CN116840469A - Immune gold mark rapid test card for single detection of multiple mycotoxins and preparation method thereof - Google Patents
Immune gold mark rapid test card for single detection of multiple mycotoxins and preparation method thereof Download PDFInfo
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 238000001514 detection method Methods 0.000 title claims abstract description 93
- 238000012360 testing method Methods 0.000 title claims abstract description 36
- 231100000678 Mycotoxin Toxicity 0.000 title claims abstract description 35
- 239000002636 mycotoxin Substances 0.000 title claims abstract description 35
- 239000010931 gold Substances 0.000 title claims abstract description 26
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 51
- 102000036639 antigens Human genes 0.000 claims abstract description 51
- 108091007433 antigens Proteins 0.000 claims abstract description 51
- 229930020125 aflatoxin-B1 Natural products 0.000 claims abstract description 46
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims abstract description 45
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 claims abstract description 28
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000012528 membrane Substances 0.000 claims abstract description 18
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 16
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 16
- 239000002245 particle Substances 0.000 claims abstract description 5
- 239000003085 diluting agent Substances 0.000 claims description 30
- 238000003908 quality control method Methods 0.000 claims description 29
- 241000283707 Capra Species 0.000 claims description 12
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 5
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 5
- 229940038773 trisodium citrate Drugs 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
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- 238000005507 spraying Methods 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000010304 firing Methods 0.000 claims description 3
- ZBKIUFWVEIBQRT-UHFFFAOYSA-N gold(1+) Chemical compound [Au+] ZBKIUFWVEIBQRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 241000416536 Euproctis pseudoconspersa Species 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 abstract description 36
- 239000002115 aflatoxin B1 Substances 0.000 abstract description 18
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 abstract description 16
- 239000003053 toxin Substances 0.000 abstract description 7
- 231100000765 toxin Toxicity 0.000 abstract description 7
- 210000004916 vomit Anatomy 0.000 abstract description 7
- 230000008673 vomiting Effects 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000011896 sensitive detection Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 239000000523 sample Substances 0.000 description 19
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 11
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 10
- -1 DON Chemical class 0.000 description 8
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- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
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- 241000233866 Fungi Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
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- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
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- 239000008399 tap water Substances 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention relates to an immune gold-labeled quick-test card for single detection of various mycotoxins and a preparation method thereof. The preparation method comprises the following steps: respectively fixing DON/ZEN/AFB1 antigen and secondary antibody on a nitrocellulose membrane; and combining the colloidal gold particles with DON/ZEN/AFB1 specific antibodies under alkaline conditions and assembling the rapid test card. The immune gold mark rapid test card can detect various mycotoxins such as vomit toxin, zearalenone and aflatoxin B1 on one card at a time. The pretreatment of the sample to be detected is completely consistent, the sample is added for only 1 time under the same condition, the sample adding amount is only 100 mu L, and the operation is simple; in addition, the detection time is extremely short (only 5-8 min is needed), and the judgment standards are uniform. Provides a rapid, on-site and sensitive detection method for the practical detection department, and is mainly applied to the detection of samples such as grain feed, food and the like.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to an immune gold-labeled rapid test card for detecting various mycotoxins at a time and a preparation method thereof.
Background
Mycotoxins are metabolites produced by fungi growing in food or feed and are extremely harmful to humans and animals. The conventional method for detecting mycotoxins is liquid chromatography, a special laboratory site is needed, special instruments and equipment are needed, the requirements on operators are high, considerable expertise, skills and operation experience are needed, the pretreatment of the detection process is complex, and the required time is long.
The gold-labeled card detection method using lateral flow as a detection principle has been applied in various fields including food safety detection and the like, and gold-labeled quick-test cards for detecting single mycotoxin have been reported, however, quick-test cards for detecting vomitoxin, zearalenone and aflatoxin B1 on one card at a time have not been disclosed.
Therefore, the technical scheme of the invention is provided based on the above.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an immune gold-labeled rapid test card for detecting various mycotoxins at a time and a preparation method thereof. The immune gold-labeled rapid test card for single detection of various mycotoxins can be used for single detection of various mycotoxins such as vomit toxin, zearalenone and aflatoxin B1 on one card. The pretreatment of the sample to be detected is completely consistent, the sample is added for only 1 time under the same condition, the sample adding amount is only 100 mu L, and the operation is simple; in addition, the detection time is extremely short (only 5-8 min is needed), and the judgment standards are uniform. Provides a rapid, on-site and sensitive detection method for the practical detection department, and is mainly applied to the detection of samples such as grain feed, food and the like.
The scheme of the invention is that the preparation method of the immune gold-labeled rapid test card for detecting various mycotoxins at a time is provided, and comprises the following steps:
(I) The preparation method of the nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C comprises the following steps:
(1) Diluting DON antigen, ZEN antigen and AFB1 antigen to obtain DON antigen diluent, ZEN antigen diluent and AFB1 antigen diluent respectively, and using the DON antigen diluent, the ZEN antigen diluent and the AFB1 antigen diluent as coating antigens of detection lines T1, T2 and T3 respectively;
(2) Diluting the goat anti-mouse IgG secondary antibody to obtain goat anti-mouse IgG secondary antibody diluent which is used as a coating antigen of the quality control line C;
(3) Taking a nitrocellulose membrane as a carrier, marking a T1 detection line by using the DON antigen diluent, a T2 detection line by using the ZEN antigen diluent, a T3 detection line by using the AFB1 antigen diluent, and a C line by using the goat anti-mouse IgG secondary anti-diluent, and taking the C line as a quality control line, and drying after finishing to obtain the nitrocellulose membrane containing the detection lines T1, T2, T3 and the quality control line C;
(II) preparation of gold-labeled pad immobilized with specific monoclonal antibody marked by colloidal gold, which comprises the following steps:
(S1) dissolving chloroauric acid in ultrapure water to prepare a gold burning solution a; dissolving trisodium citrate in ultrapure water to prepare a gold burning solution b;
(S2) stirring and mixing the gold burning solution a and ultrapure water uniformly, heating to boiling, adding the gold burning solution b, and continuing to boil to obtain colloidal gold after the completion;
(S3) regulating the pH value of the colloidal gold, respectively adding DON antibody, ZEN antibody and AFB1 antibody for room temperature reaction, and standing after the reaction is ended to respectively obtain DON labeled antibody, ZEN labeled antibody and AFB1 labeled antibody;
(S4) respectively centrifuging the DON labeled antibody, the ZEN labeled antibody and the AFB1 labeled antibody, removing supernatant, and re-dissolving the precipitate to obtain a colloidal gold labeled DON antibody solution, a colloidal gold labeled ZEN antibody solution and a colloidal gold labeled AFB1 antibody solution;
(S5) spraying the DON antibody solution marked by the colloidal gold, the ZEN antibody solution marked by the colloidal gold and the AFB1 antibody solution marked by the colloidal gold on the pretreated gold-labeled pad, and drying to obtain the gold-labeled pad fixed with the specific monoclonal antibody marked by the colloidal gold;
(III) assembling a gold mark rapid test card, wherein the method comprises the following steps:
sequentially sticking a sample pad, a gold-labeled pad fixed with a specific monoclonal antibody marked by colloidal gold, a nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C and water-absorbing filter paper on a bottom plate, cutting into 3mm wide by a slitter, and loading into a card shell to obtain the immune gold-labeled rapid test card for detecting various mycotoxins at a time.
Wherein the DON antigen is purchased from Shanghai Biotechnology Inc. (product BA 0721) and is a product obtained by coupling vomitoxin small molecules (DON) with BSA proteins; the ZEN antigen is purchased from Shanghai Biotechnology Co., ltd (product No. BA 0521) and is a product obtained by coupling zearalenone small molecule (ZEN) with BSA protein; the AFB1 antigen is purchased from Shanghai Biotechnology Inc. (product No. BA 0121) and is a product obtained by coupling aflatoxin B1 small molecule (AFB 1) with BSA protein; the goat anti-mouse IgG secondary antibody was purchased from Shanghai you long biotechnology limited.
Preferably, in step (1), the DON antigen, the ZEN antigen and the AFB1 antigen are diluted to 1.0mg/mL with PBS.
Preferably, in step (2), the goat anti-mouse IgG secondary antibody is diluted to 1.0mg/mL with PBS.
Preferably, in the step (3), the T1 detection line, the T2 detection line, the T3 detection line and the quality control line are all drawn at 1 μl/cm; the drying mode is as follows: and drying at 37 ℃ for 24 hours.
Preferably, in the step (S1), the concentration of chloroauric acid in the gold firing solution a is 1wt.%; in the gold firing solution b, the concentration of trisodium citrate is 1wt.%.
Preferably, in step (S3), K is used 2 CO 3 The pH was adjusted, the reaction time at room temperature was 40min, and the reaction was stopped by adding 10wt.% BSA.
Preferably, in step (S4), the centrifugation is performed in the following manner: centrifuging at 1500r/min, discarding precipitate formed by coagulated gold colloidal particles, and centrifuging at 8500r/min for 30min.
Preferably, in step (S4), the precipitate is reconstituted with 0.1M PBS containing 1wt.% BSA.
Based on the same technical conception, the invention also provides an immune gold standard rapid test card for detecting various mycotoxins at a time, which is prepared by the preparation method.
The immune gold mark rapid test card has the following structural characteristics:
(1) The upper layer plastic plate has two holes, namely a sample adding hole and an observation hole, wherein the size of the sample adding hole is 3mm multiplied by 5mm, and the size of the observation hole is 3mm multiplied by 18mm.
(2) The glass fiber membrane, the gold-labeled pad fixed with the specific monoclonal antibody marked by colloidal gold, the nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C and the water-absorbing filter paper are sequentially adhered side by side from the sample adding Kong Wangshang below the plastic plate. Wherein, the glass fiber membrane size is 3mm×15mm, the gold mark pad size is 3mm×3mm, the nitrocellulose membrane size is 3mm×28mm, and the water absorption filter paper size is 3mm×19mm.
(3) The amount of each monoclonal antibody on the gold-labeled pad is 5-50 ng, and the amount of antigen BSA on the nitrocellulose membrane is 0.4-1.2 mug.
(4) The drying temperature of the gold mark pad is 37 ℃ and the time is 30min.
The detection principle of the invention is as follows: the immune gold-labeled rapid test card applies the principle of competitive inhibition immune chromatography, mycotoxin in a sample is combined with a specific monoclonal antibody labeled by colloidal gold in the process of lateral movement, and the combination of the antibody and an antigen-BSA conjugate on a detection line is inhibited. If the sample contains vomitoxin, zearalenone or aflatoxin B1, the corresponding detection line does not develop color, and the result is positive; otherwise, the detection line shows red color, and the result is negative.
Based on the same technical conception, the invention further provides a using method of the immune gold mark rapid test card for detecting various mycotoxins at a time, which comprises the following steps:
and placing the immune gold-labeled quick test card horizontally, adding 100 mu L of sample solution into a sample adding hole, allowing the sample solution to permeate into a colloidal gold-labeled antibody test strip along a sample pad, competing with a specific antigen on a nitrocellulose membrane for binding the colloidal gold-labeled specific antibody, observing color change in a result observing hole after 5-8 min, and judging.
The judging standard is specifically as follows:
negative: the quality control line (C line) and all the detection lines (T1, T2 and T3 lines) are developed, and the negative detection is indicated;
positive: the quality control line (C line) develops color, and any detection line (T1, T2 and T3 line) does not develop color or develops color obviously, the detected substance is positive;
invalidation: the quality control line (C line) does not develop color.
The beneficial effects of the invention are as follows:
the immune gold-labeled rapid test card for single detection of various mycotoxins can be used for single detection of various mycotoxins such as vomit toxin, zearalenone and aflatoxin B1 on one card. The pretreatment of the sample to be detected is completely consistent, the sample is added for only 1 time under the same condition, the sample adding amount is only 100 mu L, and the operation is simple; in addition, the detection time is extremely short (only 5-8 min is needed), and the judgment standards are uniform. Provides a rapid, on-site and sensitive detection method for the practical detection department, and is mainly applied to the detection of samples such as grain feed, food and the like.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the structure of the immune gold-labeled rapid test card for detecting various mycotoxins at a time.
The reference numerals in the drawings are:
1-a viewing aperture; 2-quality control line (C line); 3-detecting line T1; 4-detecting line T2; 5-detecting line T3; 6-sample adding hole.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1
The embodiment provides a preparation method of an immune gold-labeled rapid test card for detecting various mycotoxins at a time, which comprises the following steps:
(I) The preparation method of the nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C comprises the following steps:
(1) Diluting DON antigen, ZEN antigen and AFB1 antigen to 1.0mg/mL by adopting PBS (phosphate buffer solution), respectively obtaining DON antigen diluent, ZEN antigen diluent and AFB1 antigen diluent, and respectively serving as coating antigens of detection lines T1, T2 and T3;
(2) Diluting the goat anti-mouse IgG secondary antibody to 1.0mg/mL by adopting PBS (phosphate buffer solution) to obtain goat anti-mouse IgG secondary antibody diluent which is used as a coating antigen of a quality control line C;
(3) Selecting a nitrocellulose membrane of PALL170, and marking a T1 line with DON antigen diluent with the concentration of 1.0mg/mL by using a film marking metal spraying machine at the concentration of 1.0 mu L/cm to be used as a detection line T1; marking a T2 line with ZEN antigen diluent with the concentration of 1.0mg/mL at 1.0 mu L/cm to be used as a detection line T2; marking a T3 line with the concentration of 1.0mg/mL of AFB1 antigen diluent at 1.0 mu L/cm to be used as a detection line T3; drawing a C line with a concentration of 1.0 mu L/cm as a quality control line by using goat anti-mouse IgG secondary antibody diluent with a concentration of 1.0 mg/mL; drying for 24 hours at 37 ℃ after completion to obtain a nitrocellulose membrane containing detection lines T1, T2, T3 and a quality control line C;
(II) preparation of gold-labeled pad immobilized with specific monoclonal antibody marked by colloidal gold, which comprises the following steps:
(S1) cleaning tools such as a 500mL beaker, a 20mL small beaker, a rotor, a brown bottle, a glass rod and the like, then placing the tools into an acid jar (potassium dichromate: concentrated sulfuric acid: ultrapure water=120 g:200mL:1000 mL) for soaking for 24 hours, taking out the tools, washing the tools for 3 to 4 times by tap water, washing the tools for 3 to 4 times by ultrapure water, and placing the tools into a baking oven at 37 ℃ for later use;
(S2) weighing 1g of chloroauric acid powder (purchased from sigma) in a brown bottle, adding 99mL of ultrapure water for full dissolution, and preserving at 4 ℃ in a dark place to obtain a gold burning solution a; weighing 1g of trisodium citrate (purchased from sigma) in a brown bottle, and adding 99mL of ultrapure water for full dissolution to obtain a gold burning solution b;
(S3) weighing 99mL of ultrapure water in a beaker, adding 1mL of gold burning solution a, placing on a constant-temperature magnetic stirrer for stirring and mixing uniformly, starting heating until the solution boils, rapidly adding 2.5mL of newly prepared gold burning solution b, continuing stirring and heating, gradually changing the solution into blue black, then carrying out purple black, reheating to generate red, continuing boiling to generate transparent orange red, continuing boiling for 7-10 min, naturally cooling to room temperature, adding ultrapure water to a volume of 100mL, pouring into a brown bottle, and preserving at 4 ℃ in a dark place to obtain colloidal gold (the particle size is 25 nm);
(S4) 1.5mL of the colloidal gold was taken with 0.1M K 2 CO 3 Adjusting the pH value, respectively adding corresponding amount of DON antibody, uniformly mixing, reacting at room temperature for 40min, adding 10% BSA for stopping, and standing for 30min to obtain DON labeled antibody;
(S5) centrifuging the DON labeled antibody at 1500r/min, discarding the precipitate formed by the aggregated gold colloidal particles, centrifuging at 8500r/min for 30min, carefully sucking the supernatant, re-dissolving the precipitate with 0.1M PBS (pH 7.4) containing 1% BSA, and preserving at 4 ℃ to obtain a colloidal gold labeled DON antibody solution;
(S6) preparing a gold-labeled ZEN antibody solution and a gold-labeled AFB1 antibody solution according to the operations in the steps (S4) and (S5) in a similar manner;
(S7) spraying the DON antibody solution marked by the colloidal gold, the ZEN antibody solution marked by the colloidal gold and the AFB1 antibody solution marked by the colloidal gold on the pretreated gold-labeled pad, and drying to obtain the gold-labeled pad fixed with the specific monoclonal antibody marked by the colloidal gold;
(III) assembling a gold mark rapid test card, wherein the method comprises the following steps:
sequentially sticking a sample pad, a gold-labeled pad fixed with a specific monoclonal antibody marked by colloidal gold, a nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C and water-absorbing filter paper on a bottom plate, cutting into 3mm wide by a slitter, and loading into a card shell to obtain the immune gold-labeled rapid test card for detecting various mycotoxins at a time.
Example 2
The embodiment provides an effect evaluation of an immune gold-labeled rapid test card for detecting various mycotoxins at a time, which specifically comprises the following steps:
(1) 10. Mu.L of a blank sample solution (10% by volume aqueous methanol solution) was added to the well, and after reaction at room temperature for 8 minutes, the color development was observed in the well. The result shows that the observation hole shows obvious wine red quality control line C and detection lines T1, T2 and T3, and the result is negative, namely, the observation hole does not contain vomit toxin, zearalenone and aflatoxin B1.
(2) 100. Mu.L of a vomitoxin standard (prepared from the same aqueous methanol solution as above) was added to the wells, and the procedure was followed as for the blank. The result shows that the quality control line C, the detection line T2 and the detection line T3 in the observation hole are in wine red, and the detection line T1 is colorless, so that the result is positive, namely, the vomit toxin is contained.
(3) 100. Mu.L of a zearalenone standard (prepared from the same aqueous methanol solution) was added to the sample well at a concentration of 50ng/ml, and the procedure was followed as for the blank sample. The result shows that the quality control line C, the detection line T1 and the detection line T3 in the observation hole are in wine red, and the detection line T2 is colorless, so that the result is positive, namely the zearalenone is contained.
(4) A100. Mu.L/ml aflatoxin B1 standard (prepared from the same aqueous methanol solution) was added to the wells, and the procedure was followed as for the blank. The result shows that the quality control line C, the detection line T1 and the detection line T2 in the observation hole are in wine red, and the detection line T3 is colorless, so that the result is positive, namely the aflatoxin B1 is contained.
(5) 100. Mu.L of a vomitoxin standard and 50ng/ml of a zearalenone mixed standard (prepared from the same aqueous methanol solution) were added to the wells, and the same procedure as for the blank samples was followed. The results show that the quality control line C and the detection line T3 in the observation hole are in a wine red color, the detection line T1 and the detection line T2 are colorless, and the results show that the results are positive, namely the results contain vomit toxin and zearalenone.
(6) 100. Mu.L of vomitoxin standard and 5ng/ml of aflatoxin B1 standard (prepared from the same aqueous methanol solution) were added to the wells, and the same procedure as for the blank samples was followed. The results show that the quality control line C and the detection line T2 in the observation hole are in a wine red color, and the detection line T1 and the detection line T3 are colorless, so that the results are positive, namely, the detection holes contain vomitoxin and aflatoxin B1.
(7) 100. Mu.L of zearalenone standard (50 ng/ml) and aflatoxin B1 standard (5 ng/ml) prepared from the same aqueous methanol solution were added to the wells, and the same procedure as for the blank samples was followed. The results show that the control line C and the detection line T1 in the observation hole are in a wine red color, the detection line T2 and the detection line T3 are colorless, and the results show that the results are positive, namely the corn gibberellin ketone and the aflatoxin B1 are contained.
(8) 50. Mu.L of a vomitoxin standard, 200ng/ml of zearalenone standard and 5ng/ml of an aflatoxin B1 standard (prepared from the same aqueous methanol solution) were added to the wells, and the same procedure as for the blank sample was followed. The result shows that the control line C in the observation hole is in a wine red color, and the detection line T1, the detection line T2 and the detection line T3 are colorless, so that the result is positive, namely, the result contains vomit toxin, zearalenone and aflatoxin B1.
Example 3
The embodiment provides an effect evaluation of detecting cereal feed samples by an immune gold-labeled rapid test card for detecting various mycotoxins at a time, which specifically comprises the following steps:
(1) 100. Mu.L of the negative feed extract was added to the wells, and the same procedure as for the blank samples was followed. The result shows that the quality control line C and the detection lines T1, T2 and T3 which are obviously reddish in wine are observed in the observation hole.
(2) 100 mu L of feed sample extracting solution positive for vomitoxin, zearalenone and aflatoxin B1 is added into the sample adding hole, and the same operation steps as those of a blank sample are carried out. The result shows that the quality control line C in the observation hole is in a wine red color, and the detection line T1, the detection line T2 and the detection line T3 are colorless.
The inventor uses the ELISA detection kit for detecting various mycotoxins, namely the immune gold mark rapid detection card for detecting various mycotoxins, vomitoxin, zearalenone and aflatoxin B1 simultaneously to detect 116 samples comprising 56 negative samples and 60 positive samples, and the result shows that the coincidence rate of the immune gold mark rapid detection card and the ELISA detection kit for detecting the vomitoxin, zearalenone and aflatoxin B1 is 98.1%.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. The preparation method of the immune gold-labeled rapid test card for detecting various mycotoxins at a time is characterized by comprising the following steps:
(I) The preparation method of the nitrocellulose membrane containing detection lines T1, T2 and T3 and a quality control line C comprises the following steps:
(1) Diluting DON antigen, ZEN antigen and AFB1 antigen to obtain DON antigen diluent, ZEN antigen diluent and AFB1 antigen diluent respectively, and using the DON antigen diluent, the ZEN antigen diluent and the AFB1 antigen diluent as coating antigens of detection lines T1, T2 and T3 respectively;
(2) Diluting the goat anti-mouse IgG secondary antibody to obtain goat anti-mouse IgG secondary antibody diluent which is used as a coating antigen of the quality control line C;
(3) Taking a nitrocellulose membrane as a carrier, marking a T1 detection line by using the DON antigen diluent, a T2 detection line by using the ZEN antigen diluent, a T3 detection line by using the AFB1 antigen diluent, and a C line by using the goat anti-mouse IgG secondary anti-diluent, and taking the C line as a quality control line, and drying after finishing to obtain the nitrocellulose membrane containing the detection lines T1, T2, T3 and the quality control line C;
(II) preparation of gold-labeled pad immobilized with specific monoclonal antibody marked by colloidal gold, which comprises the following steps:
(S1) dissolving chloroauric acid in ultrapure water to prepare a gold burning solution a; dissolving trisodium citrate in ultrapure water to prepare a gold burning solution b;
(S2) stirring and mixing the gold burning solution a and ultrapure water uniformly, heating to boiling, adding the gold burning solution b, and continuing to boil to obtain colloidal gold after the completion;
(S3) regulating the pH value of the colloidal gold, respectively adding DON antibody, ZEN antibody and AFB1 antibody for room temperature reaction, and standing after the reaction is ended to respectively obtain DON labeled antibody, ZEN labeled antibody and AFB1 labeled antibody;
(S4) respectively centrifuging the DON labeled antibody, the ZEN labeled antibody and the AFB1 labeled antibody, removing supernatant, and re-dissolving the precipitate to obtain a colloidal gold labeled DON antibody solution, a colloidal gold labeled ZEN antibody solution and a colloidal gold labeled AFB1 antibody solution;
(S5) spraying the DON antibody solution marked by the colloidal gold, the ZEN antibody solution marked by the colloidal gold and the AFB1 antibody solution marked by the colloidal gold on the pretreated gold-labeled pad, and drying to obtain the gold-labeled pad fixed with the specific monoclonal antibody marked by the colloidal gold;
(III) assembling an immune gold-labeled rapid test card for detecting various mycotoxins at a time.
2. The method for preparing the immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (1), the DON antigen, the ZEN antigen and the AFB1 antigen are diluted to 1.0mg/mL by PBS.
3. The method for preparing the immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (2), the goat anti-mouse IgG secondary antibody is diluted to 1.0mg/mL with PBS.
4. The method for preparing an immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (3), the T1 detection line, the T2 detection line, the T3 detection line and the quality control line are all scored at 1 μl/cm; the drying mode is as follows: and drying at 37 ℃ for 24 hours.
5. The method for preparing an immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (S1), the concentration of chloroauric acid in the gold-burning solution a is 1wt.%; in the gold firing solution b, the concentration of trisodium citrate is 1wt.%.
6. The method for preparing the immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (S3), K is adopted 2 CO 3 The pH was adjusted, the reaction time at room temperature was 40min, and the reaction was stopped by adding 10wt.% BSA.
7. The method for preparing the immune gold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in the step (S4), the centrifugation mode is as follows: centrifuging at 1500r/min, discarding precipitate formed by coagulated gold colloidal particles, and centrifuging at 8500r/min for 30min.
8. The method for preparing an immunogold-labeled rapid test card for single detection of multiple mycotoxins according to claim 1, wherein in step (S4), the precipitate is reconstituted with 0.1M PBS containing 1wt.% BSA.
9. The immune gold-labeled rapid test card for single detection of various mycotoxins prepared by the preparation method of any one of claims 1 to 8.
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