CN112881679A - Preparation method of test strip for simultaneously detecting two water-soluble mycotoxins - Google Patents
Preparation method of test strip for simultaneously detecting two water-soluble mycotoxins Download PDFInfo
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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Abstract
The invention relates to the technical field of rapid detection of mycotoxins, in particular to a preparation method of a test strip for simultaneously detecting two water-soluble mycotoxins. The antibody markers used by the test strip prepared by the invention are flower-shaped nanogold and spherical colloidal gold, the detection sensitivity of the two fungaltoxins is 5ng/mL, the sample can be diluted by 200 times, the matrix interference is eliminated, and the rapid detection of the two water-soluble fungaltoxins in the sample is realized. The test strip is suitable for food processing enterprises, quality inspection departments, feed enterprises and the like, and has wide market application prospect.
Description
Technical Field
The invention relates to the technical field of rapid detection of mycotoxins, in particular to a preparation method of a test strip for simultaneously detecting two water-soluble mycotoxins.
Background
Mycotoxin contamination is one of the prominent problems in the field of food safety, and according to the Food and Agricultural Organization (FAO) report of the united nations, about 25% of crops in the world are contaminated by molds and toxins thereof, and about 2% of crops lose nutritional and economic values due to serious pollution. There are more than ten kinds of mycotoxins seriously harmful to human, including aflatoxin B1(AFB1) Deoxynivalenol (DO N), Zearalenone (ZEN), ochratoxin and fumonisin B1(FB1) Citrinin, penicillium expansum toxin, and the like. Deoxynivalenol (DON) is one of trichothecene compounds which are toxic secondary metabolites produced by fusarium, and belongs to B-type trichothecene compounds. FumonisinsB1(Fumonisin FB1) Is another mycotoxin, which is a water-soluble metabolite produced by fusarium moniliforme. The two fungaltoxins are widely present in grain and grain products and have the effects of teratogenesis, carcinogenesis, immunosuppressive toxicity, neurotoxicity and the like, not only cause great economic losses such as yield reduction and quality reduction of crops, but also pose potential threats to the health of people and livestock.
At present, the detection methods commonly used for mycotoxin at home and abroad mainly comprise High Performance Liquid Chromatography (HPLC), enzyme linked immunosorbent assay (ELIS A) and the like, wherein the instrument detection method has the advantages of accuracy, reliability, high efficiency, high sensitivity and good reproducibility, but the existing detection method is long in time consumption, high in cost, complex in relevant equipment conditions and only suitable for laboratory detection, so that the method cannot be popularized in a large range. ELISA has the advantages of relatively simple operation, high sensitivity and the like, but also has the defects of needing a special instrument, needing a professional to operate, consuming long time and the like, and is only suitable for laboratory detection. In contrast, the gold immunochromatography is simple and rapid, does not need complex equipment and professional knowledge, and can be learned by an operator within 5 minutes, namely, the operator can use the immunochromatography to learn.
The antibody markers used by the test strip prepared by the invention are flower-shaped nanogold and spherical colloidal gold, the detection sensitivity of the two fungaltoxins is 5ng/mL, the sample can be diluted by 200 times, the matrix interference is eliminated, and the rapid detection of the two water-soluble fungaltoxins in the sample is realized. The test strip is suitable for food processing enterprises, quality inspection departments, feed enterprises and the like, and has wide market application prospect.
Disclosure of Invention
The test strip for rapidly and simultaneously detecting two water-soluble mycotoxins comprises a nitrocellulose membrane (NC membrane), a conjugate release pad, a sample pad, a bottom plate and a water absorption pad, wherein DON-BSA and FB are sprayed on the NC membrane1-a test zone (T-line) of BSA conjugate and a quality control zone (C-line) coated with goat anti-mouse IgG, said gold pad being sprayed with DON monoclonal antibody conjugate gold flower nanoparticles (blue) and FB1Monoclonal antibodies were conjugated to spherical colloidal gold (red).
In order to achieve the above object, the present invention provides a method for preparing a test strip for simultaneously detecting two water-soluble mycotoxins, wherein the method comprises the following steps:
(1) DON monoclonal antibody-flower nanoparticle marker and FB1Preparation of monoclonal antibody-15-18 nm spherical nano gold marker
Adding K into 3mL of spherical golden flower nano solution or colloidal gold solution with the particle size of 15-18nm under magnetic stirring2CO3Adjusting pH of the solution to 5.0-6.5, and collecting DON antibody and FB1Respectively diluting antibodies by 5-10 mu L, gradually adding the diluted antibodies into a golden flower nano solution or a colloidal gold solution, reacting for 60min, then respectively adding BSA and PEG-20000, reacting for 30min, centrifuging at 10000rpm for 60min, re-dissolving precipitates by using a gold seed diluent to obtain a DON monoclonal antibody-flower nano particle marker, and simultaneously preparing a compound of a secondary antibody, the colloidal gold and the golden flower according to the same method for signal amplification;
(2) treatment of gold labeled conjugate release pads
Soaking the gold-labeled conjugate release pad in gold seed diluent for 30min, vacuum drying at 37 deg.C, mixing the gold-labeled antibody complex and the secondary antibody with colloidal gold or golden flower complex, and spraying onto the gold-labeled pad;
(3) coating of nitrocellulose membranes
Spraying mycotoxin detection antigens on a nitrocellulose membrane, wherein the detection antigens are respectively used as detection lines, and goat anti-mouse Ig G is used as a quality control line;
(4) production of test paper
And (3) sticking the nitrocellulose membrane water absorption pad and the sample pad sprayed with the mycotoxin detection antigen and goat anti-mouse IgG on a PVP bottom plate, and then slitting to obtain the test strip for simultaneously detecting the two mycotoxins.
Preferably, K in said step (1)2CO3The solution was 1%, 30 mL.
Preferably, the amount of BSA added in step (1) is 10% to 100. mu.L, and the amount of PEG-20000 added is 5% to 100. mu.L.
Preferably, the gold ion diluent in the step (1) is 0.5% BSA, 5% sucrose, 0.5% PEG-20000 in PBS with a concentration of 0.01M.
Compared with the prior art, the invention has the beneficial effects that:
(1) the test strip for simultaneously detecting two water-soluble mycotoxins has the advantages of high specificity and high sensitivity, has the detection sensitivity to the two toxins of 5ng/mL and 5ng/mL, can dilute samples by 200 times, eliminates matrix interference, and has no cross reactivity to other mycotoxins.
(2) The detection result is visual and intuitive, and when the test strip displays a red line, a blue line and a purple line, the test strip shows that: the content of both water-soluble mycotoxins is lower than 5ng/mL (1000 mug/kg), and when only the quality control line shows purple color, the content of the water-soluble mycotoxins in the sample is higher than 5ng/mL (1000 mug/kg); a mycotoxin higher than 5ng/mL (1000. mu.g/kg) when only one red line and one purple line are displayed or only one blue line and one purple line are displayed; when there is no purple line, the test strip is invalid.
(3) Simple operation, and detection result can be obtained within 5-10 min.
Detailed Description
The present invention will be further described with reference to examples.
Example 1
Preparation of test strip for simultaneously and rapidly detecting two water-soluble mycotoxins
1. Preparation of mycotoxin monoclonal antibody-gold nanoflower particles and spherical colloidal gold markers
To a small beaker containing 3mL of flower-like nanogold solution or spherical colloidal gold solution was added 30. mu.L of K2CO3 (1%) with magnetic stirring to adjust the pH of the solution to 5.5. Reacting for 30min respectively with mycotoxin monoclonal antibodies. Add 100. mu.L 10% BSA and 100. mu.L 5% PEG-20000 and stir for 30 min. Centrifugation was carried out at 10000rmp for 45min at 4 ℃. And re-dissolving the precipitate with gold seed diluent to obtain two mycotoxin monoclonal antibodies, namely flower-shaped nano gold and spherical colloidal gold markers.
2. Treatment and coating of gold-labeled pads
Soaking the conjugate release pad in gold seed diluent for 10min, and oven drying at 37 deg.C. And (2) uniformly mixing the two mycotoxin monoclonal antibodies, namely the flower-shaped nanogold and the spherical colloidal gold markers obtained in the step (1), then uniformly spraying the mixture on a conjugate release pad, drying in vacuum, and standing at 4 ℃ for later use.
3. Coating of NC film
The detection antigens with the concentration of 0.1mg/mL mycotoxin coupled BSA are respectively sprayed on an NC membrane to be used as detection lines of the mycotoxins, the spraying amount is 0.74 mu L/cm, goat anti-mouse Ig G with the concentration of 0.5mg/mL is sprayed at the position which is close to one end of the absorbent paper and is 3mm away from the detection antigens to be used as a quality control line (C line), and the spraying amount is 0.74 mu L/cm. Vacuum drying, and sealing.
4. Assembly of colloidal gold test strip
Adhering the nitrocellulose membrane (NC membrane) water absorption pad and the sample pad sprayed with the two mycotoxin detection antigens and the goat anti-mouse IgG to a PVP bottom plate, and then slitting to obtain the test paper strip for simultaneously detecting the two mycotoxins.
Example 2
Simultaneous detection of two water-soluble mycotoxins in a sample
(1) Sample pretreatment and detection
Weighing 1.0g of crushed sample to be detected in a triangular flask, adding 50mL of methanol, fully shaking for 3-5min, filtering, adding 0.5mL of filtrate into 2mL of distilled water, and fully mixing to obtain a solution to be detected.
And (3) dripping the sample loading liquid into a sample adding hole of the test strip, and observing a detection result after 5 min.
(2) Analysis of results
When the test strip shows a red line, a blue line and a purple line: the content of both water-soluble mycotoxins is lower than 5ng/mL (1000 mug/kg), and when only the quality control line shows purple color, the content of the water-soluble mycotoxins in the sample is higher than 5ng/mL (1000 mug/kg); a mycotoxin higher than 5ng/mL (1000. mu.g/kg) when only one red line and one purple line are displayed or only one blue line and one purple line are displayed; when there is no purple line, the test strip is invalid.
Claims (4)
1. A preparation method of a test strip for simultaneously detecting two water-soluble mycotoxins is characterized in that: the preparation method comprises the following steps:
(1) DON monoclonal antibody-flower nanoparticle marker and FB1Preparation of monoclonal antibody-15-18 nm spherical nano gold marker
Adding K into spherical golden flower nanometer solution or colloidal gold solution with the particle size of 3mL15-18 nm under magnetic stirring2CO3Adjusting pH of the solution to 5.0-6.5, and collecting DON antibody and FB1Respectively diluting antibodies by 5-10 mu L, gradually adding the diluted antibodies into a golden flower nano solution or a colloidal gold solution, reacting for 60min, then respectively adding BSA and PEG-20000, reacting for 30min, centrifuging at 10000rpm for 60min, re-dissolving precipitates by using a gold seed diluent to obtain a DON monoclonal antibody-flower nano particle marker, and simultaneously preparing a compound of a secondary antibody, the colloidal gold and the golden flower according to the same method for signal amplification;
(2) treatment of gold labeled conjugate release pads
Soaking the gold-labeled conjugate release pad in gold seed diluent for 30min, vacuum drying at 37 deg.C, mixing the gold-labeled antibody complex and the secondary antibody with colloidal gold or golden flower complex, and spraying onto the gold-labeled pad;
(3) coating of nitrocellulose membranes
Spraying mycotoxin detection antigens on a nitrocellulose membrane, wherein the detection antigens are respectively used as detection lines, and goat anti-mouse Ig G is used as a quality control line;
(4) production of test paper
And (3) sticking the nitrocellulose membrane water absorption pad and the sample pad sprayed with the mycotoxin detection antigen and goat anti-mouse IgG on a PVP bottom plate, and then slitting to obtain the test strip for simultaneously detecting the two mycotoxins.
2. The method of claim 1, wherein: k in the step (1)2CO3The solution was 1%, 30 mL.
3. The method of claim 1, wherein: in the step (1), the adding amount of BSA is 10% and 100 mu L, and the adding amount of PEG-20000 is 5% and 100 mu L.
4. The method of claim 1, wherein: the gold ion diluent in the step (1) is 0.5% BSA, 5% sucrose and 0.5% PEG-20000 PBS solution with the concentration of 0.01M.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113607944A (en) * | 2021-08-26 | 2021-11-05 | 深圳市亚辉龙生物科技股份有限公司 | Colloidal gold chromatography reagent strip, preparation method and neocorona antigen detection kit |
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