CN103792358A - Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board - Google Patents
Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board Download PDFInfo
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- CN103792358A CN103792358A CN201210435166.7A CN201210435166A CN103792358A CN 103792358 A CN103792358 A CN 103792358A CN 201210435166 A CN201210435166 A CN 201210435166A CN 103792358 A CN103792358 A CN 103792358A
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Abstract
The invention discloses a colloidal gold immunochromatograohic assay detection board capable of semi-quantitatively detecting aflatoxin G1 in feed, belonging to the field of biological and food safety. The detection board is characterized in that a shell internally comprises a test strip; a water absorption pad, a detection pad, a gold mark pad and a sample pad are pasted on one side of the test strip sequentially from top to bottom; adjacent pads are overlapped and connected at the joint; in the detection board, a nitrocellulose membrane is used as a base pad; the nitrocellulose membrane is provided with a transverse quality control line (a C line) and a detection line (a T line) from top to bottom; the detection line coats an aflatoxin G1-bovine serum albumin conjugate (AFG1-BSA), and the quality control line coats a rabbit antimouse polyclonal antibody; the gold mark pad is transversally sprayed with a nanogold-marked aflatoxin G1 monoclonal antibody. The detection board is used for detecting the aflatoxin G1 and has the characteristics of quick detection, simplicity in operation and high sensitivity.
Description
Technical field
The invention belongs to biology and food safety detection technical field, be specifically related to a kind of preparation and detection method thereof of aflatoxin G 1 collaurum check-out console.
Background technology
Aflatoxin (Aflatoxins) is to be grown in aspergillus flavus (Aspergillus flavus) in food and feed and one group of similar product of chemical constitution of aspergillus parasiticus metabolism.Aflatoxin is the extremely strong extremely toxic substance of a kind of toxicity, and people and animal's liver tissue are had to destruction, when serious, can cause liver cancer even dead.Wherein aflatoxin B1 carcinogenicity and toxicity are the strongest.The toxicity of aflatoxin G 1 only this in aflatoxin B1.Human and animal's health is had to very serious harm.
To aflatoxin, the residue limits in food all has regulation in various countries.Nineteen ninety-five, in the food that the World Health Organization (WHO) formulates, aflatoxin maximum permissible concentration is 15 μ g/kg.Aspergillus flavus poison content in Federal Government relevant laws and regulations human consumption's food and milk cow forage can not exceed 15 μ g/kg, and the content in other animal feeds can not exceed 300 μ g/kg.China's regulation, in corn and shelled peanut goods (by raw material conversion), aspergillus flavus poison content is no more than 20 μ g/kg.In rice, other edible oils, must not exceed 10 μ g/kg, must not exceed 5 μ g/kg in other grains, beans, fermented food, in baby's milk powder substitute, must not detect, other food can be carried out with reference to above standard.
The method of measuring at present aflatoxin mainly contains thin-layered chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, mass spectroscopy, radioimmunoassay.These detection methods have been brought into play good effect at different times, but there is sample pre-treatments complex steps, time and effort consuming, instrument is expensive, the shortcoming that needs professional to operate etc., and colloidal gold immunity chromatography has high specificity, sample pre-treatments is simple, cost is low, little and can be simultaneously to advantages such as great amount of samples detect to the contamination hazard of experimenter and environment.It is a kind of fast detecting product applicable to large-scale plant, feed factory etc.
Summary of the invention
The object of this invention is to provide a kind of aflatoxin G 1 sxemiquantitative speed and survey preparation and the using method of collaurum check-out console, opened up application prospect widely for realizing the fast detecting of aflatoxin G 1 in feed.
Technical scheme of the present invention: a kind of aflatoxin G 1 sxemiquantitative speed is surveyed the preparation method of check-out console, check-out console is made up of shell, sample pad, gold mark pad, nitrocellulose filter and adsorptive pads four parts; Gold mark pad is: make golden labeling antibody with collaurum and the combination of aflatoxin G 1 antibody, cohesive process is: be 1 x 10 by 50 mL concentration
-4the chlorauric acid solution of g/mL is heated to boiling, then add rapidly l.5 mL trisodium citrate, continue heating stirring reaction 15 min and obtain colloidal gold solution, then antibody-solutions is dropwise joined in colloidal gold solution with the ratio of 1:1 volume ratio, limit edged stirs, react after 5 min, obtain golden labeling antibody solution, by the golden labeling antibody furnishing A preparing
540=1.0 concentration, is dispersed on glass fiber filter, is gold mark pad after natural drying for subsequent use; Check-out console used is: with automatic stroke film instrument, nitrocellulose filter is detected to reagent linear coated, lower end horizontal line is coated with aflatoxin G 1 antigen A FG1-B SA, concentration is that 0.5 mg/mL is detection line, upper end horizontal line is coated with sheep anti-mouse igg, concentration is that 1 mg/mL is control line, the PBS solution of the pH 7.4 of the natural drying rear BSA with 0.l mol/L of nitrocellulose filter after coated detection line and control line soaks 30 min and seals processing, dry rear for subsequent use; Sample pad, gold mark pad, nitrocellulose filter and adsorptive pads four parts are mutually superposeed and are pasted in agent plate successively, survey check-out console to be aflatoxin G 1 sxemiquantitative speed after certain width slitting.
Direct extraction method: feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable.Take the sample that 3.0 g crush, add (the extract preparation: 50 mL methyl alcohol+50 mL water of 9 mL sample extracting solutions, add 4 g sodium chloride and mix and obtain sample extracting solution, can configure as required), fully be uniformly mixed (10 min), gentle aspiration supernatant after standing 20 min, carries out sample detection (this method can survey the feed of 30 μ g/kgs) after mixing in 1:1 ratio with sample dilution;
Dry/dry up facture: feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable.Take the sample that 3.0 g crush, add (the extract preparation: first will add 4 g sodium chloride with 20 mL water and dissolve of 9 mL sample extracting solutions, add again 80 mL methyl alcohol, obtain sample extracting solution, can configure as required), fully be uniformly mixed (3 min), leave standstill 20 min, gentle aspiration supernatant 1 mL, be put in 110 ℃ of baking ovens and dry that (approximately 1 h) or 50～60 ℃ of water-bath nitrogen dries up, and after fully dissolving, can carry out sample detection (this method can be surveyed the feed of 20 μ g/kg) with 0.5 mL 10% methyl alcohol.
Beneficial effect of the present invention is:
(1) detect aflatoxin G 1 in feed.The antibody that aflatoxin G 1 colloidal gold immunochromatographimethod check-out console provided by the invention uses is for aspergillus flavus resisting toxin G1 monoclonal antibody specific, for specificity fast detecting aflatoxin G 1;
(2) simple to operate.While detection with this aflatoxin G 1 colloidal gold immunochromatographimethod check-out console, only sample solution dropwise need be added in the sample pad of test strips, for single step operation, simple, convenient;
(3) testing process does not need aflatoxin G 1 standard solution as positive control.Aflatoxin G 1 colloidal gold immunochromatographimethod check-out console provided by the invention does not need to use aflatoxin G 1 standard solution as positive control while detecting sample, and only need be with blank sample as negative control;
(4) detection sensitivity is high.Aflatoxin G 1 colloidal gold immunochromatographimethod check-out console provided by the invention is limited to 20 ng/mL to the lowest detection of aflatoxin G 1 in sample.
Accompanying drawing explanation
Fig. 1: aflatoxin G 1 colloidal gold immunochromatographimethod check-out console outside drawing (wherein 1, get stuck, 2, nitrocellulose filter, 3, well, 4, nature controlling line, 5, detection line).
Fig. 2: test strips in aflatoxin G 1 check-out console, Fig. 2 a be positive, 2b be side (wherein 1, adsorptive pads, 2, nature controlling line, 3, detection line, 4, gold mark pads, 5, sample pad).
Fig. 3: aflatoxin G 1 sxemiquantitative colloidal gold immunochromatographimethod testing result (the wherein positive result of Fig. 3 a, Fig. 3 b is negative, Fig. 3 c is null result).
Embodiment
The preparation of aflatoxin G 1 sxemiquantitative collaurum fast testing plate is as described in technique scheme.
Sample pre-treatments:
(1) feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable;
(2) take the sample that 3.0 g crush, add 9 mL sample extracting solutions (extract preparation: 50 mL methyl alcohol+50 mL water, add 4 g sodium chloride and mix and obtain sample extracting solution, can configure as required), be fully uniformly mixed (10 min);
(3) gentle aspiration supernatant after standing 20 min, carries out sample detection after mixing in 1:1 ratio with sample dilution.
The using method of aflatoxin G 1 sxemiquantitative collaurum fast testing plate:
Slowly vertically drip 2 samples (approximately 50 μ L) with Dispette and, in well, after application of sample, start timing.After application of sample, be sure not mobile check-out console, 8-10 min sentence read result.
Testing result:
Negative (-): T line (detection line, near well one end) colour developing, shows in sample that aflatoxin G 1 concentration is lower than 30 μ g/kg or not containing aflatoxin G 1; Positive (+): T line, without colour developing, shows in sample that aflatoxin G 1 concentration is higher than 30 μ g/kg; Null result: not occurring C line (control line), may be that misoperation is applied new check-out console and retested.
Sample pre-treatments:
(1) feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable;
(2) take the sample that 3.0 g crush, add (the extract preparation: first will add 4 g sodium chloride with 20 mL water and dissolve, then add 80 mL methyl alcohol of 9 mL sample extracting solutions, obtain sample extracting solution, can configure as required), be fully uniformly mixed (3 min), leave standstill 20 min;
(3) gentle aspiration supernatant 1 mL, is put in 110 ℃ of baking ovens and dries that (approximately 1 h) or 50～60 ℃ of water-bath nitrogen dries up, and after fully dissolving, can carry out sample detection with 0.5 mL 10% methyl alcohol.
The using method of aflatoxin G 1 sxemiquantitative collaurum fast testing plate:
Slowly vertically drip 2 samples (approximately 50 μ L) with Dispette and, in well, after application of sample, start timing.After application of sample, be sure not mobile check-out console, 8-10 min sentence read result.
Testing result:
Negative (-): T line (detection line, near well one end) colour developing, shows in sample that aflatoxin G 1 concentration is lower than 20 μ g/kg or not containing aflatoxin G 1; Positive (+): T line, without colour developing, shows in sample that aflatoxin G 1 concentration is higher than 20 μ g/kg; Null result: not occurring C line (control line), may be that misoperation is applied new check-out console and retested.
Claims (3)
1. aflatoxin G 1 colloidal gold immunochromatographimethod check-out console, is characterized in that: in shell, comprise a test strips, the one side of test strips is pasted adsorptive pads, detecting pad, gold mark pad and sample pad, the overlapping connection in junction of adjacent each pad from top to bottom successively; Gold mark pad: make golden labeling antibody with collaurum and the combination of aflatoxin G 1 antibody; Cohesive process: be 1 x 10 by 50 mL concentration
-4the chlorauric acid solution of g/mL is heated to boiling, then add rapidly 1.5 mL trisodium citrates, continue heating stirring reaction 15 min and obtain colloidal gold solution, then antibody-solutions is dropwise joined in colloidal gold solution with the ratio of 1:1 volume ratio, limit edged stirs, react after 5 min, obtain golden labeling antibody solution, by the golden labeling antibody furnishing A preparing
540=1.0 concentration, is dispersed on glass fiber filter, is gold mark pad after natural drying for subsequent use; Check-out console used is: with stroke film instrument, nitrocellulose filter is detected to reagent linear coated, lower end horizontal line is coated with aflatoxin G 1 antigen A FG1-BSA, concentration is that 0.5 mg/mL is detection line, upper end horizontal line is coated with sheep anti-mouse igg, concentration is that 1 mg/mL is control line, the PBS solution of the pH 7.4 of the BSA of natural drying rear use 0.1 mol/L of nitrocellulose filter after coated detection line and nature controlling line soaks 30 min and seals processing, dry rear for subsequent use; Sample pad, gold mark pad, nitrocellulose filter and adsorptive pads four parts are mutually superposeed and are pasted in agent plate successively, survey check-out console to be aflatoxin G 1 sxemiquantitative speed after certain width slitting.
2. according to the preparation method of the aflatoxin G 1 immunochromatography check-out console described in claim 1, it is characterized in that: comprise the following steps:
(1) preparation of adsorptive pads
Thieving paper is cut out and is obtained adsorptive pads;
(2) preparation of detecting pad
Being coated with of detection line (T line):
The conjugate of aflatoxin G 1-bovine serum albumin(BSA) is mixed with to the solution A of 0.5 mg/mL; On apart from nitrocellulose filter along being the position of 10-15 mm, with automatically drawing film instrument, solution A is laterally coated in and on nitrocellulose filter, obtains detection line, on detection line, the package amount of the conjugate of required aflatoxin G 1-bovine serum albumin(BSA) (AFG1-BSA) is 215 ng/cm, then dry 5～20 min under 37～40 ℃ of conditions;
Being coated with of nature controlling line (C line):
Anti-rabbit mouse polyclonal antibody is mixed with to the solution B of 1 mg/mL; In the position apart from detection line 5～10 mm, with automatically drawing film instrument, solution B is laterally coated on nitrocellulose filter, obtain nature controlling line; On nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 300 ng/cm, then dry 5～20 min under 37～40 ℃ of conditions;
(3) preparation of gold mark pad
Make golden labeling antibody with collaurum and the combination of aflatoxin G 1 antibody, cohesive process is: be 1 x 10 by 50 mL concentration
-4the chlorauric acid solution of g/mL is heated to boiling, then add rapidly l.5 mL trisodium citrate, continue heating stirring reaction 15 min and obtain colloidal gold solution, then antibody-solutions is dropwise joined in colloidal gold solution with the ratio of 1:1 volume ratio, limit edged stirs, react after 5 min, obtain golden labeling antibody solution, by the golden labeling antibody furnishing A preparing
540=1.0 concentration, is dispersed on glass fiber filter, is gold mark pad after natural drying for subsequent use;
(4) assembling of check-out console
Select the test card shell matching with above-mentioned test strips, in test strips is inserted and got stuck, sample pad is just to well, and nitrocellulose filter is just to detection window.
3. the aflatoxin G 1 sxemiquantitative speed that as claimed in claim 1 prepared by method is surveyed the application process of check-out console, it is characterized in that:
Direct extraction method (can survey the feed of 30 μ g/kg)
(1) feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable;
(2) take the sample that 3.0 g crush, add 9 mL sample extracting solutions (extract preparation: 50 mL methyl alcohol+50 mL water, add 4 g sodium chloride and mix and obtain sample extracting solution, can configure as required), be fully uniformly mixed (10 min);
(3) gentle aspiration supernatant after standing 20 min, carries out sample detection after mixing in 1:1 ratio with sample dilution;
Dry/dry up facture (can survey the feed of 20 μ g/kg)
(1) feed, cereal, Fructus Hordei Germinatus sample are pulverized so that 75% sample can pass through 20 mesh sieves, size particles and the instant coffee of sample are suitable;
?(2) take the sample that 3.0 g crush, add (the extract preparation: first will add 4 g sodium chloride with 20 mL water and dissolve, then add 80 mL methyl alcohol of 9 mL sample extracting solutions, obtain sample extracting solution, can configure as required), be fully uniformly mixed (3 min), leave standstill 20 min;
?(3) gentle aspiration supernatant 1 mL, is put in 110 ℃ of baking ovens and dries that (approximately 1 h) or 50～60 ℃ of water-bath nitrogen dries up, and after fully dissolving, can carry out sample detection with 0.5 mL 10% methyl alcohol.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226503A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
| CN112946286A (en) * | 2021-03-08 | 2021-06-11 | 河南工业大学 | Two detection line immunochromatography test paper strips based on biotin-avidin system semi-quantitatively detect aflatoxin B1Preparation of |
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Patent Citations (5)
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| EP1787700A1 (en) * | 2005-11-17 | 2007-05-23 | Vicam, L.P. | Multi-analyte affinity column for analyzing aflatoxin, ochratoxin , zearalenone and deoxynivalenol |
| CN101109735A (en) * | 2007-05-14 | 2008-01-23 | 劲牌有限公司 | Fluorescence photometry for immune affinity column of aflatoxin in paddy |
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|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226503A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
| CN112946286A (en) * | 2021-03-08 | 2021-06-11 | 河南工业大学 | Two detection line immunochromatography test paper strips based on biotin-avidin system semi-quantitatively detect aflatoxin B1Preparation of |
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Application publication date: 20140514 |