CN114660294B - Kit for synchronously detecting six mycotoxins and application thereof - Google Patents

Kit for synchronously detecting six mycotoxins and application thereof Download PDF

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CN114660294B
CN114660294B CN202011556600.8A CN202011556600A CN114660294B CN 114660294 B CN114660294 B CN 114660294B CN 202011556600 A CN202011556600 A CN 202011556600A CN 114660294 B CN114660294 B CN 114660294B
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nanogold
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CN114660294A (en
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陈义强
金永鹏
刘颖
张婉君
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China Agricultural University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a kit for synchronously detecting six mycotoxins and application thereof, the kit comprises a detection test strip and a detection reagent, wherein the detection test strip is provided with 6 detection lines and a quality control line, each detection line is coated with 1 mycotoxin antigen, the detection reagent is six nano gold complexes formed by combining with monoclonal antibodies of six mycotoxin markers, and the six mycotoxins are aflatoxins B respectively 1 Ochratoxins, zearalenone, fumonisins B 1 T-2toxin and deoxynivalenol. The test strip is used for detecting six mycotoxins in samples such as corn, wheat, compound feed and the like, the detection limit of each mycotoxin is lower than the sanitary standard limit requirement of feed in China, and meanwhile, the test strip also has the advantages of low detection cost, high detection speed and the like, and can be suitable for on-site rapid detection of the mycotoxins.

Description

Kit for synchronously detecting six mycotoxins and application thereof
Technical Field
The invention relates to the technical field of colloidal gold detection, in particular to a kit for synchronously detecting six mycotoxins and application thereof.
Background
Mycotoxins are a class of toxic metabolites produced by toxic fungi under suitable environmental conditions, and are widely found in crops. Mycotoxins can be a serious hazard to human and animal health due to their potential toxic effects such as carcinogenicity, nephrotoxicity, neurotoxicity, immunosuppression, and estrogenic effects. The contamination of mycotoxins in crops has become a global problem. About 25% of the crops worldwide suffer from mycotoxin pollution every year, and about 2% of the crops lose nutrition and economic value due to serious pollution, and the direct and indirect economic losses reach hundreds of billions of dollars. Feed raw materials are mainly derived from crops, so mycotoxins are one of the common pollutants in feeds. The main mycotoxins in the feed are aflatoxin B1 (AFB) 1 ) Ochratoxin (OTA), zearalenone (ZEA), fumonisin B 1 (FB 1 ) Intake of mycotoxin-contaminated feed such as T-2toxin (T-2) and Deoxynivalenol (DON) can cause reduction of livestock and fowl intake, reduction of feed conversion rate, hypoimmunity, and reduction of production and reproductive performanceSymptoms, thus severely reducing the production efficiency and economic return of livestock and poultry. Meanwhile, contamination of mycotoxins in feeds can cause their accumulation in animal-derived foods such as meat, eggs, milk, thus potentially damaging the health of consumers. Therefore, monitoring the contamination level of mycotoxins in feed is of great importance to ensure animal and consumer health.
At present, the analysis methods for detecting mycotoxins at home and abroad mainly fall into two main categories: instrumental and immunoassay methods. The methods of the instrument mainly comprise liquid chromatography, gas chromatography, capillary electrophoresis, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and the like, which have high accuracy and precision, are commonly used for quantitative and confirmatory analysis of mycotoxins, but generally need to be operated in a laboratory environment, and require expensive instruments and professional technicians, and require complicated pretreatment of samples before the instrument analysis is performed, which usually takes several hours or tens of hours. Thus, the instrumental methods are not suitable for rapid detection of mycotoxins in both the primary laboratory and the field environment.
The immunoassay method mainly comprises a fluorescence photometry method, a fluorescence polarization immunoassay method, an enzyme-linked immunoassay method, a colloidal gold immunochromatography method and the like, and is widely used for screening mycotoxins at present due to the characteristics of simple and rapid operation, low detection cost, no need of expensive instruments and the like. However, these methods can detect only one mycotoxin at a time, and cannot detect a plurality of mycotoxins simultaneously. The feed raw material sources are wide, and a plurality of mycotoxins can be polluted in the same feed sample, so that the research and development of the multi-component immunoassay method for mycotoxins can obviously improve the detection efficiency, reduce the detection cost and provide a more effective means for monitoring the pollution of mycotoxins in the feed.
Disclosure of Invention
The invention aims to provide a kit for simultaneously detecting six mycotoxins and application thereof, which are used for rapidly, accurately and sensitively detecting aflatoxin B in food or feed 1 (AFB 1 ) Ochratoxin (OTA), zearalenone (ZEA), T-2Toxin (T-2), fumonisins (FB) 1 ) And Deoxynivalenol (DON).
The invention firstly provides a kit for synchronously detecting the six mycotoxins, which comprises a detection test strip and a detection reagent; the detection test strip is a lateral flow immunochromatography detection test strip and comprises a sample pad, a nitrocellulose membrane, an absorption pad and a lining plate; 6 detection lines and 1 quality control line are arranged on the nitrocellulose membrane, and each detection line is coated with 1 antigen; the detection reagent is a mixed solution of nano gold-antibody complexes of the monoclonal antibodies of the six mycotoxins respectively combined with nano gold.
In the kit, antigens coated on the detection line are antigens of six mycotoxin antigens respectively coupled with carrier proteins, wherein the carrier proteins are BSA, OVA or KLH; and the quality control line is sprayed with goat anti-mouse secondary antibody.
In the kit of the invention, antigens coated on 6 detection lines are AFB respectively 1 BSA, OTA-BSA, T2-OVA, ZEA-BSA, DON-BSA and FB 1 -OVA。
The experiment of the invention shows that the OTA-BSA and FB are removed 1 In addition to OVA, the detection sensitivity can be significantly improved if other antigens are replaced with heterologous coating sources. Therefore, in the kit of the invention, the antigens coated on the 6 detection lines are aflatoxin M respectively 1 -BSA(AFM 1 -BSA), OTA-BSA, HT-2 toxin-OVA (HT) 2 -OVA), alpha-zearalanol-BSA (alpha-ZAL-BSA), nivalenol-BSA (NIV-BSA) and FB 1 -OVA. Removal of OTA-BSA and FB 1 Other antigens are heterologous coating antigens, except OVA.
In the kit, the distribution condition that 6 detection lines on the detection test paper strip are far from the sample pad is as follows:
(1) Detection line 1: coating AFB 1 -a detection line for BSA antigen;
(2) Detection line 2: a detection line coated with OTA-BSA antigen;
(3) Detection line 3: a detection line coated with T2-OVA antigen;
(4) Detection line 4: a detection line coated with ZAA-BSA antigen;
(5) Detection line 5: a detection line coated with DON-BSA antigen;
(6) And (6) detecting line: coating FB 1 -detection line for OVA antigen.
Preferably, each antigen is dissolved in 50mM carbonate buffer, pH9.5, and the coated antigen concentrations are: AFB 0.2mg/mL 1 BSA, 0.4mg/mL OTA-BSA, 0.3mg/mL T2-OVA, 0.3mg/mL ZEA-BSA, 0.6mg/mL DON-BSA and 0.8mg/mL FB 1 -OVA. Spraying the detection lines on the nitrocellulose membrane respectively to obtain 6 detection lines.
More preferably, the distribution of 6 detection lines from far to near to the sample pad is as follows:
(1) Detection line 1: coating AFM 1 -a detection line for BSA antigen;
(2) Detection line 2: a detection line coated with OTA-BSA antigen;
(3) Detection line 3: a detection line coated with HT2-OVA antigen;
(4) Detection line 4: a detection line coated with an alpha-ZAL-BSA antigen;
(5) Detection line 5: a detection line coated with NIV-BSA antigen;
(6) And (6) detecting line: coating FB 1 -detection line for OVA antigen.
Further preferably, each antigen is dissolved in 50mM carbonate buffer, pH9.5, and the coated antigen concentrations are respectively: 0.4mg/mL AFM 1 -BSA, 0.4mg/mL OTA-BSA, 0.4mg/mL HT2-OVA, 0.5mg/mL alpha-ZAL-BSA, 0.9mg/mL NIV-BSA and 0.8mg/mL FB 1 -OVA. Spraying the detection lines on the nitrocellulose membrane respectively to obtain 6 detection lines.
The length of the nitrocellulose membrane of the detection test strip in the kit is 4cm; the interval distance of 6 detection lines on the nitrocellulose membrane is 3mm respectively.
In the detection reagent of the kit, the OD of the nano gold-antibody complex 532 Are all 2.5, AFB according to the order of monoclonal antibodies 1 ：OTA：T-2：ZEA：DON：FB 1 Mix =2:1:2:4:2:3.
The preparation method of the nano gold in the kit comprises the following steps: 100mL of 0.01% (m/v) chloroauric acid prepared by ultrapure water is placed in a clean triangular flask, heated to boiling, and then 1.0, 1.6 or 2.5mL of 1.0% trisodium citrate (w/v) is rapidly added and stirred continuously; after 20 minutes of reaction, the solution was cooled, ultrapure water was added to the initial volume, and 0.05% sodium azide was replenished;
the nano gold-antibody complex is prepared by the following method: with 0.1. 0.1M K 2 CO 3 Aqueous solution 10.0mL OD 530nm The pH value of the gold nano-solution with the concentration of 0.8 is regulated to 7.0-8.5, the gold nano-solution is continuously stirred, and then 1.0mL of 10-100 mu g/mL of anti-AFB is respectively added 1 OTA, T2, ZEA, DON and FB 1 Is a monoclonal antibody of (a); after 30 minutes incubation at room temperature, 1.0mL of 10% bsa containing 2mm ph8.5 borate buffer was added and the solution was vortexed continuously for 15 minutes; the mixture was centrifuged at 5000g for 10min and the precipitate of the nanogold-antibody complex was resuspended in 10.0mL of 2mM borate buffer, pH8.5, 0.10% BSA; the centrifugation and re-suspension steps were repeated twice, and finally the pellet was re-suspended in 1% BSA, 3% sucrose and 0.5% Tween-20; the optical density of all the nanogold-antibody complexes at 532nm was adjusted to 2.5, and then 6 nanogold-antibody complexes AFB were used 1 ：OTA：T-2：ZEA：DON：FB 1 Mix at a ratio of 2:1:2:4:2:3, add 200 μl per well to microwell plate; freeze drying the solution in the micropores for standby.
Immobilization method of capture antigen (mycotoxin-protein conjugate): AFB was performed using 4cm wide nitrocellulose membranes 1 -BSA (0.2 mg/mL), OTA-BSA (0.4 mg/mL), T2-OVA (0.3 mg/mL), ZEA-BSA (0.3 mg/mL), DON-BSA (0.6 mg/mL) and FB 1 OVA antigen (0.8 mg/mL) (both dissolved in 50mM carbonate buffer, pH 9.5) was sprayed onto nitrocellulose membrane as six detection lines, respectively, in order from top to bottom; AFM respectively 1 -BSA (0.4 mg/mL), OTA-BSA (0.4 mg/mL), HT2-OVA (0.4 mg/mL), alpha-ZAL-BSA (0.5 mg/mL), NIV-BSA (0.9 mg/mL) and FB 1 OVA (0.8 mg/mL) antigen (both dissolved in 50mM carbonate buffer, pH 9.5) was sprayed onto nitrocellulose membrane as six detection lines, respectively, in order from top to bottom (FIG. 1). Spraying goat anti-mouse secondary antibody (0.1-0.5 mg/mL) on nitric acidThe upper end of the cellulose membrane is used as a quality control line. The spray volumes were 1. Mu.L/cm. After antigen immobilization, nitrocellulose membranes were dried at 37 ℃ for 4 hours and stored for use under 4 ℃ drying conditions.
Assembling a test strip: as shown in fig. 1, the six-in-one immunochromatographic test strip is composed of four parts: sample pad, nitrocellulose membrane, absorbent pad and backing plate. The test strip assembly procedure is as follows: a nitrocellulose membrane scored with capture antigen and secondary antibody was adhered to the center of the liner, and then one end of the sample pad was adhered to the liner with a partial cover (2 mm) of nitrocellulose membrane. The absorbent pad was attached to the other end of the backing sheet and partially overlapped (2 mm) with the nitrocellulose membrane. Finally, the assembled test strip was cut to the appropriate size (4 mm wide).
The invention provides application of the kit in detecting mycotoxin in feed or food, wherein the mycotoxin is aflatoxin M 1 Ochratoxins, zearalenone, T-2 toxins, fumonisins B 1 And deoxynivalenol; the method comprises the following steps:
(1) Pretreatment of a sample to be detected: weighing 5g of a sample to be detected, adding 1g of sodium chloride, adding 25mL of 70% methanol-water, extracting for 3min by vortex, centrifuging for 10min 5000g, taking 1mL of supernatant, and fully and uniformly mixing with 4mL of phosphate buffer solution containing 0.5% Triton-100 for later use;
(2) The kit provided by the invention is used for detecting a sample to be detected: and adding 200 mu L of sample solution into micropores containing the freeze-dried detection reagent, uniformly mixing, incubating for 3min at room temperature, immersing the detection test strip into the incubated reagent for reaction, taking out the test strip, judging the test result by naked eyes, and taking the complete disappearance of the red detection line as a judgment standard of a positive sample to generate a red detection line and a negative sample of one quality control line.
The detection principle of the six-component immunochromatographic test strip of the kit is shown in figure 1. Six different antigens were sprayed on different positions of the nitrocellulose membrane as detection lines. Monoclonal antibodies of six different mycotoxins are combined with nano gold to be used as detection reagents. If the sample extracting solution does not contain mycotoxin, the specific nano gold-antibody complex is combined with the corresponding coating antigen on the nitrocellulose membrane to form a macroscopic red detection line. However, if one of the mycotoxins is present in the sample extract, it competes with the capture antigen for a limited amount of nanogold-antibody complex. Thus, fewer nanogold-antibody complexes will be captured by the corresponding coating antigen on the nitrocellulose membrane, and the corresponding detection line will be lighter in color. If the mycotoxin concentration is sufficiently high, the detection reagent is completely prevented from binding to the respective capture antigen, so that a positive sample does not produce a macroscopic red detection line at the corresponding location of the nitrocellulose membrane. Thus, the complete disappearance of the red detection line was considered as a positive result of the corresponding mycotoxins.
The invention greatly improves the detection sensitivity and specificity by skillfully setting the positions of 6 different antigen detection lines and adopting the heterologous antigen as the coating antigen, and can be used for six mycotoxins aflatoxin B in samples such as corn, wheat, compound feed and the like 1 Ochratoxins, zearalenone, fumonisins B 1 The synchronous and rapid screening of the T-2toxin and the deoxynivalenol has the advantages that the detection limit of the mycotoxins is lower than the limit requirement of the national feed sanitation standard, the test strip has the advantages of low detection cost, high detection speed and the like, and the test strip can be suitable for on-site rapid monitoring of the mycotoxins.
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FIG. 1 is a schematic diagram of the operation of the test strip and test reagent in the kit of the present invention.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Unless otherwise indicated, the technical means used in the examples are conventional means and conventional detection methods well known to those skilled in the art. All reagent consumables in the examples are commercially available unless otherwise specified.
The following examples all employ a method of repeating the experiment 5 times, and the results show that the difference between the data of the parallel experimental results of each batch of the repeated experiment is not significant, and the final average value is listed in the examples.
Six mycotoxin monoclonal antibodies are all obtained in the laboratory through the steps of mouse immunization, cell fusion, hybridoma screening, ascites preparation, antibody purification and the like by a conventional method. Mycotoxin Antigen (AFB) as described in the examples 1 -BSA、AFM 1 BSA, OTA-BSA, T2-OVA, HT2-OVA, alpha-ZAL-BSA, ZEA-BSA, DON-BSA, NIV-BSA and FB1-OVA. Other antigens except OTA-BSA and FB1-OVA are heterologous coating antigen) and are prepared by the methods disclosed in the prior art.
AFB 1 BSA antigen, AFM 1 -BSA antigen synthesis method reference: chu, F.S, ueno, i.,1977.Production of antibody against aflatoxin B1.Appl.Environ.Microbiol.33,1125-1128.
ZEA-BSA antigen, alpha-ZAL-BSA antigen Synthesis methods reference: thouvant, d., morfin, R.F.,1983.Radioimmunoassay for zearalenone and zearalanol in human serum:production,properties,and use of porcine antibodies.Appl.Environ.Microbiol.45,16-23.
FB 1 -BSA antigen, FB1-OVA antigen synthesis methods reference: yu, f.y., chu, F.S.,1996.Production and characterization of antibodies against fumonisin B1,J.Food Prot.59,992-997.
DON-BSA antigen, NIV-BSA antigen Synthesis methods reference: maragos, C.M., mccormick, S.P.,2000.Monoclonal Antibodies for the Mycotoxins Deoxynivalenol and 3-Acetyl-deoxynivalenol. Food Agr. Immunol.12,181-192.
T-2 toxin-BSA antigen, HT2-OVA antigen Synthesis methods reference: chu, F.S., grossman, S., wei, R., mirocha, C.J.,1979.Production of Antibody Against T-2Toxin. Appl. Environ. Microbiol.37,104-108.
OTA-BSA antigen, OTA-BSA antigen Synthesis methods reference: liu, B.H., tsao, Z.J., wang, J.J., yu, F.Y.,2008.Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip.Anal.chem.80,7029-7035.
Example 1 preparation method of gold nanoparticle
100mL of 0.01% (m/v) chloroauric acid prepared with ultrapure water was placed in a clean triangular flask, heated to boiling, and 1.0, 1.6 or 2.5mL of 1.0% trisodium citrate (w/v) was then rapidly added and stirred continuously. After 20 minutes of reaction, the solution was cooled and ultrapure water was added to the initial volume. The gold nano solution obtained by supplementing 0.05% of sodium azide can be stabilized for several months at 4 ℃.
Example 2 preparation of detection reagent (nanogold-antibody Complex)
With 0.1. 0.1M K 2 CO 3 Aqueous solution 10.0mL of gold nanosolution (OD 530nm =0.8) was adjusted to pH 7.0-8.5 with constant stirring, and then 1.0mL10-100 μg/mL of each anti-mycotoxin monoclonal antibody was added. After incubation for 30 min at room temperature, 1.0mL of 10% BSA with 2mM borate buffer (pH 8.5) was added and the solution was vortexed continuously for 15 min. Next, the mixture was centrifuged at 5000g for 10 minutes, and the precipitate of the nanogold-antibody complex was resuspended in 10.0mL of 2mM borate buffer (pH 8.5) in 0.10% BSA. The centrifugation and re-suspension steps were repeated twice and finally the pellet was re-suspended in 1% BSA, 3% sucrose and 0.5% Tween-20. The optical density of all nanogold-antibody complexes at 532nm was adjusted to 2.5. The 6 nanogold-antibody complexes were then combined at 2:1:2:4:2:3 (AFB 1 ：OTA：T-2：ZEA：DON：FB 1 ) Experiments show that under the condition of the mixing proportion, the color of each mycotoxin detection line under the condition of a negative sample is basically consistent, and 200 mu L of the mixture is added into a microplate per well. And finally, freeze-drying the solution in the micropores for later use.
Example 3 selection and immobilization of Capture antigen (mycotoxin-protein conjugate)
The invention synthesizes 6 homologous coating origins of mycotoxins and 4 heterologous coating origins of mycotoxins by referring to the prior art, and simultaneously adjusts the synthesis process of micromolecular hapten and carrier proteinThe feeding ratio is used to obtain the homologous coating source or the heterologous coating source with different coupling ratios. The various coating sources are respectively fixed on nitrocellulose membranes to be assembled into test strips, and the sensitivity of detection is measured. The results are shown in Table 1. The results show that the coating materials with different coupling ratios can have a significant effect on the sensitivity, but the effect on the sensitivity is less than 2 times. Compared with the homologous coating source, the sensitivity of the generation of 4 heterologous coating sources can be improved by 2-5 times. Thus, the final selection yields the 4 heterologous coating precursors and 2 homologous coating precursors with the highest sensitivity for subsequent experiments. The selected coating source names and coupling ratios are as follows: AFM (atomic force microscope) 1 -BSA(1.9:1)、HT2-OVA(2.2:1)、α-ZAL-BSA(2.1:1)、NIV-BSA(4:7:1)、OTA-BSA(2.5:1)、FB 1 -OVA(4.9:1)。
Table 1: sensitivity contrast of the homologous or heterologous coating origins of different coupling ratios (sensitivity is expressed in terms of detection limit of analytical standard solution, ng/mL)
Figure BDA0002856081580000081
Figure BDA0002856081580000091
And (3) respectively spraying mycotoxin antigens (4 heterologous coating sources and 2 homologous coating sources) on different detection line positions (figure 1) of the nitrocellulose membrane by adopting a nitrocellulose membrane with the width of 4cm, and performing immunochromatography analysis to obtain the sensitivity. The results (Table 2) show that AFM 1 The sensitivity obtained by BSA, OTA-BSA, HT2-OVA and alpha-ZAL-BSA at locations remote from the sample pad is higher than the sensitivity obtained at locations close to the sample pad, NIV-BSA and FB 1 The sensitivity of OVA at different positions of the detection line is not very different. The AFM of each mycotoxin antigen is finally determined by combining the limit level requirements of each mycotoxin in the feed hygiene standard 1 BSA, OTA-BSA, HT2-OVA, alpha-ZAL-BSA, NIV-BSA and FB 1 OVA antigen (0.1-1 mg/mL in 50mM carbonate buffer, pH 9.5) was sprayed onto nitrocellulose separately in order from top to bottomSix detection lines were provided on the membrane (fig. 1). Spraying goat anti-mouse secondary antibody (0.1-0.5 mg/mL) on the upper end of the nitrocellulose membrane as a quality control line. The spray volumes were 1. Mu.L/cm. After antigen immobilization, nitrocellulose membranes were dried at 37 ℃ for 4 hours and stored for use under 4 ℃ drying conditions.
Table 2: sensitivity of each mycotoxin antigen coating at different detection line positions
(sensitivity is expressed in terms of detection limit of analytical standard solution, ng/mL)
Detection target Coating antigen Detection line 1 Detection line 2 Detection line 3 Detection line 4 Detection line 5 Detection line 6
AFB 1 AFM 1 -BSA 0.1 0.1 0.2 0.2 0.3 0.3
OTA OTA-BSA 0.5 0.6 0.6 0.8 0.9 1.0
T-2 HT2-OVA 0.3 0.4 0.4 0.5 0.6 0.7
ZEA α-ZAL-BSA 0.8 0.8 1.0 1.1 1.2 1.6
DON NIV-BSA 4.8 4.8 5.0 5.0 5.2 5.2
FB 1 FB 1 -OVA 14 14 14 15 15 15
Example 4 Assembly of the kit
(1) Preparation of nitrocellulose membranes
Six antigens coupled to the carrier protein (AFM) were subjected to the following the results of the different antigen selections of example 3 on the detection limit 1 Four kinds of heterologous antigens, namely-BSA, HT2-OVA, alpha-ZAL-BSA and NIV-BSA, OTA-BSA and FB 1 -OVA is a homologous antigen), goat anti-mouse secondary antibody (0.5 mg/mL) was dissolved in carbonic acid buffer (0.05M, pH 9.5) respectively, and then 6 detection lines and 1 quality control line were sprayed on nitrocellulose membrane at a speed of 1. Mu.L/cm and a pitch of 3mm using a film-drawing instrument, respectively. Finally, the nitrocellulose membrane was dried at 37 ℃ for 6 hours and sealed dry at room temperature for use.
(2) Assembly of test strips
As shown in fig. 1, the six-in-one immunochromatographic test strip is composed of four parts: sample pad, nitrocellulose membrane, absorbent pad and backing plate. The test strip assembly procedure is as follows: a nitrocellulose membrane scored with capture antigen and secondary antibody was adhered to the center of the liner, and then one end of the sample pad was adhered to the liner with a partial cover (2 mm) of nitrocellulose membrane. The absorbent pad was attached to the other end of the backing sheet and partially overlapped (2 mm) with the nitrocellulose membrane. Finally, the assembled test strip was cut to the appropriate size (4 mm wide). The six-in-one immunochromatography test strip is called a detection test strip for short, and the six-in-one immunochromatography test strip and the detection reagent prepared in the embodiment 2 of the invention form the kit for synchronously detecting six mycotoxins.
EXAMPLE 5 establishment of immunoassay method for detecting six mycotoxins in feed
(1) Sample pretreatment: respectively weighing 5g of feed raw materials (corn, wheat) and feed samples (compound feed for pigs and chickens), adding 1g of sodium chloride, adding 20mL of 70% methanol-water, performing vortex extraction for 3min, centrifuging 5000g for 10min, taking 1mL of supernatant, and fully and uniformly mixing with 4mL of Phosphate Buffer Solution (PBS) containing 0.5% Triton-100 for later use.
(2) The measurement principle is as follows: example 4 in the assembled kit, the principle of measurement of the six-component immunochromatographic test strip is shown in FIG. 1. Six different antigens were sprayed on different positions of the nitrocellulose membrane as detection lines. Monoclonal antibodies of six different mycotoxins are combined with nano gold to be used as detection reagents. If the sample extracting solution does not contain mycotoxin, the specific nano gold-antibody complex is combined with the corresponding coating antigen on the nitrocellulose membrane to form a macroscopic red detection line. However, if one of the mycotoxins is present in the sample extract, it competes with the capture antigen for a limited amount of nanogold-antibody complex. Thus, fewer nanogold-antibody complexes will be captured by the corresponding coating antigen on the nitrocellulose membrane, and the corresponding detection line will be lighter in color. If the mycotoxin concentration is sufficiently high, the detection reagent is completely prevented from binding to the respective capture antigen, so that a positive sample does not produce a macroscopic red detection line at the corresponding location of the nitrocellulose membrane. Thus, the complete disappearance of the red detection line was considered as a positive result of the corresponding mycotoxins.
(3) The measuring step comprises the following steps: the assay was performed using the kit prepared in example 4 using the microwell method. 200. Mu.L of the sample solution was added to the microwells containing the lyophilized detection reagent, the detection reagent was dissolved, and thoroughly mixed. After incubation for 3 minutes at room temperature, the immunochromatographic strip was immersed in the sample well and the solution was allowed to flow toward the absorbent pad. After 8 minutes, the test result can be judged by naked eyes, the corresponding detection line completely disappears to be used as a judging standard of a positive sample, and the corresponding mycotoxin detection limit is determined.
(4) Sensitivity of the immunoassay test strip: through measurement, the immunochromatography test strip is used for measuring AFB in feed raw materials and feeds 1 、OTA、T-2、ZEA. DON and FB 1 The detection limits of the formula (I) are respectively 2-5 mug/kg, 12-16 mug/kg, 8-12 mug/kg, 20-30 mug/kg, 100-160 mug/kg and 300-450 mug/kg, which are lower than the corresponding limit standard requirements of the feed hygiene standard in China.
Example 6 actual sample analysis
4 samples of corn, wheat, pig compound feed, chicken compound feed and the like are collected, pretreatment is carried out by adopting the steps in the example 5 and detection is carried out by adopting the kit in the example 4, and analysis is carried out simultaneously by adopting a liquid chromatography mass spectrometry analysis method, so that the reliability of the analysis result of the kit is examined. The results (Table 3) show that: ZEA, DON, FB in all samples detected by the kit 1 All positive, part of the samples AFB 1 The OTA or T-2 toxins are positive, and the positive results are determined to contain a certain amount of corresponding mycotoxins through liquid chromatography mass spectrometry analysis, so that the detection result of the kit has no false positive result; meanwhile, the kit provided by the invention is used for determining AFB in corn, pig feed and chicken feed 1 Negative, the content is determined to be lower than the detection limit through liquid chromatography mass spectrometry analysis, and the detection result of the kit is indicated to have no false negative result; the kit determines that the OTA and the T-2 in the wheat are negative results, and the liquid chromatography mass spectrometry determines that the kit contains a certain amount of OTA and T-2, but the content is lower than the detection limit of the kit and the national feed sanitation standard.
The results show that the detection result of the kit disclosed by the invention is basically consistent with the liquid chromatography mass spectrometry method, and meanwhile, the detection limit of the detection of each mycotoxin by the kit disclosed by the invention is consistent with the actual sample analysis result. But the cost of the analysis of the kit is less than 30 yuan/sample and is far lower than that of liquid chromatography mass spectrometry; meanwhile, the detection time of the kit is less than 20 minutes/sample, and is also far less than the time of liquid chromatography mass spectrometry analysis. Therefore, the kit disclosed by the invention can be used for synchronously and rapidly screening 6 mycotoxins in feed and feed raw materials, and has a good application prospect.
TABLE 3 analysis results of actual samples
Figure BDA0002856081580000121
While the invention has been described in detail in terms of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications or improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (9)

1.A kit for synchronously detecting six mycotoxins, wherein the six mycotoxins are respectively: aflatoxin B 1 Ochratoxins, zearalenone, T-2 toxins, fumonisins B 1 And deoxynivalenol; the kit is characterized by comprising a detection test strip and a detection reagent; the detection test strip is a lateral flow immunochromatography detection test strip and comprises a sample pad, a nitrocellulose membrane, an absorption pad and a lining plate; 6 detection lines and 1 quality control line are arranged on the nitrocellulose membrane, and each detection line is coated with 1 antigen; the detection reagent is a mixed solution of nano gold-antibody complexes of the monoclonal antibodies of the six mycotoxins respectively combined with nano gold;
the antigens coated on the 6 detection lines are respectively: AFM (atomic force microscope) 1 -BSA、OTA-BSA、HT 2 -OVA, alpha-ZAL-BSA, NIV-BSA and FB 1 -OVA；
The distribution condition that 6 detection lines are far from the sample pad is as follows:
(1) Detection line 1: a detection line coated with AFM1-BSA antigen;
(2) Detection line 2: a detection line coated with OTA-BSA antigen;
(3) Detection line 3: a detection line coated with HT2-OVA antigen;
(4) Detection line 4: a detection line coated with an alpha-ZAL-BSA antigen;
(5) Detection line 5: a detection line coated with NIV-BSA antigen;
(6) And (6) detecting line: detection line coated with FB1-OVA antigen.
2. The kit of claim 1, wherein the antigens coated on the detection line are six mycotoxin antigens coupled to a carrier protein, respectively, and the carrier protein is BSA, OVA or KLH; and the quality control line is sprayed with goat anti-mouse secondary antibody.
3. The kit of any one of claims 1-2, wherein the nitrocellulose membrane has a length of 4cm; the interval distance of the 6 detection lines on the nitrocellulose membrane is 3mm respectively.
4. The kit of any one of claims 1-2, wherein the detection reagent comprises an OD of a nanogold-antibody complex 532 Are all 2.5, AFB according to the order of monoclonal antibodies 1 ：OTA：T-2：ZEA：DON：FB 1 Mix =2:1:2:4:2:3.
5. The kit according to claim 3, wherein the detection reagent has an OD of the nanogold-antibody complex 532 Are all 2.5, AFB according to the order of monoclonal antibodies 1 ：OTA：T-2：ZEA：DON：FB 1 Mix =2:1:2:4:2:3.
6. The kit of any one of claims 1-2, 5, wherein the method for preparing the nanogold comprises the following steps: preparing 0.01% (m/v) chloroauric acid with ultrapure water, placing 100mL in a clean triangular flask, heating to boil, then rapidly adding 1.0, 1.6 or 2.5mL of 1.0% trisodium citrate (w/v) and continuously stirring; after 20 minutes of reaction, the solution was cooled, ultrapure water was added to the initial volume, and 0.05% sodium azide was replenished;
the nano gold-antibody complex is prepared by the following method: with 0.1M K 2 CO 3 Aqueous solution 10.0mL OD 530nm The pH value of the gold nano-solution with the concentration of 0.8 is regulated to 7.0-8.5, the gold nano-solution is continuously stirred, and then 1.0mL of 10-100 mu g/mL of anti-AFB is respectively added 1 OTA, T2, ZEA, DON and FB 1 Is a monoclonal antibody of (a); after incubation for 30 minutes at room temperature, add1.0mL of 10% BSA with 2mM borate buffer, pH8.5, was added and the solution was vortexed continuously for 15 minutes; the mixture was centrifuged at 5000g for 10min and the precipitate of the nanogold-antibody complex was resuspended in 10.0mL of 2mM 0.10% BSA in pH8.5 borate buffer; the centrifugation and re-suspension steps were repeated twice, and finally the pellet was re-suspended in 1% BSA, 3% sucrose and 0.5% Tween-20; the optical density of all the nanogold-antibody complexes at 532nm was adjusted to 2.5, and then 6 nanogold-antibody complexes AFB were used 1 ：OTA：T-2：ZEA：DON：FB 1 Mix at a ratio of 2:1:2:4:2:3, the mixture was added to the microplate at 200 μl per well; freeze drying the solution in the micropores for standby.
7. The kit of claim 3, wherein the method for preparing the nanogold comprises the following steps: preparing 0.01% (m/v) chloroauric acid with ultrapure water, placing 100mL in a clean triangular flask, heating to boil, then rapidly adding 1.0, 1.6 or 2.5mL of 1.0% trisodium citrate (w/v) and continuously stirring; after 20 minutes of reaction, the solution was cooled, ultrapure water was added to the initial volume, and 0.05% sodium azide was replenished;
the nano gold-antibody complex is prepared by the following method: with 0.1M K 2 CO 3 Aqueous solution 10.0mL OD 530nm The pH value of the gold nano-solution with the concentration of 0.8 is regulated to 7.0-8.5, the gold nano-solution is continuously stirred, and then 1.0mL of 10-100 mu g/mL of anti-AFB is respectively added 1 OTA, T2, ZEA, DON and FB 1 Is a monoclonal antibody of (a); after 30 minutes incubation at room temperature, 1.0mL of 10% BSA containing 2mM ph8.5 borate buffer was added and the solution was vortexed continuously for 15 minutes; the mixture was centrifuged at 5000g for 10min and the precipitate of the nanogold-antibody complex was resuspended in 10.0mL of 2mM 0.10% BSA in pH8.5 borate buffer; the centrifugation and re-suspension steps were repeated twice, and finally the pellet was re-suspended in 1% BSA, 3% sucrose and 0.5% Tween-20; the optical density of all the nanogold-antibody complexes at 532nm was adjusted to 2.5, and then 6 nanogold-antibody complexes AFB were used 1 ：OTA：T-2：ZEA：DON：FB 1 At 2:1:2:4:2:3Mixing in proportions, the mixture being added to the microplate at 200 μl per well; freeze drying the solution in the micropores for standby.
8. The kit of claim 4, wherein the method for preparing the nanogold comprises the following steps: preparing 0.01% (m/v) chloroauric acid with ultrapure water, placing 100mL in a clean triangular flask, heating to boil, then rapidly adding 1.0, 1.6 or 2.5mL of 1.0% trisodium citrate (w/v) and continuously stirring; after 20 minutes of reaction, the solution was cooled, ultrapure water was added to the initial volume, and 0.05% sodium azide was replenished;
the nano gold-antibody complex is prepared by the following method: with 0.1M K 2 CO 3 Aqueous solution 10.0mL OD 530nm The pH value of the gold nano-solution with the concentration of 0.8 is regulated to 7.0-8.5, the gold nano-solution is continuously stirred, and then 1.0mL of 10-100 mu g/mL of anti-AFB is respectively added 1 OTA, T2, ZEA, DON and FB 1 Is a monoclonal antibody of (a); after 30 minutes incubation at room temperature, 1.0mL of 10% BSA containing 2mM ph8.5 borate buffer was added and the solution was vortexed continuously for 15 minutes; the mixture was centrifuged at 5000g for 10min and the precipitate of the nanogold-antibody complex was resuspended in 10.0mL of 2mM 0.10% BSA in pH8.5 borate buffer; the centrifugation and re-suspension steps were repeated twice, and finally the pellet was re-suspended in 1% BSA, 3% sucrose and 0.5% Tween-20; the optical density of all the nanogold-antibody complexes at 532nm was adjusted to 2.5, and then 6 nanogold-antibody complexes AFB were used 1 ：OTA：T-2：ZEA：DON：FB 1 Mix at a ratio of 2:1:2:4:2:3, the mixture was added to the microplate at 200 μl per well; freeze drying the solution in the micropores for standby.
9. Use of a kit according to any one of claims 1 to 8 for detecting mycotoxins in feed or food, said mycotoxins being aflatoxin M 1 Ochratoxins, zearalenone, T-2 toxins, fumonisins B 1 And deoxynivalenol; the method is characterized by comprising the following steps of:
(1) Pretreatment of a sample to be detected: weighing a sample 5 to be measured g, adding 1g sodium chloride, adding 25ml70% methanol-water, extracting for 3min by vortex, centrifuging for 10min by 5000g, taking supernatant 1mL, and fully and uniformly mixing with 4mL phosphate buffer solution containing 0.5% Triton-100 for later use;
(2) Performing a test sample test using the kit of any one of claims 1-8: and adding 200 mu L of sample solution into micropores containing the freeze-dried detection reagent, uniformly mixing, incubating for 3min at room temperature, immersing the detection test strip into the incubated reagent for reaction, taking out the test strip, judging the test result by naked eyes, and taking the complete disappearance of the red detection line as a judgment standard of a positive sample to generate a red detection line and a negative sample of one quality control line.
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