxynivalenol monoclonal antibody marked by the colloidal gold, the higher the content of the deoxynivalenol in the sample is, the fewer the anti-deoxynivalenol monoclonal antibody marked by the colloidal gold and capable of being combined with the antigen on the detection line is, and the lighter the color of a color development band formed on the detection line is; when the sample contains T-2 toxin, the T-2 toxin is combined with the colloidal gold-labeled anti-T-2 toxin monoclonal antibody on the gold-labeled pad and swims upwards together, when the T-2 toxin reaches a detection line III fixed with T-2 toxin-bovine serum albumin conjugate (T-2-BSA) antigen, the antigen competes with the T-2 toxin to be combined with limited antigen combining sites on the colloidal gold-labeled anti-T-2 toxin monoclonal antibody, the higher the content of the T-2 toxin in the sample is, the fewer the colloidal gold-labeled anti-T-2 toxin monoclonal antibodies which can be combined with the antigen on the detection line are, and the lighter the color developing band formed on the detection line is.

When the number of the corresponding antibodies of the colloidal gold labels combined by the antigens on the three detection lines is less than a certain number, no red line appears at the three detection lines. Regardless of whether the sample contains the three mycotoxins or not, the colloidal gold-labeled anti-mycotoxin antibody or the conjugate of the colloidal gold-labeled anti-mycotoxin antibody and the mycotoxins, which are not captured by the antigen on the detection line, move to the quality control line, are bound with the rabbit anti-mouse polyclonal antibody on the quality control line, and are enriched and developed. According to the method, the detection line coated with the coupling substance of sickle knife enol diacetate and bovine serum albumin on the detection test strip, the detection line coated with the coupling substance of deoxynivalenol-bovine serum albumin (DON-BSA) and the detection line coated with the coupling substance of T-2 toxin-bovine serum albumin (T-2-BSA) are subjected to color development contrast with the corresponding detection line color on the contrast test strip, so that the mixed pollution condition of three kinds of fungal toxins, namely the sickle enol diacetate, the deoxynivalenol and the T-2 toxin in a sample can be obtained.

The invention has the beneficial effects that:

(1) rapidly and synchronously detecting fusarium oxydianum, deoxynivalenol and T-2 toxin. The colloidal gold immunochromatographic test strip provided by the invention can realize synchronous and rapid detection of three mycotoxins, namely, the ribes diacetate fusarenol, the deoxynivalenol and the T-2 toxin, on one test strip, and all the used antibodies are monoclonal antibodies, particularly, the used ribes diacetate fusarenol monoclonal antibody has good specificity (the cross reaction to toxins such as T2 toxin and vomitoxin (DON) is less than 0.01 percent), and the sensitivity is high, so that the detection of each mycotoxin can be ensured to be free from interference, and the detection is simple and rapid.

(2) The sensitivity is high. The colloidal gold immunochromatographic test strip provided by the invention has the lowest detection limit of the sickle knife enol diacetate in the detection solution reaching 5ng/mL, the lowest detection limit of the sickle knife enol diacetate in the detection solution reaching 10ng/mL, the lowest detection limit of the T-2 toxin reaching 5ng/mL, and the detection limit can meet the limit requirements of European Union on the three mycotoxins in food.

(3) The sample pretreatment method is simple. The sample pretreatment only needs to add the methanol water extracting solution into the sample for ultrasonic extraction for 5-10 minutes, then stand for 5-10 minutes, and take the supernatant for dilution to carry out detection, and the whole sample pretreatment process is simple and rapid.

Drawings

FIG. 1 is a front view of a colloidal gold immunochromatographic test strip for synchronously detecting mixed contamination of sickle knife enol diacetate, deoxynivalenol and T-2 toxin according to the present invention;

FIG. 2 is a graph showing the results of determination of the wheat samples (numbers 001, 002 and 003, respectively) in example 2;

in the figure: 1, a paper board, 2 water absorption pads, 3 detection pads, 4 gold mark pads, 5 sample pads, 6 quality control lines, 7 detection lines III, 8 detection lines II, 9 detection lines I, 10 control test strips and 11 detection test strips;

fig. 3 is affinity measurement data of a sickle enol monoclonal antibody based on ribes diacetate provided by the present invention;

fig. 4(a) shows the cross reaction result between the ribes diacetate fusarenol monoclonal antibody and other mycotoxins provided by the present invention; (b) the invention provides a standard curve of a ribes diacetate fusarenol enzyme-linked immunosorbent assay method established by the ribes diacetate fusarenol monoclonal antibody.

Detailed Description

Example 1: obtaining of anti-diacetyl ribes fusarenol monoclonal antibody

The anti-diacetyl ribes fusarium enol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO. C201881, and the preparation method comprises the following steps:

injecting a hybridoma cell strain DAS5G11E7 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the concrete operations are as follows: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. Centrifuging at 12000r/min at 4 deg.C for more than 30min, discarding precipitate, filtering the obtained supernatant with double-layer filter paper, 1/10 adding phosphate buffer solution with molarity of 0.1mol/L and pH of 7.4, adjusting pH of the mixture to 7.4 with 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in ice bath to final concentration of 0.277g/mL, standing at 4 deg.C for more than 2 hr, then 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate with phosphate buffer solution with the molar concentration of 0.01mol/L, pH of 7.4 of the volume of original ascites volume 1/10, filling into a dialysis bag, dialyzing for two days with 0.01mol/LPBS, dialyzing for two days with PB again, taking out the protein solution in the dialysis bag, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing at-70 ℃, and freeze-drying in a freeze-dryer. Collecting freeze-dried powder, namely the purified ribes diacetate resistant sickle enol monoclonal antibody;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water into 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate.

The subtype of the ribes diacetate resisting sickle enol monoclonal antibody secreted by the hybridoma cell strain DAS5G11E7 is identified to be IgG2b by using a commercial subtype identification kit.

The titer of the antibody obtained by ascites purification of the mice can reach 3.2 multiplied by 10 measured by the conventional non-competitive enzyme-linked immunosorbent assay (ELISA)⁵I.e. 3.2X 10 dilution of antibody⁵The solution test result is positive. The sensitivity of the method to the sickle enol diacetate is 3.08ng/mL by using the conventional indirect competition ELISA. The cross-reactions with other mycotoxins, T2 toxin, HT2 toxin, vomitoxin, 3-acetyl deoxynivalenol, ochratoxin, fumonisin were all less than 0.01% (Table 1; FIG. 4). The specificity of the antibody can be evaluated by the cross-reactivity. Detecting DAS5G11E7 monoclonal antibody by indirect competition ELISA method, and detecting DAS, T2 toxin, HT2 toxin, DON, 3-ACDON, OTA and FB₁Preparing standard solutions with series concentrations, respectively adding the standard solutions and an antibody with the same volume into an ELISA plate, incubating for 1h, and performing other steps with an indirect competitive ELISA method. And (3) drawing a competitive inhibition curve by taking the concentration of the toxin standard substance as an abscissa and an OD value B/B0 measured by an enzyme-labeling instrument at 450nm as an ordinate, and determining the cross reaction rate by calculating the ratio of the IC50 values of the DAS to other toxins. The calculation formula is as follows:

CR% (IC50DAS/IC50 other toxins) × 100.

Table 1 cross-reactivity of DAS5G11E7 with other toxins.

The affinity of DAS5G11E7 was determined using an indirect non-competitive ELISA. Coating an enzyme label plate with DAS-OVA according to the concentration of 1.0, 0.5, 0.25 and 0.125 mu g/mL, 100 mu L/hole, 37 ℃ and 2 h; after blocking with blocking solution for 1h, the antibody (dilution factor 1:2) diluted with PBS was added to the ELISA plate, and the rest steps were performed in the same manner as in the indirect non-competitive ELISA method.With the measured OD450 values as ordinate and the logarithm of the antibody concentration (mol/L) as abscissa, 4S-shaped curves of 4 concentrations were made. The maximum OD value at the top of each S-curve, i.e., ODmax, was found, and the antibody concentration corresponding to 50% ODmax of each curve was found. Any two of the 4 concentrations are combined into one group according to the formula Ka ═ n-1)/2(n [ Ab']t- [Ab]t) calculating the affinity constant of the antibody, wherein [ Ab']t、[Ab]t is the antibody concentration corresponding to the two 50% maximum OD values in each group, n is the multiple of the envelope antigen concentration in each group (including three ratios of 1:2, 1: 4, 1: 8), and 6 Ka values are obtained. The six obtained Ka values are averaged to obtain the anti-diacetyl-grass fusarenol mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method with the affinity of 5.4 multiplied by 10⁸L/moL (FIG. 3).

Screening of hybridoma cell line DAS5G11E7

1. Animal immunization

A BALB/c mouse of 6-7 weeks old is immunized by using a sickle knife enol complete antigen DAS-BSA prepared in a laboratory. The first immunization is that after the diacetyl sickle enol complete antigen and equivalent volume of Freund's complete adjuvant are emulsified, subcutaneous multi-point injection is carried out on the back of the neck of a mouse. The second immunization is carried out after 4 weeks, and the second immunization is emulsified by a Freund incomplete adjuvant and isovolumetric sickle enol complete antigen and injected into the abdominal cavity of a mouse. The third immunization was carried out at 4 weeks intervals from the second immunization in the same manner as the first immunization, and the fourth immunization was carried out 3 weeks after the third immunization in the same manner as the second immunization, which was also administered intraperitoneally. The 4 immunization doses were identical and were 70. mu.g per mouse. And (3) collecting blood from tail veins 8-10 days after each immunization of the first 3 times, separating serum, and detecting the serum titer of the mice by adopting indirect ELISA. 8 days after 3 rd immunization, blood is collected by breaking the tail, and mice corresponding to serum with relatively high titer and sensitivity are selected for carrying out the last boosting immunization, wherein the immunization dose is 2 times of that of the mice.

2. Cell fusion

After 3 days of boosting immunity, adopting PEG with 50% of polyethylene glycol and molecular weight of 1450 as fusion agent, and making cell fusion according to conventional method, and its concrete steps are: killing the mouse by removing neck under aseptic condition, taking out spleen, crushing spleen with homogenizer, separating spleen cells with filter screen, mixing with mouse myeloma cell SP2/0 at a ratio of 5: 1, centrifuging, resuspending mixed cells with RPMI-1640 basic culture solution, centrifuging, and discarding supernatant. Adding 1-2mL of 50% PEG, adding 10-20mL of RPMI-1640 basic culture solution in an adherent manner for 1 minute in a common use, centrifuging, discarding the supernatant, re-suspending the fused cells at the bottom of the tube by using 20mL of cell complete culture medium containing 1% HAT, adding the suspended cells into 80mL of semisolid culture medium, uniformly mixing, adding the mixture onto a 6-well cell culture plate, culturing at 1.5 mL/well, and placing the mixture in a carbon dioxide incubator at 37 ℃. The complete cell culture medium containing 1% HAT contains 20% (volume percent) of fetal calf serum, 75% (volume percent) of RPMI-1640 basic culture solution, 1% (weight percent) of L-glutamine, 1% (volume percent) of HEPES, 1% (volume percent) of diabody (10000 units per milliliter of penicillin and 10000 micrograms per milliliter of streptomycin), 2% (volume percent) of growth factor (HFCS) and 1% (weight percent) of hypoxanthine-aminopterine-thymidine, namely HAT and methylcellulose, which are purchased from sigma-Aldrich company.

Screening and cloning of cell lines

And (3) picking out the clone from the culture medium by using a micropipette when the cell colony grows to be visible by naked eyes 2-3 weeks after the cells are fused, transferring the clone to a 96-hole cell culture plate, culturing by using HAT liquid, and sucking culture supernatant for detection when the cells grow to the bottom of 2/3 holes. Adopting a two-step screening method, wherein in the first step, an indirect ELISA method is adopted to screen out positive holes which resist sickle knife enol diacetate but not resist carrier protein BSA; and in the second step, detecting the positive holes screened in the first step by adopting an indirect competition ELISA method, using sickle enol diacetate as a competitor, and selecting holes with higher light absorption value and sensitivity (the holes with higher light absorption value indicate that the competitor is 0, namely the final measured value of the positive control hole is higher, and the higher sensitivity indicates that the concentration of the competitor is also IC when the inhibition rate is 50%₅₀Smaller value), carrying out subcloning by using a limiting dilution method, carrying out detection by using the same two-step method after subcloning, and repeating the subcloning for 4-5 times to obtain a hybridoma cell strain DAS5G11E 7. The hybridoma cell strain has been preserved in Chinese typical culture in 2018, 4 months and 3 daysThe preservation center (CCTCC) has the preservation address of China, Wuhan and Wuhan university and the preservation number is CCTCC NO: C201881.

Determination of variable region sequence of anti-ribes diacetate fusarenol monoclonal antibody hybridoma cell strain DAS5G11E7 antibody

(1) Extracting total RNA: extracting total RNA capable of generating hybridoma cell strains DAS5G11E7 by adopting a total RNA extraction kit of Tiangen company according to an instruction;

(2) synthesis of cDNA: taking the total RNA obtained in the step 1 as a template, and oligo (dT)15 as a primer according to SuperScript^TM-2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT)15 was purchased from Invitrogen;

(3) the PCR method is to design primers according to conserved sites of a mouse antibody gene sequence in GENBANK, amplify the heavy chain and light chain variable region genes of the antibody by using CDNA as a template, wherein the PCR program comprises the steps of amplifying the heavy chain and light chain variable region genes by using CDNA as a template, amplifying the genes for 30 cycles at 94 ℃, 58 ℃ for 45S and 72 ℃ for 1min, finally extending the products for 10min at 72 ℃, separating the PCR products by agarose gel electrophoresis at 1 percent (weight percent), purifying and recovering DNA fragments by using a kit, connecting the DNA fragments to a vector pMD18-T, transforming escherichia coli DH5 α competent cells, picking up positive clones, and sending the positive clones to Hippocampus Sangnie Biotech limited for sequencing, wherein the sequences of the primers are respectively 5 '-CAG GTS MAR CTG MAG GAG TCW G-3' (22mer) and 5'-CAG GGG CCAGTG GAT AGA CAG ATG GGG G-3' (28mer) of the heavy chain variable region primer, wherein S, M, R and W are merged bases, M ═ A/C, R ═ A/G, S ═ G/C, W ═ A/T, and light chain variable region primer 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24 mer).

Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 351bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 117 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence is 324bp long, the sequence is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 108 amino acids, and the sequence is shown as SEQ ID NO. 4.

The anti-deoxynivalenol monoclonal antibody is preferably an anti-deoxynivalenol monoclonal antibody with the IC50 less than or equal to 15 ppb. For example, the anti-deoxynivalenol monoclonal antibody produced by santong dungdu biotechnology limited, etc., the anti-deoxynivalenol monoclonal antibody specifically used in this example was the anti-deoxynivalenol monoclonal antibody produced by santong dungdu biotechnology limited, and the sensitivity IC50 was 12 ppb.

The anti-T-2 toxin monoclonal antibody is preferably an anti-T-2 toxin monoclonal antibody with an IC50 of 2ppb or less (2ng/mL), such as the T-2 toxin monoclonal antibody produced by Shandong Lloyd Biotech, Inc., the T-2 toxin monoclonal antibody of Shandong Lloyd Biotech, Inc. is specifically used in this example, and the detection sensitivity IC50 is 0.8 ng/mL.

Example 2

The preparation method of the immunochromatographic test strip for synchronously detecting mixed pollution of sickle knife enol diacetate, deoxynivalenol and T-2 toxin comprises the following steps:

(1) preparation of absorbent pad

Cutting the absorbent paper into pieces with the length of 16mm and the width of 4mm to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

preparing a coating solution of 0.4mg/mL of T-2 toxin-bovine serum albumin conjugate (OTA-BSA) by using a coating buffer solution, coating the coating solution on a nitrocellulose membrane at a position 15mm away from the nitrocellulose membrane by using a dot spraying mode to obtain a detection line III, preparing a coating solution of 0.4mg/mL of T-2 toxin-bovine serum albumin conjugate (T-2-BSA) required on each cm of the detection line III at a position 2mm away from the detection line III, coating the coating solution on the nitrocellulose membrane by using a deoxynivalenol-bovine serum albumin conjugate (DON-BSA) at a position 2mm away from the detection line III to obtain a detection line II, coating the coating solution of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA) required on each cm of the detection line II at a position of 200ng, and preparing a coating solution of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA) required on the detection line II by using a dot spraying mode (DAS-BSA) is prepared into 0.5mg/mL solution by using a coating buffer solution, the solution is coated on a nitrocellulose membrane at a position 2mm away from a detection line II by using a dot spraying mode to obtain a detection line I, the coating amount of the sickle knife ketoenol diacetate-bovine serum albumin conjugate (DAS-BSA) required on each centimeter of the detection line I is 200ng, and then the drying is carried out for 30 minutes at the temperature of 37 ℃;

coating of quality control line:

preparing a coating buffer solution for the rabbit anti-mouse polyclonal antibody into a coating solution of 0.25mg/mL, transversely coating the coating solution on a nitrocellulose membrane at a position 3mm away from a detection line III by a dot spraying mode to obtain a quality control line, wherein the coating amount of the rabbit anti-mouse polyclonal antibody required on each centimeter of the quality control line is 80ng, and then drying for 1 hour at 37 ℃;

the coating buffer solution is as follows: 0.1g bovine serum albumin, 0.08g sodium chloride, 0.029g disodium hydrogen phosphate dodecahydrate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate, and water to a constant volume of 10 mL.

The nitrocellulose membrane is 24mm long and 4mm wide.

(3) Preparation of sample pad

Cutting the glass fiber membrane into pieces with length of 12mm and width of 4mm, soaking in sealing solution, taking out, drying at 37 deg.C for 8 hr to obtain sample pad, and storing in a desiccator at room temperature.

The confining liquid is as follows: adding water into 1g of egg white albumin, 2g of sucrose, 0.02g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate to keep the volume constant to 100 mL.

(4) Preparation of gold label pad

Cutting a glass fiber membrane into a specification with the length of 10mm and the width of 4mm, putting the glass fiber membrane into the sealing liquid in the step (3) for soaking, taking out, drying the glass fiber membrane for 8 hours at 37 ℃, and spraying a mixed solution of a colloidal gold-labeled anti-sickelen diacetate monoclonal antibody solution, a colloidal gold-labeled anti-deoxynivalen monoclonal antibody solution and a colloidal gold-labeled anti-T-2 toxin monoclonal antibody solution on the dried glass fiber membrane in a point spraying manner, wherein: the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by the spraying length per centimeter is 150ng, the dosage of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody required by the spraying length per centimeter is 120ng, the dosage of the T-2 toxin monoclonal antibody required by the spraying length per centimeter is 150ng, and then the spraying length per centimeter is subjected to vacuum freeze drying for 2 hours and is stored in a dryer at room temperature;

the colloidal gold labeled anti-ribes diacetate fusarenol monoclonal antibody solution is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercially available colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value by using 0.4mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 2mL of 0.1mg/mL aqueous solution of anti-sickle enol diacetate under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, removing supernatant, re-suspending the precipitate with labeled washing and preserving solution to obtain 5.0mL of concentrate, and placing the concentrate in a refrigerator at 4 ℃ for later use, wherein the mass concentration of the colloidal gold labeled anti-sickle enol diacetate monoclonal antibody solution is 0.04 mg/mL;

the colloidal gold labeled anti-deoxynivalenol monoclonal antibody is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercial colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value by using 0.4mL0.1mol/L potassium carbonate aqueous solution, slowly adding 1.5mL0.1mg/mL of an anti-deoxynivalenol monoclonal antibody aqueous solution in a stirring state, and continuously stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing and preserving solution to obtain 5.0mL of concentrate, and placing in a refrigerator at 4 ℃ for later use, wherein the mass concentration of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody solution is 0.03 mg/mL;

the colloidal gold labeled anti-T-2 toxin monoclonal antibody solution is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercial colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value with 0.425mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 2.5mL of 0.1mg/mL of anti-T-2 toxin monoclonal antibody aqueous solution under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing and preserving solution to obtain 5.0mL of concentrate, and placing in a refrigerator at 4 ℃ for later use, wherein the mass concentration of the colloidal gold labeled anti-T-2 toxin monoclonal antibody solution is 0.05 mg/mL;

the particle size of the colloidal gold in the colloidal gold solution is 15 nm;

(5) Assembly of test strips

Paste the pad that absorbs water, detect pad, gold mark pad and sample pad in proper order from the top down at the one side of cardboard, adjacent each pad is in the junction overlap connection, and overlap length is 1 ~ 3mm, and the immunochromatography test paper strip that obtains synchronous detection ribes grass sickle enol diacetate, deoxynivalenol, T-2 toxin mixed contamination is shown in figure 1 and figure 2.

The immunochromatography test strip for synchronously detecting the mixed pollution of the sickle knife enol diacetate, the deoxynivalenol and the T-2 toxin is applied to the detection of corn samples:

weighing a wheat sample to be detected (the number is 001, 002 and 003 respectively), adding 70% methanol aqueous solution, mixing uniformly, carrying out ultrasonic extraction for 10 minutes in a water bath at 50 ℃, standing for 10 minutes, enabling the final volume concentration of methanol in a diluent to be 14% by passing supernatant liquid, namely an extracting solution, through a 0.45-micron organic filter membrane to obtain a sample solution, taking 100 microliter of the diluted sample solution as a detection solution, dropwise adding a sample pad of an immunochromatographic test strip for synchronously detecting mixed pollution of sickle enol diacetate, deoxynivalenol and T-2 toxin, taking 100 microliter of an aqueous solution with the methanol concentration of 14% as a negative control solution, dropwise adding another sample pad of the immunochromatographic test strip for synchronously detecting mixed pollution of sickle enol diacetate, deoxynivalenol and T-2 toxin, it served as a control test strip and the results were read after 15 minutes.

And (3) detection results: the quality control line of the 001 detection test strip shows a red strip, the colors of the detection line I and the detection line II are close to those of the detection line I and the detection line III in the comparison test strip, the detection line II does not develop color, and the detection line II is shown in 2-1 in figure 2, so that the judgment is carried out: the content of ribes diacetate sickle knife enol in a 001 sample solution to be detected is lower than 5 ng/mL; the content of T-2 toxin is lower than 5 ng/mL; the content of deoxynivalenol is equal to or higher than 200 ng/mL; the contents of the ribes diacetate fusarenol and the T-2 toxin in the 001 sample to be detected are lower than 25ng/g through conversion, and the content of the deoxynivalenol is equal to or higher than 1000 ng/g.

The quality control line of the 002 detection test strip shows a red strip, the color of the detection line I is close to that of the detection line I in the comparison test strip, and the color of the detection line II and the color of the detection line III are lighter than that of the detection line II and the color of the comparison test strip, which is shown as 2-2 in figure 2, so that the detection is judged: 002 the content of the sickle knife enol diacetate in the sample solution to be detected is lower than 5ng/mL, the content of the deoxynivalenol is higher than 10ng/mL and lower than 200ng/mL, the content of T-2 toxin is higher than 5ng/mL and lower than 80ng/mL, the conversion can be carried out to obtain 002 the content of the sickle knife enol diacetate in the sample to be detected is lower than 25ng/g, and the content of the deoxynivalenol is higher than 50ng/g and lower than 1000 ng/g; the content of T-2 toxin is equal to or higher than 25ng/g and lower than 400 ng/g.

The quality control line of the 003 test strip shows a red strip, the color of the detection line I, the color of the detection line II and the color of the detection line III are close to the color of the detection line in the control test strip, which is shown as 2-3 in figure 2, and shows that: the content of ribes diacetate sickle knife enol in the sample solution to be detected is lower than 5 ng/mL; the content of deoxynivalenol is lower than 10 ng/mL; the content of T-2 toxin is lower than 5 ng/mL; the content of the sickle knife enol diacetate in the sample to be detected is equal to or higher than 25ng/g through conversion; the content of deoxynivalenol is lower than 50 ng/g; the content of T-2 toxin is less than 25 ng/g.

Example 3

(1) preparation of absorbent pad

Cutting the absorbent paper into pieces with the length of 18mm and the width of 3mm to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

preparing 0.5mg/mL coating solution from sickle cell enol diacetate-bovine serum albumin conjugate (DAS-BSA) by using a coating buffer solution, coating the solution on a nitrocellulose membrane at a position 18mm away from the nitrocellulose membrane by using a dot spraying method to obtain a detection line I, preparing 0.4mg/mL coating solution from sickle cell enol diacetate-bovine serum albumin conjugate (DAS-BSA coating amount is 200 ng) required on each cm detection line I, coating the solution on the nitrocellulose membrane by using a coating buffer solution at a position 3mm away from the detection line I to obtain a detection line II, and coating the solution from sickle cell enol diacetate-bovine serum albumin conjugate (DON-BSA) required on each cm detection line II by using a dot spraying method to obtain 200ng T-2-BSA) is prepared into a 0.25mg/mL solution by using a coating solution, the solution is coated on a nitrocellulose membrane at a position 3mm away from a detection line II by a dot spray mode to obtain a detection line II, the coating amount of a T-2 toxin-bovine serum albumin conjugate (T-2-BSA) required on each cm of the detection line II is 300ng, and then the solution is dried for 30 minutes at the temperature of 40 ℃;

coating of quality control line:

preparing a coating buffer solution for the rabbit anti-mouse polyclonal antibody into a coating solution of 0.25mg/mL, transversely coating the coating solution on a nitrocellulose membrane at a position 6mm away from a detection line I by a dot spraying mode to obtain a quality control line, wherein the coating amount of the rabbit anti-mouse polyclonal antibody required on each centimeter of the quality control line is 80ng, and then drying for 1 hour at 37 ℃;

the coating buffer solution is as follows: 0.2g bovine serum albumin, 0.08g sodium chloride, 0.029g disodium hydrogen phosphate dodecahydrate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate, and water to a constant volume of 10 mL.

The length of the nitrocellulose membrane is 30mm, and the width of the nitrocellulose membrane is 3 mm.

(3) Preparation of sample pad

Cutting the glass fiber membrane into pieces with length of 15mm and width of 3mm, soaking in sealing solution, taking out, drying at 37 deg.C for 6 hr to obtain sample pad, and storing in a desiccator at room temperature.

The confining liquid is as follows: adding water into 2g of egg white albumin, 4g of sucrose, 0.05g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate to keep the volume constant to 100 mL.

(4) Preparation of gold label pad

Cutting a glass fiber membrane into a specification with the length of 12mm and the width of 3mm, putting the glass fiber membrane into the sealing liquid obtained in the step (3) for soaking, taking out, drying the glass fiber membrane for 6 hours at 40 ℃, and spraying a mixed solution of a colloidal gold-labeled anti-sickle enol diacetate monoclonal antibody solution, a colloidal gold-labeled anti-deoxynivalenol monoclonal antibody solution and a colloidal gold-labeled anti-T-2 toxin clone antibody solution on the dried glass fiber membrane in a point spraying manner, wherein: the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by the spraying length per centimeter is 200ng, the dosage of the colloidal gold labeled anti-T-2 toxin monoclonal antibody required by the spraying length per centimeter is 200ng, the dosage of the required anti-deoxynivalenol monoclonal antibody is 400ng, and then the obtained product is subjected to vacuum freeze drying for 2 hours and is stored in a dryer at room temperature;

the preparation method of the colloidal gold labeled anti-ribes diacetate fusarium enol monoclonal antibody solution, the colloidal gold labeled anti-T-2 toxin monoclonal antibody solution and the colloidal gold labeled anti-deoxynivalenol monoclonal antibody solution is as in example 2, and the difference lies in that; the grain size of the colloidal gold in the used colloidal gold solution is 20 nm;

(5) assembly of test strips

Paste the pad that absorbs water, detect pad, gold mark pad and sample pad in proper order from the top down at the one side of cardboard, adjacent each pad is in junction overlap connection, and overlap length is 1mm, obtains the immunochromatography test paper strip of synchronous detection ribes grass sickle-knife enol diacetate, deoxynivalenol, T-2 toxin mixed contamination.

Weighing a ground wheat sample to be detected, adding a methanol aqueous solution with the volume concentration of 70%, uniformly mixing, carrying out ultrasonic extraction for 10 minutes in a water bath at 50 ℃, standing for 10 minutes to enable the final volume concentration of methanol in a diluent to be 14% to obtain a sample solution, taking 100 mu L of the diluted sample solution as a detection solution, dropwise adding a sample pad of an immunochromatographic test strip for synchronously detecting mixed pollution of sickle enol diacetate, deoxynivalenol and T-2 toxin, wherein the sample pad is taken as a detection test strip, taking 100 mu L of the aqueous solution with the methanol concentration of 14% as a negative control solution, dropwise adding another sample pad of the immunochromatographic test strip for synchronously detecting mixed pollution of sickle enol diacetate, deoxynivalenol and T-2 toxin, wherein the sample pad is taken as a control test strip, and reading a result after 20 minutes.

And (3) detection results: the quality control line of the detection test strip shows a red strip, and the colors of the detection line I, the detection line II and the detection line III are respectively close to the colors of the detection lines in the comparison test strip, so that the detection test strip is judged: the content of T-2 toxin in the sample solution to be detected is lower than 5 ng/mL; the content of deoxynivalenol is lower than 10ng/mL, and the content of ribes diacetate sickle-knife-enol is lower than 5 ng/mL; the content of the T-2 toxin in the sample to be detected is lower than 25ng/g through conversion; the content of deoxynivalenol is lower than 50ng/g, and the content of ribes diacetate sickle-knife-enol is lower than 25 ng/g.

< 110 > institute of oil crops of Chinese academy of agricultural sciences

< 120 > synchronous detection of ribes diacetate sickle knife fungus enol, deoxynivalenol, T-2 toxin colloidal gold immunochromatographic test strip

＜160＞ 4

＜210＞ 1

＜211＞ 351bp

＜212＞ DNA

< 213 > mice

＜400＞ 1

gaagtgcaac tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60

tcctgttcag cctccggatt cactttcaat tactatggca tgtcttgggt tcgccagact 120

ccagacaacc tcctggagtg ggtcgcaggc attagtagtg gtggttctta cacctattat 180

tctgacagtg tgaagggacg attcaccatc tccagagaca gtgccacgaa caccctgtac 240

ctgcaaatga ccagtctgaa gtctcaagac acagccatgt attattgtat tagactcccg 300

tttgggtcta tggactattg gggtcaagga accgcagtca ccgtctcctc a 351

＜210＞ 1

＜211＞ 324bp

＜212＞ DNA

< 213 > mice

＜400＞ 2

caggctgttg tgactcagga acctgcactc accacatcac ctggtgaaac agtcacactc 60

acttgtcgct caagtactgg ggctgtaaca actggtaatt atgtcaactg ggtccaagag 120

aaaccagatc atttattcag tggtctaata ggtaatacca ataaccgagc tccaggtgtt 180

cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacagggaca 240

cagactgagg atgaggcaat atatttctgt gctctatggt acaccgacca tttggtgttc 300

ggtggaggaa ccaaattgac tgtc 324

＜210＞ 1

＜211＞ 117

＜212＞ PRT

< 213 > mice

＜400＞ 3

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Asn Tyr Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Asn Leu Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Thr Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Ser Gln Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ile Arg Leu Pro Phe Gly Ser Met Asp Tyr Trp Gly Gln Gly Thr Ala

100 105 110

Val Thr Val Ser Ser

115

＜210＞ 1

＜211＞ 108

＜212＞ PRT

< 213 > mice

＜400＞ 4

Gln Ala Val Val Thr Gln Glu Pro Ala Thr Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Gly

20 25 30

Asn Tyr Val Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Ser Gly

35 40 45

Leu Ile Gly Asn Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Thr

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Thr Asp

85 90 95

His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105

Claims

1. Detect colloidal gold immunochromatography test paper strip of ribes diacetate sickle enol, deoxynivalenol, T-2 toxin in step, its characterized in that: the detection device comprises a bottom plate, wherein a water absorption pad, a detection pad, a gold mark pad and a sample pad are sequentially adhered to one surface of the bottom plate from top to bottom, the adjacent pads are connected at the joints in an overlapping manner, the detection pad takes a nitrocellulose membrane as a base pad, quality control lines and detection lines are transversely arranged on the nitrocellulose membrane, the detection lines are positioned below the quality control lines, the number of the detection lines is three, the detection lines are distributed at intervals, the three detection lines are respectively coated with a fusarenol diacetate-bovine serum albumin conjugate (DAS-BSA), a fusarenol-bovine serum albumin conjugate (DON-BSA) and a T-2 toxin-bovine serum albumin conjugate (T-2-BSA), and the quality control lines are coated with a rabbit anti-mouse polyclonal antibody; the gold label pad is transversely sprayed with a colloidal gold-labeled anti-sickle cell enol diacetate monoclonal antibody, a colloidal gold-labeled anti-deoxynivalenol monoclonal antibody and a colloidal gold-labeled anti-T-2 toxin monoclonal antibody; the anti-ribes diacetate fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881.

2. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the water absorption pad is 16-18 mm long and 3-4 mm wide, and the detection pad is 18-30 mm long and 3-4 mm wide; the gold mark pad is 10-12 mm long and 3-4 mm wide; the sample pad is 12-15 mm long, and 3-4 mm wide, and the overlap length of each adjacent pad is 1-3 mm.

3. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the absorbent pad is absorbent paper.

4. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the distance between every two adjacent detection lines on the detection pad is 2-3mm, the distance between the detection line close to the quality control line and the upper edge of the nitrocellulose membrane is 15-20 mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7 mm.

5. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the coating amount of the sickle knife enol diacetate-bovine serum albumin conjugate (DAS-BSA) required per centimeter on a detection line of the sickle knife enol diacetate-bovine serum albumin conjugate (DAS-BSA) coated by the detection pad is 100-300 ng; the coating amount of the deoxynivalenol-bovine serum albumin conjugate (DON-BSA) required by each centimeter on a detection line coated with the deoxynivalenol-bovine serum albumin conjugate is 100-300 ng; the coating amount of the T-2 toxin-bovine serum albumin conjugate (T-2-BSA) required by each centimeter on a detection line coated with the T-2 toxin-bovine serum albumin conjugate (T-2-BSA) is 100-300 ng; the coating amount of the rabbit anti-mouse polyclonal antibody required by each centimeter on the quality control line is 50-200 ng.

6. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the particle size of colloidal gold used in the gold-labeled pad is 15-20 nm; the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by the spraying length per centimeter on the gold label pad is 100-200 ng, the dosage of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody is 100-200 ng, and the dosage of the colloidal gold labeled anti-T-2 toxin monoclonal antibody is 200-400 ng.

7. The colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the IC50 of the anti-deoxynivalenol monoclonal antibody is less than or equal to 15 ppb; the anti-T-2 toxin monoclonal antibody has an IC50 of 2ppb or less.

8. The method for preparing the immunochromatographic test strip for synchronously detecting sickle knife enol, deoxynivalenol and T-2 toxin of ribes diacetate in claim 1, which is characterized in that: the method comprises the following steps:

(1) preparation of absorbent pad

Cutting the absorbent paper to obtain the absorbent pad;

(2) preparation of detection pad

Coating of detection lines:

coating of quality control line:

(3) preparation of sample pad

(4) preparation of gold label pad

Soaking a glass fiber membrane in a sealing solution, taking out, drying for 6-10 hours at 37-40 ℃, and spraying a mixed solution of colloidal gold-labeled anti-sickle enol diacetate monoclonal antibody solution, colloidal gold-labeled anti-deoxynivalenol monoclonal antibody solution and colloidal gold-labeled anti-T-2 toxin monoclonal antibody solution onto the dried glass fiber membrane in a spot spraying manner, wherein: the dosage of the colloidal gold labeled anti-sickle cell enol diacetate monoclonal antibody required by each centimeter of spraying length is 100-200 ng, the dosage of the colloidal gold labeled anti-deoxynivalenol monoclonal antibody required by each centimeter is 100-200 ng, the dosage of the T-2 toxin monoclonal antibody required by each centimeter is 200-400 ng, and then the coating is subjected to vacuum freeze drying for 2-4 hours and is stored in a dryer at room temperature; the anti-ribes diacetate fusarenol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881;

(5) assembly of test strips

9. The method for preparing the immunochromatographic test strip according to claim 8, characterized in that: the coating buffer contains the following components in 10 mL: bovine serum albumin 0.1-0.2 g, sodium chloride 0.08g, disodium hydrogen phosphate dodecahydrate 0.029g, potassium chloride 0.002g and potassium dihydrogen phosphate 0.002 g;

the blocking solution in the step (3) and the step (4) contains the following components in each 100 mL: 1-2 g of egg white albumin, 2-5 g of sucrose, 0.02-0.05 g of sodium azide, 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate;

the colloidal gold labeled anti-ribes diacetate fusarenol monoclonal antibody solution is prepared by adopting an unsaturated labeling method, and the specific method comprises the following steps: taking 50.0mL of a commercially available colloidal gold solution with the mass concentration of 0.01%, adjusting the pH value with 0.4mL of 0.1mol/L potassium carbonate aqueous solution, slowly adding 2mL of 0.1mg/mL aqueous solution of a ribes diacetate sickle enol monoclonal antibody under the stirring state, and continuing stirring for 30 min; adding 10% bovine serum albumin aqueous solution until the final mass concentration of bovine serum albumin is 1%, and continuing stirring for 30 min; standing at 4 deg.C for 2 hr, centrifuging at 1500r/min for 15min, collecting supernatant, and removing precipitate; centrifuging the supernatant at 12000r/min for 30min, discarding the supernatant, and adding 40.0mL of labeled washing and preserving fluid; centrifuging at 12000r/min for 30min, discarding supernatant, resuspending the precipitate with labeled washing preservation solution to obtain 5.0mL concentrate, and placing in 4 deg.C refrigerator;

10. The application of the immunochromatographic test strip for synchronously detecting sickle knife enol, deoxynivalenol and T-2 toxin of ribes diacetate as shown in claim 1, which is characterized in that: the application method comprises the following steps: weighing a levigated sample to be detected, adding a methanol aqueous solution with volume concentration of 60-80%, uniformly mixing, carrying out ultrasonic extraction for 5-10 minutes in a water bath at 50-60 ℃, standing for 5-10 minutes, diluting a supernatant, namely an extracting solution, with water to enable the final volume concentration of methanol in the diluting solution to be 20-30% to obtain a sample solution to be detected, dropwise adding 80-150 microliter of the sample solution to be detected as a detection solution onto a sample pad of an immunochromatographic test strip for synchronously detecting mixed pollution of sethoxydim diacetate, deoxynivalenol and T-2 toxin, taking another methanol aqueous solution with the same volume of methanol concentration as a negative control solution, dropwise adding another sample pad of the immunochromatographic test strip for synchronously detecting mixed pollution of sethoxydim diacetate, deoxynivalenol and T-2 toxin, the test strip is used as a control test strip, and the detection test strip and the control test strip are subjected to color development control after 15-20 minutes: