CN106153929A - By test strips and the method for fluorescence immune chromatography technology for detection mycotoxin - Google Patents
By test strips and the method for fluorescence immune chromatography technology for detection mycotoxin Download PDFInfo
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- CN106153929A CN106153929A CN201510147228.8A CN201510147228A CN106153929A CN 106153929 A CN106153929 A CN 106153929A CN 201510147228 A CN201510147228 A CN 201510147228A CN 106153929 A CN106153929 A CN 106153929A
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Abstract
The present invention relates to the test strips with fluorescence immune chromatography technology for detection mycotoxin and method, concrete, disclose the test strips of a kind of fluorescence immune chromatography technology for detection mycotoxin, including: the labeling pad of the mycotoxin antigen containing fluorescent microsphere labelling, the capture film containing mycotoxin detection antibody.A kind of method that the invention also discloses fluorescence immune chromatography technology for detection mycotoxin, including: the solution containing mycotoxin is added drop-wise in the sample pad of aforementioned test strips, after reaction a period of time, detects fluorescence signal with fluorescence detector.
Description
Technical field
The present invention relates to the fields such as nano material technology, immunological technique, Sidestream chromatography technology.More specifically, the present invention relates to the test strips of a kind of fluorescence immune chromatography technology for detection mycotoxin；Moreover, it relates to a kind of method that fluorescence immune chromatography technology detects mycotoxin.
Background technology
Grain feed be not fully dried when results or in transporting procedures temperature or humidity too high, band dye fungus on grain feed will be made to mushroom out, fungus grow in food or feedstuff produced by metabolite mycotoxin.Mycotoxin includes AFB1, Aflatoxins M1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, Trichothecenes fusarium toxin (T-2 toxin), deoxynivalenol (vomitoxin), ochratoxin A, fumonisin, patulin, ZER etc..Many countries the most once found aflatoxin at Semen Maydis, Semen arachidis hypogaeae and Oleum Arachidis hypogaeae semen, peanut cake, peanut butter, cottonseed cake, Semen Caryae Cathayensis, Fructus Fici, Semen coryli heterophyllae, cacao bean, Semen Armeniacae Amarum, milk powder, milk, buffalo milk, Yoghourt, cheese, rice etc..Mycotoxin is the most harmful to human and animal, as aflatoxin or 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone can make people carcinogenic or infertile, miscarriage.Fusarium toxin acute poisoning can cause general spasticity, HF mortality, chronic poisoning often can cause gastritis, feel sick, mouth, nose, pharynx and digestive tract hemorrhage etc..Therefore, it is necessary to feedstuff, flour, rice, Semen sojae atricolor, the raw material of milk and converted products all to be carried out the monitoring of these mycotoxins.
The method of detection mycotoxin mainly has chromatography, mass spectrography, enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography at present.Wherein chromatography, mass spectrography, ELISA will use expensive equipment, need professional and technical personnel to operate in the lab, and operating procedure is many, the longest.Colloidal gold immunity chromatography is the most simple, convenient, quick and cheap, but its sensitivity is relatively low, and can not detection by quantitative.It is therefore desirable to provide a kind of quick, convenient, simple and highly sensitive mycotoxin quantitative detecting method.
Summary of the invention
Present invention seek to address that the above-mentioned various problems in this area and realize following target.Specifically, present invention aim to provide a kind of test strips and method easily and fast and detecting the mycotoxin in sample in high sensitivity.
Concrete, the invention provides the test strips of mycotoxin in a kind of fluorescence immune chromatography technology for detection sample.
In one embodiment, the invention provides the test strips of a kind of immunochromatography technique detection mycotoxin, including labeling pad and capture film, it is characterised in that, described labeling pad contains the mycotoxin antigen of fluorescent microsphere labelling, and described capture film contains mycotoxin detection antibody.
In further embodiment, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
In further embodiment, it is characterised in that described time-resolved fluorescence microsphere is the microsphere containing europium complex.
In further embodiment, it is characterised in that described mycotoxin is aflatoxin.
In further embodiment, it is characterised in that described mycotoxin is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone.
In further embodiment, it is characterised in that described mycotoxin is vomitoxin.
In further embodiment, it is characterised in that described mycotoxin is ochratoxin A.
In further embodiment, it is characterised in that described mycotoxin is T-2 toxin.
In further embodiment, it is characterised in that described mycotoxin is fumonisin.
In another embodiment, the method that the invention provides a kind of immunochromatography technique detection mycotoxin, it is characterised in that the dropping sample containing mycotoxin in the test strips sample pad described in claim 1, detects fluorescence signal after reaction.
Describe in detail
Show with test strips such as Fig. 1 of mycotoxin in immunochromatography technique detection sample, including sample pad 1, labeling pad 2, capture film 3, adsorptive pads 4, support baseboard 5.Containing C line 6 and T line 7 on capture film 3.
Wherein sample pad is generally glass fibre, filter paper or hemofiltration film.Labeling pad can be glass fibre, filter paper or polyester film.Can combine when sample pad is identical with labeling pad material, complete the function of sample pad and labeling pad with a glass fibre, filter paper or polyester film.Described labeling pad contains the mycotoxin antigen of fluorescent microsphere labelling, it is also possible to the reference protein containing fluorescent microsphere labelling or antibody, such as streptavidin or rabbit antibody (rabbit igg).
Capture film can be nitrocellulose filter (NC film), nylon membrane etc., the T line of described capture film contains mycotoxin detection antibody, C line is positive control, C line contains the albumen for test strips quality control or antibody, such as biotin labeled bovine serum albumin or ovalbumin (containing streptavidin in labeling pad), or goat anti-rabbit antibody (antibody Han rabbit in labeling pad).
By the method for mycotoxin in immunochromatography technique detection sample, as Fig. 1 shows, after sample solution is added drop-wise in the sample pad 1 of test strips, liquid will move along the horizontal direction of arrow, arrive first and reach labeling pad 2, mycotoxin in sample moves forward together with resisting with the mycotoxin antigen of fluorescent microsphere labelling, the rabbit of fluorescent microsphere labelling in labeling pad 2, when solution moves to reach the T line of capture film, mycotoxin, ' mycotoxin antigen～fluorescent microsphere ' in solution will compete and be fixed on the mycotoxin antibodies on T line together.If the concentration of mycotoxins in sample is high, the mycotoxin being then mainly in sample and the mycotoxin antibodies on T line, form ' mycotoxin-mycotoxin detection antibody ' complex, in labeling pad, ' mycotoxin antigen～the fluorescent microsphere ' of release then captures less, and T line position fluorescence signal is more weak；If the concentration of mycotoxins in sample is relatively low, then the mycotoxin in sample and the detection of the mycotoxin on T line antibodies are less, and ' mycotoxin antigen～the fluorescent microsphere ' of the mycotoxin antibody capture on T line is more, defining more ' mycotoxin antibody-mycotoxin antigen～fluorescent microsphere ' complex, T line position fluorescence signal is stronger.Rabbit igg ~ the fluorescent microsphere of labeling pad release then continues to move to, can be by can be in conjunction with goat-anti rabbit two anti-binding of rabbit igg at C line.Detect the fluorescence signal on T line with detector subsequently, and compare with fluorescence signal-concentration of mycotoxins standard curve and calculate the concentration of mycotoxin in testing sample.
Owing at T line, the fluorescent microsphere of mycotoxin antibody capture includes a lot of fluorescence molecule, so the strongest fluorescence signal can be detected at T line, so that the method for this immunochromatography technique detection mycotoxin has the highest sensitivity.When fluorescence molecule is time-resolved fluorescence complex, the fluorescence lifetime of hundreds of microsecond to several milliseconds is had owing to launching light, optical signal can be launched to the detection of hundreds of microseconds by tens of microseconds after exciting light removes, now exciting light and background fluorescence have disappeared, the fluorescence signal that only fluorescence molecule is special, so the specificity of detection and sensitivity are all greatly improved.
' mycotoxin ' in the present invention refers to that fungus grows produced metabolite in food or feedstuff, including AFB1, Aflatoxins M1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, Trichothecenes fusarium toxin (T-2 toxin), deoxynivalenol (vomitoxin), ochratoxin A, fumonisin, patulin, ZER etc..
' microsphere ' in the present invention can be silicon dioxide microsphere, polystyrene microsphere or other polymer microspheres, and microsphere diameter is between 10nm to 10 μm, and its surface can be with functional groups such as carboxyl, amino or ketone groups.
' fluorescent microsphere ' in the present invention refers to embed in spheroid the microsphere of fluorescence molecule.Described ' fluorescence molecule ' includes lanthanide series complex fluorescence molecule and the phosphorescent molecules such as platinum complexes, palladium complex such as the fluorescence molecule or derivatives thereof such as fluorescein, rhodamine, europium complex, samarium complex, terbium complex, wherein europium complex be europium with ligand sequestration after the complex that formed with other organic molecules again, platinum complexes is the complex that platinum is formed with part (including porphyrin, porphin, pyridine and its derivatives) chelating, wherein europium complex is again time-resolved fluorescence complex, and platinum complexes is again phosphorescence complex.
' the mycotoxin antigen of fluorescent microsphere labelling ' in the present invention refers to the mycotoxin antigen coupled with fluorescent microsphere.This mycotoxin antigen is the couplings of mycotoxin and albumen, and this albumen can be BSA, ovalbumin or other albumen.
' mycotoxin antibody ' in the present invention refers to the mycotoxin antibody being combined on test strips capture film.More specifically, mycotoxin antibody is can be with the specific binding antibody of mycotoxin with occur in animal blood after the mycotoxin immune animal with bovine serum albumin (BSA), ovalbumin or other protein couplings.
In the present invention, ' the T line ' on test strips capture film refers to the region at mycotoxin antibody place, namely the mycotoxin antigen of fluorescent microsphere labelling and the region of mycotoxin antibodies.
In the present invention, ' C line ' on test strips capture film refers to the rabbit igg of fluorescent microsphere labelling and the region of goat-anti rabbit two anti-binding, or other are as the antibody of test strips quality control or the region at antigen place.
Because mycotoxin toxicity is very big, even if so feedstuff or food contain a small amount of mycotoxin, also health can be produced injury.And the present invention can detect the mycotoxin in sample convenient by immunochromatographic method, simply, quickly and in high sensitivity, it is to avoid feedstuff or food containing mycotoxin are eaten by human or animal and health is produced injury.
Accompanying drawing explanation
According to one or more different embodiments, with reference to drawings below to the present invention have been described in detail.The merely illustrative purpose of accompanying drawing and provide, and the embodiment that the present invention is typical or exemplary is only described.These accompanying drawings are provided to promote reader's the understanding of the present invention, are not to be regarded as limiting the width of the present invention, scope or application.
Fig. 1 is the test strips schematic diagram of fluorescence immune chromatography detection mycotoxin in the present invention.In Fig. 1, reference 1-7 illustrates: 1-sample pad；2-labeling pad；3-captures film；4-adsorptive pads；5-support baseboard；6-C line；7-T line.
Fig. 2 is the relation curve in embodiment by fluorescence immune chromatography method gained fluorescence intensity with concentration of mycotoxins.
Detailed description of the invention
Embodiment 1, with the preparation of the test strips of Aflatoxins M1 in fluorescence immune chromatography detection sample
The test strips preparation method with immunochromatography detection Aflatoxins M1 shown in Fig. 1 is as follows:
1, the SiO containing europium complex of carboxyl modified2Microsphere is provided by Zhi Ang bio tech ltd, Changchun, and Aflatoxins M1 antibody and Aflatoxins M1 antigen are provided for Kang Huihua Bioisystech Co., Ltd by Beijing.
2, fluorescent microsphere and Aflatoxins M1 antigen, the coupling of rabbit igg
By 200 μ l
20mM PBS, pH7.4, the fluorescent microsphere of solid content 1% and 6 milligrams of carbodiimides mixing, shaken at room temperature 30 minutes, 12,000rpm are centrifuged 10 minutes, clean 2 times with borate buffer solution, are resuspended in by the fluorescent microsphere of activation in 300 μ l borate buffer solutions.The Aflatoxins M1 antigen of 200 μ l 1mg/ml, at room temperature oscillating reactions 2.5 hours is added in microsphere suspensions.By reacted solution in 12,000rpm is centrifuged 10 minutes, adds 400 μ l 0.25M ethanolamine and vibrates 30 minutes in room temperature again.2 times will be washed with PBS again after reacted solution centrifugal.The BSA adding 300 μ l 2mg/ml after centrifugation is resuspended, and room temperature vibrates after 30 minutes and washes 3 times with PBS, then with 200 μ l 50mM PBS,
The Aflatoxins M1 antigen of pH7.4,0.1% Tween 20,5% sucrose further fluorescent microsphere labelling.
The Rhizoma Nelumbinis of rabbit igg and fluorescent microsphere is in conjunction with upper method.
3, the Aflatoxins M1 antigen of fluorescent microsphere labelling, the rabbit igg of fluorescent microsphere labelling process labeling pad, Aflatoxins M1 antibody and goat anti-rabbit antibody (Sigma Co., USA) and process capture film
Being mixed with the rabbit igg solution equal-volume of fluorescent microsphere labelling by the Aflatoxins M1 antigenic solution of fluorescent microsphere labelling, then be sprayed onto in labeling pad (Millipore company) with BioDot spray film instrument, spray speed is 9 μ l/cm.By 1mg/ml Aflatoxins M1 antibody and 1mg/ml goat anti-rabbit antibody BioDot company spray film instrument line capture film (Millipore company NC film), line speed is 2 μ l/cm.Then the labeling pad sprayed and capture film are placed in 37 ° of C calorstats and are dried 1 hour, then the part of the Aflatoxins M1 antigen containing fluorescent microsphere labelling in labeling pad is cut down, obtain 5mm width, the band of 30cm length.
4, the assembling of test strips kilocalorie and cutting
According in Fig. 1 shown in the structure of test strips, first capture film is pasted onto on support baseboard (Millipore company), by T line side successively binding mark thing pad and sample pad (Millipore company), adsorptive pads (Millipore company) is being pasted by C line side, then cutting into the wide test strips of 5mm with BioDot company cutting machine, in aluminium foil bag, Seal and preservation is standby.
Embodiment 2, with the Aflatoxins M1 in fluorescence immune chromatography method detection sample
By 100 μ l containing 0 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, the Aflatoxins M1 solution of 50 μ g/L is added drop-wise on immuno-chromatographic test paper strip respectively, with fluorescence detector (Shang Haihai jump mold company limited) detection fluorescence signal after reacting 15 minutes, then maps to obtain fluorescence intensity standard curve such as Fig. 2 to Aflatoxins M1 concentration relative to Aflatoxins M1 concentration by fluorescence intensity.
The 100 μ l milk containing Aflatoxins M1 is added drop-wise in the sample pad of immuno-chromatographic test paper strip, fluorescence signal is detected after reacting 15 minutes, it is 0.45 μ g/L that fluorescence intensity combined standard curve calculates Aflatoxins M1 concentration, consistent with the result detected by ELISA method, illustrate that the method for immunochromatography detection by quantitative Aflatoxins M1 is reliable.
Embodiment 3, with the 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone in fluorescence immune chromatography method detection sample
The test strips preparation method of detection 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone is with embodiment 1, aflatoxin antigenic shift therein is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antigen, aflatoxin antibody is replaced by 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone antibody, and the antigen of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and antibody are also provided for Kang Huihua Bioisystech Co., Ltd by Beijing.
By 100 μ l containing 0 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.5 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, the 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone solution of 50 μ g/L is added drop-wise on immuno-chromatographic test paper strip respectively, with detector (Shang Haihai jump mold company limited) detection fluorescence signal after reacting 15 minutes, then maps to obtain the fluorescence intensity standard curve to 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone concentration relative to 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone concentration by fluorescence intensity.
Take the 100 μ l sample solution containing 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone to be added drop-wise in the sample pad of immuno-chromatographic test paper strip, fluorescence signal is detected after reacting 15 minutes, it is 0.4 μ g/L that fluorescence intensity combined standard curve calculates 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone concentration, consistent with the result detected by ELISA method, illustrate that the method for immunochromatography quantitative detection of zearalenone is reliable.
Although the application describes specific embodiment in detail, but the application is not limited to these embodiments, on the contrary, its intention contains the various amendments in the spirit and scope being included in embodiment and equivalent, in some particular cases, can lack and increase certain or some technical characteristics without departing from the spirit and scope of the present invention, scope of the present application is limited only by the appended claims.
Claims (10)
1., by a test strips for fluorescence immune chromatography technology for detection mycotoxin, including labeling pad and capture film, it is characterised in that described labeling pad contains the mycotoxin antigen of fluorescent microsphere labelling, described capture film contains mycotoxin detection antibody.
2. test strips as claimed in claim 1, it is characterised in that described fluorescent microsphere is time-resolved fluorescence microsphere.
3. test strips as claimed in claim 2, it is characterised in that described time-resolved fluorescence microsphere is the microsphere containing europium complex.
4. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is aflatoxin.
5. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone.
6. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is vomitoxin.
7. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is ochratoxin A.
8. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is T-2 toxin.
9. the test strips as described in any one of claim 1-3, it is characterised in that described mycotoxin is fumonisin.
10. the method with fluorescence immune chromatography technology for detection mycotoxin, it is characterised in that the dropping sample containing mycotoxin in the test strips sample pad described in claim 1, detects fluorescence signal after reaction.
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| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN109085336A (en) * | 2018-08-29 | 2018-12-25 | 郑州工程技术学院 | A kind of immune chromatography test paper detecting fumonisins B1 |
| CN110988339A (en) * | 2019-12-30 | 2020-04-10 | 江南大学 | Time-resolved immune quantitative test strip for detecting aflatoxin M1 in milk |
| CN111007246A (en) * | 2019-11-15 | 2020-04-14 | 中国农业科学院油料作物研究所 | Time resolution fluorescence kit for synchronously detecting ribes diacetate sickle enol, deoxynivalenol and T-2 toxin |
| CN114235807A (en) * | 2021-12-20 | 2022-03-25 | 中国农业科学院油料作物研究所 | Intelligent instant quantitative detection device for mycotoxin quantum dot test paper strips |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106932370A (en) * | 2017-03-07 | 2017-07-07 | 中国农业科学院油料作物研究所 | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application |
| CN106932370B (en) * | 2017-03-07 | 2019-08-13 | 中国农业科学院油料作物研究所 | The synchronous chromatography time-resolved fluorescence kit for detecting five kinds of mycotoxins and application |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN109085336A (en) * | 2018-08-29 | 2018-12-25 | 郑州工程技术学院 | A kind of immune chromatography test paper detecting fumonisins B1 |
| CN109085336B (en) * | 2018-08-29 | 2021-03-02 | 郑州工程技术学院 | Immunochromatographic test paper for detecting fumonisin B1 |
| CN111007246A (en) * | 2019-11-15 | 2020-04-14 | 中国农业科学院油料作物研究所 | Time resolution fluorescence kit for synchronously detecting ribes diacetate sickle enol, deoxynivalenol and T-2 toxin |
| CN111007246B (en) * | 2019-11-15 | 2021-09-21 | 中国农业科学院油料作物研究所 | Time resolution fluorescence kit for synchronously detecting ribes diacetate sickle enol, deoxynivalenol and T-2 toxin |
| CN110988339A (en) * | 2019-12-30 | 2020-04-10 | 江南大学 | Time-resolved immune quantitative test strip for detecting aflatoxin M1 in milk |
| CN114235807A (en) * | 2021-12-20 | 2022-03-25 | 中国农业科学院油料作物研究所 | Intelligent instant quantitative detection device for mycotoxin quantum dot test paper strips |
| CN114235807B (en) * | 2021-12-20 | 2022-11-01 | 中国农业科学院油料作物研究所 | Intelligent instant quantitative detection device for mycotoxin quantum dot test paper strips |
| US11802840B2 (en) | 2021-12-20 | 2023-10-31 | Institute of Oilseed Crops, Chinese Academy of Agricultural Sciences | Intelligent instant quantitative testing device for mycotoxin quantum dot test strip |
| CN115999542A (en) * | 2023-01-03 | 2023-04-25 | 东南大学 | Europium-silver cluster enzyme and preparation method and application thereof |
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Application publication date: 20161123 |