An immuno-chromatographic method for simultaneously detecting multiple mycotoxins in grain and oil

Abstract

This invention discloses an immunochromatographic method for the simultaneous detection of multiple mycotoxins in grains and oils. The method includes: pulverization and weighing: pulverizing the grain sample and weighing the pulverized sample into a centrifuge tube; adding purified water and a formulation packet, sealing the tube, vigorously shaking at high speed to mix, centrifuging to obtain the supernatant; collecting the supernatant; adding it separately to a reactor; inserting test strips for detecting different mycotoxins into the reactor and waiting for the colorimetric reaction to complete the detection. The formulation packet for the water extraction detection of multiple mycotoxins, based on 100 samples, includes the following components: 7-9 parts bovine serum albumin, 3-5 parts HLB packing material, and 3-5 parts PAS packing material. This invention is based on the principle of traditional chromatographic detection methods, utilizing water extraction by combining different formulation packets with the analytes for extraction, optimizing the experimental steps and reducing the use of organic solvents.

Description

Technical Field

[0001] This invention belongs to the field of fungal detection technology, specifically relating to an immunochromatographic method for the simultaneous detection of multiple mycotoxins in grains and oils. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by fungi. They can enter the bodies of humans and animals through feed or food, causing acute or chronic poisoning and endangering human and animal health. The growth of molds is closely related to region, climate, and season; therefore, the contamination levels of feed and feed ingredients with mycotoxins vary depending on the region, time, and season. Aflatoxin, vomitoxin (deoxynivalenol), F-2 toxin (zearalenone), T-2 toxin, fumonisin, and ochratoxin are six toxins that often contaminate crops and their processed products simultaneously.

[0003] Currently, the main methods for detecting mycotoxins include thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), gas chromatography (GC), and high-performance liquid chromatography (HPLC). TLC and ELISA methods cannot accurately quantify mycotoxins, and while GC has high sensitivity, sample pretreatment is complex and prone to interference from impurities. HPLC is currently widely accepted for mycotoxin detection. After purification of the extracted sample using an immunoaffinity column, impurities are reduced, and the detection limit is low. Through experimental research, an immunoaffinity column-HPLC method has been established, which offers high sensitivity, high recovery, and good repeatability. However, HPLC is technically challenging to operate. Current market methods primarily focus on single-toxin detection, failing to achieve comprehensive detection. Furthermore, the operation requires specialized personnel and involves the use of organic solvents for extraction, which can lead to solvent waste and contamination. Summary of the Invention

[0004] This invention proposes an immunochromatographic method for the simultaneous detection of multiple mycotoxins in grains and oils. By using different formulations, the mycotoxins bind to their functional groups to form complexes, thereby extracting the mycotoxins and adsorbing impurities to achieve sample separation and purification. In liquid samples, the mixture is shaken and centrifuged, allowing the substances in the formulation to adsorb impurities while simultaneously binding with water, thus extracting the analytes. This method achieves rapid separation and purification, reduces sample matrix interference, and improves detection sensitivity. It solves the problem of significant interference in traditional chromatographic detection methods and eliminates the need for complex pretreatment processes.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An immunochromatographic method for simultaneous detection of multiple mycotoxins in grains and oils, the method comprising the following steps:

[0007] S1 Crushing and Weighing: Crush the grain sample and weigh the crushed sample into a centrifuge tube;

[0008] S2 Mix and centrifuge: Add purified water and the formula packet, cover and mix by high-speed vigorous shaking and centrifugation to obtain the supernatant;

[0009] S3 Sampling and Addition: Take the supernatant and add it to the reactor separately;

[0010] S4 inserts the test strips for detecting different fungal toxins into the reactor and waits for the colorimetric reaction;

[0011] The detection of multiple mycotoxins in grains and oils can be completed simultaneously.

[0012] Preferably, the detection device used in the method includes test strips, a reactor, and a tray. The reactor is placed on the tray and consists of several reaction tubes. A through hole is provided in the center of the reactor, and the reaction tubes are arranged around the through hole in sequence. The height of the through hole is lower than the height of the reaction tubes.

[0013] More preferably, the bottom of the reaction tube is conical so that the test paper can stand upright in the reaction tube; the reaction tubes sequentially form a polygon around the perimeter of the through hole.

[0014] More preferably, the bottom of the reaction tube is conical so that the test paper can stand upright in the reaction tube; the reaction tubes sequentially form a polygon around the perimeter of the through hole.

[0015] More preferably, the test strip includes a backing plate, absorbent paper laminated at one end of the backing plate, a nitrocellulose film attached to the backing plate, and absorbent glass fibers overlapping the nitrocellulose film and close to the absorbent paper. At least one detection line (T line) and one control line (C line) are provided on the nitrocellulose film. Absorbent filter paper is provided on the side close to the control line (C line).

[0016] More preferably, antibodies against various fungal toxins are immobilized on a nitrocellulose membrane as the detection line (T line), and goat anti-mouse IgG antibodies are immobilized on a control line as the control line (C).

[0017] The absorbent glass fiber contains a solid phase of colloidal gold-monoclonal antigen conjugate.

[0018] More preferably, the colloidal gold-monoclonal antigen conjugate with a solid phase disposed on the absorbent glass fiber includes the following steps:

[0019] (1) Colloidal gold solvent: Put pure water into anti-boiling glass beads, heat to boiling, add trisodium citrate solution, boil, add chloroauric acid solution, cool, and obtain colloidal gold solvent.

[0020] (2) Adjust the pH of the colloidal gold solvent obtained in (1) to 7.4, add a number of fungal toxin antigens, stir, add bovine serum albumin, stir until the reaction is complete, and obtain the initial colloidal gold antigen-labeled solution.

[0021] (3) Centrifuge the initial colloidal gold antigen-labeled solution to remove unbound antigens and obtain colloidal gold antigen-labeled precipitate. Dissolve the precipitate in the original volume of 0.02 mol / L TBS (pH 8.2, containing 1% BSA and 0.05% sodium azide). Repeat centrifugation 2-3 times. Dissolve the precipitate in 1 mL TBS, add 50% glycerol, and store at -20℃. Spray the gold-labeled antigen solution onto glass fiber, dry at room temperature, and then paste and assemble.

[0022] Further preferably, in step (2), the mycotoxins include aflatoxin monoclonal antigen, zearalenone monoclonal antigen, vomitoxin monoclonal antigen, T-2 toxin monoclonal antigen, fumonisin monoclonal antigen, or ochratoxin A monoclonal antigen or others; in step (2), the amount of bovine serum albumin added is 1.2-1.5 times the mass of the antigen.

[0023] Preferably, the formulation package used in the method comprises the following components based on 100 parts by sample weight:

[0024] 7-9 parts bovine serum albumin, 3-5 parts filler.

[0025] More preferably, the formulation package further includes the following components:

[0026] 3-5 parts of graphitized carbon; the filler is HLB filler, PAS filler, or NH2 filler.

[0027] More preferably, the formulation comprises the following components in parts by weight:

[0028] 8 parts bovine serum albumin, 4 parts filler, and 4 parts graphitized carbon.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention is based on the principles of traditional chromatography detection methods. It utilizes water extraction by combining different formulation packages with the analyte for extraction, optimizing the experimental steps and reducing the difficulty of experimental operation. The original experimental operation required the use of organic solvents, which could easily cause environmental pollution. The current formulation package extraction reduces the use of organic solvents and thus reduces organic solvent pollution. The addition of multiple toxin antigens to the microporous gold enables high-throughput detection of multiple toxins.

[0031] 2. The formula package includes bovine serum albumin, a globulin found in bovine serum. Albumin is composed of 581 amino acid residues, including 35 cysteine ​​residues forming 17 disulfide bonds, and a free sulfhydryl group at position 34 of the peptide chain. Albumin can bind to various cations, anions, and other small molecules. It can carry metal ions and fatty acids. Using bovine serum albumin to bind with mycotoxins allows for the extraction of mycotoxins via water extraction, reducing the use of organic reagents and making it more environmentally friendly.

[0032] 3. Test Strips: Six antibodies are immobilized on a nitrocellulose membrane as the test line (T), and goat anti-mouse IgG antibody is immobilized on the control line as the control line (C). The front end of each test strip is conjugated with colloidal gold containing the six antibodies. If the sample contains toxins, after thorough mixing, the toxins and the conjugate complex move forward on the sample pad to the test line. Because the toxins bind to the antibodies on the test line, forming a conjugate complex, the colloidal gold occupies the detection site, and the test line shows varying shades of red. This result is considered positive, and the toxin concentration can be determined based on the color intensity. However, if the sample does not contain fungal toxins, the colloidal gold conjugated with the six antibodies cannot replace the antibody sites on the test line, and no color develops on the test line. This result is considered negative.

[0033] 4. It requires a small sample size, is highly efficient, and can perform multiple tests simultaneously, saving testing time.

[0034] 5. The detection device used in the method includes test strips, a reactor, and a tray. The reactor is placed on the tray and consists of several reaction tubes. A through hole is provided in the center of the reactor, and the reaction tubes are arranged sequentially around the through hole. The height of the through hole is lower than the height of the reaction tubes. The volume of the reaction tubes is constant and they do not interfere with each other. Each reaction tube overflows after reaching the required volume. The bottom of each reaction tube is conical to facilitate the suspension of the test strip in the middle. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the detection device of the present invention. Figure 1 'a' is the top view. Figure 1 b is a side view. Figure 1 c Test state diagram.

[0036] Figure 2 This is a schematic diagram of the bottom of the reaction tube.

[0037] Figure 3 This is a schematic diagram of the test strip of the present invention.

[0038] The components include: test strip 1, reactor 2, tray 3, reaction tube 4, through hole 5, back plate 101, absorbent paper 102, nitrocellulose membrane 103, absorbent glass fiber 104, detection line T line 105, control line C line 106, and absorbent filter paper 107.

[0039] Figure 4 This is a schematic diagram of the reaction steps of the present invention. Detailed implementation method:

[0040] The present invention will be further described below with reference to specific embodiments. The present invention will be described in further detail, but is not limited to these embodiments.

[0041] Example 1

[0042] An immunochromatographic method for simultaneous detection of multiple mycotoxins in grains and oils, the method comprising the following steps:

[0043] S1 Grinding and Weighing: Grind the grain sample and weigh 5g of the ground sample into a 50ml centrifuge tube; S2 Mixing and Centrifuging: Add 25mL of purified water and the formula packet, cover and shake vigorously at high speed for 5min;

[0044] Centrifuge at 4000 rpm for 5 min to obtain the supernatant;

[0045] S3 Sampling and Addition: Take 300 μL of supernatant (equivalent to 0.06 g of sample); add it to the sample;

[0046] S4 Insert the reagent strip into the sample cell, close the outer cover, and wait for the test results.

[0047] Preferably, the detection device used in the method includes a test strip 1, a reactor 2 and a tray 3. The reactor 2 is placed on the tray 3 and consists of several reaction tubes 4. A through hole 5 is provided in the center of the reactor 2, and the reaction tubes 4 are arranged around the through hole 5 in sequence. The height of the through hole 5 is lower than the height of the reaction tubes 4.

[0048] More preferably, the bottom of the reaction tube 4 is conical so that the test paper 1 can stand upright in the reaction tube 4; the reaction tube 4 forms a polygon around the through hole 5.

[0049] More preferably, the bottom of the reaction tube 4 is conical so that the test paper 1 can stand upright in the reaction tube 4; the reaction tube 4 forms a polygon around the through hole 5.

[0050] More preferably, the test strip 1 includes a backing plate 101, absorbent paper 102 laminated at one end of the backing plate 101, a nitrocellulose film 103 attached to the backing plate 101, and absorbent glass fibers 104 overlapping the nitrocellulose film 103 and close to the absorbent paper 102. The nitrocellulose film 103 is provided with at least one detection line T line 105 and a control line C line 106. Absorbent filter paper 107 is provided on the side close to the control line C line 106.

[0051] More preferably, antibodies against various fungal toxins are immobilized on a nitrocellulose membrane as the detection line T line 105, and goat anti-mouse IgG antibodies are immobilized on a control line as the control line (C).

[0052] The absorbent glass fiber 104 has a solid phase of colloidal gold-monoclonal antigen conjugate.

[0053] More preferably, the water-absorbing glass fiber 104 containing a solid-phase colloidal gold-monoclonal antigen conjugate comprises the following steps:

[0054] (1) Colloidal gold solvent: Accurately measure 1L of pure water into a round-bottom flask, put in anti-boiling glass beads, heat to boiling, add 15ml of 1% trisodium citrate solution, boil for 1 minute, then add 10ml of 1% chloroauric acid solution, and continue heating for 8 minutes after the color stabilizes. After cooling, add pure water to make up to 1L.

[0055] (2) Adjust the pH of the colloidal gold solvent obtained in (1) to 7.4, and then add aflatoxin monoclonal antigen, zearalenone monoclonal antigen, vomitoxin monoclonal antigen, T-2 toxin monoclonal antigen, fumonisin monoclonal antigen, and ochratoxin A monoclonal antigen. After stirring for 20 minutes, add bovine serum albumin (the amount of bovine serum albumin added is 1.3 times the mass of the antigen) and stir until the reaction is complete to obtain the initial colloidal gold antigen labeling solution.

[0056] (3) Centrifuge the initial colloidal gold antigen-labeled solution to remove unbound antigen and obtain colloidal gold antigen-labeled precipitate. Dissolve the precipitate in the original volume of 0.02 mol / L TBS (pH 8.2, containing 1% BSA and 0.05% sodium azide), repeat centrifugation 2-3 times, dissolve the precipitate in 1 mL TBS, add 50% glycerol, store at -20℃, spray the gold-labeled antigen solution onto glass fiber, dry at room temperature, and then paste and assemble.

[0057] Feed samples were mixed with different concentrations of aflatoxin standard solution, zearalenone standard solution, vomitoxin standard solution, T-2 toxin standard solution, fumonisin standard solution, and ochratoxin A standard solution to prepare six feed toxins. Six toxin-containing feeds and a blank control were prepared and numbered. Feeds 1-2 contained aflatoxin B1 of 50 and 200 μg / kg, respectively. Feeds 4-5 contained zearalenone of 1 μg / kg, respectively. Samples 7-8 contained 500 μg / kg of vomitoxin and 1000 μg / kg of fumonisin, respectively; samples 10-11 contained 100 μg / kg of T-2 toxin and 500 μg / kg of fumonisin, respectively; samples 13-14 contained 100 μg / kg of fumonisin and 500 μg / kg of fumonisin, respectively; samples 16-17 contained 100 μg / kg of ochratoxin A and 500 μg / kg of ochratoxin A; and samples 3, 6, 9, 12, 15, and 18 served as blank controls. Pretreatment experiments were conducted using different formulation packages. The detection system of this invention was used to test 18 samples using the above-described detection method, with each sample tested in triplicate.

[0058] Component 1 of the formulation package: The formulation package, based on the sample (5g) mass as 100%, contains the following amounts: bovine serum albumin 8%, NH2 filler 4%, and PAS filler 4%.

[0059] The two components of the formulation package are as follows: the amount added to the formulation package is 8% bovine serum albumin, 4% NH2 filler, and 4% HLB filler, based on the sample (5g) mass as 100%.

[0060] The formulation package consists of three components: the amount of each component added is as follows, based on the sample (5g) mass as 100%: bovine serum albumin 8%, graphitized carbon 4%, and PAS filler 4%.

[0061] The formulation package consists of 4 components: the amount added based on the sample (5g) mass as 100% is as follows: bovine serum albumin 8%, graphitized carbon 4%, and HLB filler 4%.

[0062] The formulation package contains 5 components: the amounts added to the formulation package based on the sample (5g) mass as 100% are: bovine serum albumin 8%, HLB filler 4%, and PAS filler 4%.

[0063] The formulation package contains 6 components: based on the sample (5g) mass as 100%, the added amounts are: bovine serum albumin 8%, NH2 filler 4%, and graphitized carbon 4%. The results are shown in Table 1.

[0064] In addition, feed samples were mixed with different concentrations of aflatoxin standard solution, zearalenone standard solution, vomitoxin standard solution, T-2 toxin standard solution, fumonisin standard solution, and ochratoxin A standard solution to prepare six feed toxins. Six toxin-containing feeds and a blank control were prepared and numbered. Feeds 1-2 contained aflatoxin B1 of 50 and 200 μg / kg, respectively. Feeds 4-5 contained zearalenone of the following concentrations. Samples 7-8 contained vomitoxin at concentrations of 500 and 1000 μg / kg, respectively; samples 10-11 contained T-2 toxin at concentrations of 100 and 500 μg / kg, respectively; samples 13-14 contained fumonisin at concentrations of 100 and 500 μg / kg, respectively; samples 16-17 contained ochratoxin A at concentrations of 100 and 500 μg / kg, respectively; and samples 3, 6, 9, 12, 15, and 18 served as blank controls. Toxin detection cards were used to test 18 samples using the above methods, with each sample tested in triplicate. Specific procedures for water extraction detection are as follows:

[0065] S1 Crushing and Weighing: Crush the grain sample and weigh 5g of the crushed sample into a 50ml centrifuge tube;

[0066] S2 Mix and centrifuge: Add 25mL of purified water, cover and shake vigorously at high speed for 5min; centrifuge at 4000rpm for 5min to obtain the supernatant;

[0067] S3 Sampling and Addition: Take 300 μL of supernatant (equivalent to 0.06 g sample) and add it to the sample cell;

[0068] S4 Insert the reagent strip into the sample cell and wait for the test results. The results are shown in Table 1.

[0069] Specific operating procedures for ethanol testing:

[0070] S1 Crushing and Weighing: Crush the grain sample and weigh 5g of the crushed sample into a 50ml centrifuge tube;

[0071] S2 Mix and centrifuge: Add 25 mL of ethanol, cover and shake vigorously at high speed for 5 min; centrifuge at 4000 rpm for 5 min to obtain the supernatant;

[0072] S3 Sampling and Addition: Take 300 μL of supernatant (equivalent to 0.06 g sample) and add it to the micropores of the colloidal gold powder;

[0073] S4. Use a pipette to mix thoroughly, at least 3 times.

[0074] S5 Insert the reagent strip into the microwell of the colloidal gold powder, close the outer cap, and wait for the test results. The results are shown in Table 1.

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079] Formula Pack 7: Based on Formula Pack 6, the difference is that the formula does not include graphitized carbon. Otherwise, it is the same as in Example 1. The test results are shown in Table 3.

[0080] Formula Pack 8: Based on Formula Pack 6, the difference is that the formula does not include NH2 filler. Otherwise, it is the same as in Example 1. The test results are shown in Table 3.

[0081] Formula Pack 9: Based on Formula Pack 6, the difference is that bovine serum albumin is not included in the formula. Otherwise, it is the same as in Example 1. The test results are shown in Table 3.

[0082] Table 3

[0083]

[0084]

[0085] The standard range for spiked recovery rate is specified in GB / T 27404-2008 - Laboratory Quality Control Standard - Food Physicochemical Testing, and 80-120 is considered a relatively optimal range.

[0086] As shown in Table 1-3, by using bovine serum albumin in combination with packing material, a unified pretreatment can be achieved to extract mycotoxins from water, reducing the use of chemical reagents. Different packing material combinations can also improve the recovery rate of mycotoxins, with the combination of bovine serum albumin, NH2 packing material, and graphitized carbon yielding the best recovery rate.

[0087] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. An immunochromatographic method for simultaneous detection of multiple mycotoxins in grain and oil, characterized in that, The method comprises the following steps: S1: crushing and weighing: crushing the grain sample, and weighing the crushed sample in a centrifuge tube; S2: mixing and centrifuging: adding purified water and a formula bag, covering the cap, mixing and centrifuging at high speed, obtaining supernatant, wherein the formula bag comprises the following components in terms of 100 parts of sample quality: 7-9 parts of bovine serum albumin, 3-5 parts of NH2 filler, and 3-5 parts of graphitized carbon; S3: sampling and adding: taking the supernatant and adding it into a reactor; S4: inserting a detection test paper for detecting different mycotoxins into the reactor and waiting for color development reaction; The method for simultaneously detecting multiple mycotoxins in grains and oils is completed. The detection device used in the method comprises a detection test paper (1), a reactor (2), and a tray (3), the reactor (2) is arranged on the tray (3), the reactor (2) is composed of a plurality of reaction tubes (4), a through hole (5) is arranged at the center of the reactor (2), the reaction tubes (4) are arranged around the through hole (5) in sequence, the height of the through hole (5) is lower than the height of the reaction tubes (4), and the bottom of the reaction tube (4) is conical so that the detection test paper (1) can stand in the reaction tube (4).

2. The method according to claim 1, wherein the method is for simultaneous detection of multiple mycotoxins in grain and oil. The reaction tubes (4) are arranged around the through hole (5) in sequence to form a polygon.

3. The immuno-chromatographic method for simultaneous detection of multiple mycotoxins in grain and oil according to claim 1, characterized in that, The detection test paper (1) comprises a back plate (101), a water absorption paper (102) arranged at one end of the back plate (101), a nitric acid fiber membrane (103) attached to the back plate (101), a water absorption glass fiber (104) lapped on the nitric acid fiber membrane (103) and close to the water absorption paper (102), at least one detection line T line (105) and a control line C line (106) arranged on the nitric acid fiber membrane (103), and a water absorption filter paper (107) arranged on one side close to the control line C line (106).

4. The immuno-chromatographic method for simultaneous detection of multiple mycotoxins in grain and oil according to claim 3, characterized in that, Antibodies of multiple mycotoxins are fixed on the nitrocellulose membrane as the detection line T line (105), and IgG antibodies of sheep anti-mouse are fixed on the quality control line as the control line (C). The water absorption glass fiber (104) is provided with a solid-phase colloidal gold-monoclonal antigen combination.

5. The immuno-chromatographic method for simultaneous detection of multiple mycotoxins in grain and oil according to claim 3, characterized in that, The water absorption glass fiber (104) provided with the solid-phase colloidal gold-monoclonal antigen combination comprises the following steps: (1) Colloidal gold solvent: pure water is put into anti-bumping glass beads, heated to boiling, and then trisodium citrate solution and chloroauric acid solution are added, and then cooled to obtain the colloidal gold solvent; (2) The colloidal gold solvent obtained in (1) is adjusted to pH 7.4, and then a plurality of mycotoxin antigens are added, stirred, and then bovine serum albumin is added, and stirred until the reaction is complete to obtain colloidal gold antigen labeling primary solution; (3) The colloidal gold antigen labeling primary solution is centrifuged to remove unbound antigens, and then a colloidal gold antigen labeling precipitate is obtained, which is dissolved in the original volume of 0.02 mol / L TBS, and the pH of the TBS is 8.2, and the TBS contains 1% BSA and 0.05% sodium azide, and the precipitate is repeatedly centrifuged for 2-3 times, and then dissolved in 1 mL of TBS, and then 50% glycerol is added, and then stored at -20 ℃, and then the gold-labeled antigen is sprayed on the water absorption glass fiber, and then dried at room temperature, and then pasted and combined.

6. The immuno-chromatographic method for simultaneous detection of multiple mycotoxins in grain and oil according to claim 5, characterized in that, The step (2) is that several mycotoxins include aflatoxin monoclonal antibody, corn smut monoclonal antibody, emetic monoclonal antibody, T-2 toxin monoclonal antibody, fumonisin monoclonal antibody or ochratoxin A monoclonal antibody or others; the step (2) is that the amount of bovine serum albumin is 1.2-1.5 times of the mass of antigen.

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