CN101231290A - Kit and method for quantitative determination of ochratoxin A content in food product - Google Patents
Kit and method for quantitative determination of ochratoxin A content in food product Download PDFInfo
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- CN101231290A CN101231290A CNA2007100565581A CN200710056558A CN101231290A CN 101231290 A CN101231290 A CN 101231290A CN A2007100565581 A CNA2007100565581 A CN A2007100565581A CN 200710056558 A CN200710056558 A CN 200710056558A CN 101231290 A CN101231290 A CN 101231290A
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Abstract
The invention belongs to the immunity analysi technical field, in particular to a reagent box for qualitatively detecting the content of ochratoxin A in food and the detecting method thereof. The reagent box comprises a test paper strip and reagent, wherein, the test paper strip is of a solid support which takes a joining enveloped with the ochratoxin A and egg-albumin as a detecting area A of the test pater, and takes a goat anti-rabbit secondary antibody as an effective standard area B of the test paper strip. The invention has the advantages of simple operation, high detecting sensitivity, strong specificity, good veracity, low detecting cost, better stability, etc., in addition, the range of application is wider. The invention is mainly used for the qualitative detection of the content of the ochratoxin A in the food and the provision.
Description
Technical field
The invention belongs to the immuno analytical method field, ochratoxin A content kit and detection method thereof in especially a kind of qualitative detection food.
Background technology
Ochratoxin is that the toxic metabolite product that is produced behind grain, food, feed and other agricultural byproducts is infected in some toxigenic bacterium strain in Aspergillus ochraceus and the Penicillium notatum.Ochratoxin A is a kind of that the ochratoxin toxic is the strongest, output is the highest, and humans and animals is had strong toxic action, can cause nephrarctia, fetal anomaly, miscarriage, the death of animal, and has the carcinogenicity of height.OTA exists in most of cereal, mainly comprises barley, wheat, oat, corn and coffee etc.Simultaneously, the poultry of raising with these cereal also can be polluted.So ochratoxin is another toxin that causes World Focusing behind aflatoxin.1993, international cancer research institute (The International Agency for Researchon Cancer) was decided to be 2B group carcinogen (may be human carcinogenic substance) with ochratoxin.Because ochratoxin A is to the toxic and side effect of human body, various countries have formulated the limit standard of ochratoxin A in the cereal one after another, the 56th FAO/WHO food additives joint specialist executive session carried out hazard assessment to OTA and formulated that OTA maximum residue limit standard is 5 μ g/kg in the cereal foods, and China and European Union and member state thereof are defined in also that maximum residue limit is 5 μ g/kg in the cereal.
The assay method of present ochratoxin A has multiple, as: thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrography methods such as (LC/MS).What wherein, research both at home and abroad and application were more is the HPLC method, but this method pre-treatment process is loaded down with trivial details, complicated operation, and need the professional to operate usually, the instrument and equipment costliness, testing process is consuming time and expense is higher, is unsuitable for the field quick detection of a large amount of samples.Enzymoimmunoassay ELISA is owing to its high specificity, and highly sensitive, accuracy is good, and is easy and simple to handle, and the advantages such as detection that are suitable for batch samples more and more are subjected to people's favor.Though the scientific research personnel of a lot of scientific research institutions and universities and colleges studies ochratoxin A enzyme-linked immuno assay detection method, the sensitivity that detects and the stable aspect of kit also have certain distance from practical application.
Immobilon-p immunoassays (solid phase membrane-based immuoassay) are similar with solid-phase enzyme immunoassay (ELISA), are characterized in microporous barrier as solid phase carrier.Label available enzyme and various coloured particulate are as color latex, collaurum, electroselenium etc., the most commonly used with the collaurum of redness.The characteristics of immobilon-p are its poriness, as filter paper.Immobilon-p can be passed outflow by liquid, and liquid also can be divided a word with a hyphen at the end of a line forward on film by capillarity.Solid phase carrier film commonly used is cellulose nitrate (nitrocellulose, NC) film, nylon (nylon) film etc.Colloid gold label immunoassay technology (immunogold labelling technique) be with collaurum as the trace labelling thing, be applied to a kind of novel immunolabelling technique of antigen-antibody reaction.Kind of the commercialization colloidal gold kit of having had an appointment both at home and abroad at present surplus in the of 20 is sold, detect test paper, gonorrhoea detection box etc. as modal very early pregnancy, but except that drugs and excitant, the research of the residue detection aspect of other micromolecular compound and the rarely found report of application detect almost blank for micromolecular biotoxin.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of simple to operate, detection sensitivity is high, high specificity, accuracy are good, detect that cost is low, stability ochratoxin A content kit and detection method thereof in the qualitative detection food preferably.
The present invention is achieved through the following technical solutions:
Ochratoxin A content kit in a kind of qualitative detection food, be made of test strips and reagent set, it is characterized in that: this test strips is to be coated with ochratoxin A and ovalbumin connector as this test strips detection zone A and goat-anti rabbit two anti-solid phase carriers as this test strips validity standard regions B.
And described solid phase carrier is a nitrocellulose filter.
And described reagent set comprises:
(1) dilution: for containing the pH9.0 phosphate buffer of methyl alcohol and tween; Dilute according to 1: 5 with secondary water during use;
(2) colloid gold label antibody 0.5mL.
And the point sample amount of described ochratoxin A and ovalbumin connector is 0.5-2 μ g, and the test strips specification is 1.0-2.2cm * 4.5-9.0cm, and the width in A, two zones of B equates, is all 3-5mm, and two zones of A, B are at a distance of 10-15cm.
A kind of detection method of using ochratoxin A content kit in the qualitative detection food: may further comprise the steps:
(1) sample pre-treatments;
(2) get colloidal gold mark test paper, golden labelled antibody and sample diluting liquid were mixed in 1: 5 by volume, after competitive reaction 8-12 minute, draw 100 these reaction mixtures of μ L and join and be coated with haptenic zone;
(3) centre between the zone of the detection line on the control stripes bar and effective control line zone drips the blank mixed liquor of 100 μ L, in test strip with control stripes bar validity control zone redness all occurs and surveyed area occurs under the situation of red lines, if the comparison of test strip surveyed area color is shallow according to test strips, contain ochratoxin A in the interpret sample.
And described sample pre-treatments is that sample powder essence is powdered, 40-60 ℃ of oven dry in drying box, and sample thief adds dilution, and ultrasonic extraction 20min is centrifugal, gets supernatant.
Beneficial effect of the present invention and advantage are:
1. the present invention has simple to operate, characteristics such as detection sensitivity is high, high specificity, accuracy is good, the detection cost is low, stability is better, and the scope of application is wider.Mainly be applicable to the qualitative detection of the ochratoxin A content in food and the grain.
2. method for quick provided by the invention is easy and simple to handle, quick, and it is short to finish whole detecting operation process times, and the degree of accuracy height that detects, and detection sensitivity can reach μ g/kg level and be fit to very much the on-the-spot needs that detect.
3. the present invention is with low cost, detection efficiency is high, easy and simple to handle, not only have an economic benefit but also social benefit is arranged, be ochratoxin A content kit and detection method thereof in the higher qualitative detection food that has a good application prospect of a kind of novelty.
Description of drawings
Fig. 1 the present invention gold mark test paper is to the testing result of variable concentrations OTA.
Embodiment
The present invention is described in further detail by following examples, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Ochratoxin A content kit in a kind of qualitative detection food, constitute by test strips and reagent set, wherein this test strips is to be coated with ochratoxin A and ovalbumin connector as this test strips detection zone A and goat-anti rabbit two anti-solid phase carriers as this test strips validity standard regions B, this solid phase carrier is a nitrocellulose filter, and the point sample amount of ochratoxin A and ovalbumin connector is 1 μ g, the test strips specification is 1.0 * 4.5cm, the width in A, two zones of B equates, be all 5mm, two zones of A, B are at a distance of 10cm.
Concrete operations are as follows:
(on the 1.5cm * 10cm), the point sample amount is 1 μ g, every long-pending 1 μ L of point sample belt body, every point sample belt length 5mm in nitrocellulose filter with the connector point sample of OTA-OVA with semi-automatic point sample instrument.Quality control band (control line) bag is anti-with the goat-anti rabbit two of 50 times of PBS dilutions by 0.5-1 μ L.Then film is cut with a knife and be slit into a certain size (1.5cm * 1.0cm).Earlier fixing 15min in 37 ℃ of constant temperature ovens is immersed in 1%BSA/PBS sealing 30min again.Film after the sealing with dilution flushing three times, is blotted redundant moisture with filter paper, after 37 ℃ of dryings, 4 ℃ of airtight preservations down.
(preparation of colloid gold label antibody is referring to No. 200510013371.4 patented claims).
Dilution: be pH 9.0 phosphate buffers that contain methyl alcohol and tween; Dilute according to 1: 5 with secondary water during use;
Use the detection method of ochratoxin A content kit in the qualitative detection food: may further comprise the steps:
(1) sample pre-treatments: sample powder essence is powdered, 50 ℃ of oven dry in drying box, sample thief adds dilution, ultrasonic extraction 20min, centrifugal, it is standby to get supernatant;
(2) get colloidal gold mark test paper, golden labelled antibody and sample diluting liquid were mixed in 1: 5 by volume, competitive reaction is after 10 minutes, draws 100 these reaction mixtures of μ L and joins and be coated with haptenic zone;
(3) centre between the zone of the detection line on the control stripes bar and effective control line zone drips the blank mixed liquor of 100 μ L, in test strip with control stripes bar validity control zone redness all occurs and surveyed area occurs under the situation of red lines, if the comparison of test strip surveyed area color is shallow according to test strips, contain ochratoxin A in the interpret sample.
Below by an instantiation, effect of the present invention is narrated:
The qualitative detection of the ochratoxin A content in the barley:
(1). sample pre-treatments: barley is powdered through the abundant powder essence of comminutor, 50 ℃ of oven dry in drying box ,-20 ℃ store for future use.Get each 5g of barley sample respectively, in various mark-on samples, add the 10mL dilution respectively, ultrasonic Extraction 20min, centrifugal, it is standby to get supernatant.
(2). get colloidal gold mark test paper, golden labelled antibody and sample diluting liquid were mixed in 1: 5 by volume, after the competitive reaction 10 minutes, drawing 100 these reaction mixtures of μ L joins and is coated with haptenic zone, centre between the zone of the detection line on the control stripes bar and effective control line zone drips the blank mixed liquor of 100 μ L, observe the relatively color of surveyed area, redness all appears in test strip and control stripes bar validity control zone, and surveyed area occurs under the situation of red lines, the color that compares two test strips surveyed areas, if the comparison of test strip surveyed area color is shallow according to test strips, illustrates in the barley sample and contain ochratoxin A.
The mark-on test:
In the mark-on test, in sample, add the ochratoxin A of variable concentrations respectively, with the abundant mixing of mark-on sample, room temperature standing over night.All the other processing procedures are the same.In detection to the barley sample, respectively 2.5,5 and the 10ng level carry out the mark-on recovery test, visual observation all is positive, and gradient is obvious.
Principle of work of the present invention:
Adopt the colloid gold label quick detection test paper bar qualitative detection ochratoxin A in the immunoassay detection technique, the primary dcreening operation that is used for sample, bag is anti-by ochratoxin A-ovalbumin (OA) and goat-anti rabbit two on nitrocellulose filter, with ochratoxin A antibody is immune detection antibody, collaurum is as the detection reaction label, adopt indirect competitive immunological detection method, by the mode that the reaction mixture vertical current is crossed or chromatography spreads, finish immune response at short notice, the testing result that acquisition can Direct observation.And the present invention mainly adopts the immunoassay detection technique to detect ochratoxin A, to reach purpose to the ochratoxin A qualitative detection in food and the cereal crops, and kit is simple in structure, easy to use, inexpensive, highly sensitive, and detection sensitivity can reach μ g/kg level.
Claims (6)
1. ochratoxin A content kit in the qualitative detection food, be made of test strips and reagent set, it is characterized in that: this test strips is to be coated with ochratoxin A and ovalbumin connector as this test strips detection zone A and goat-anti rabbit two anti-solid phase carriers as this test strips validity standard regions B.
2. ochratoxin A content kit in the qualitative detection food according to claim 1 is characterized in that: described solid phase carrier is a nitrocellulose filter.
3. ochratoxin A content kit in the qualitative detection food according to claim 1, it is characterized in that: described reagent set comprises:
(1) dilution: for containing the pH9.0 phosphate buffer of methyl alcohol and tween; Dilute according to 1: 5 with secondary water during use;
(2) colloid gold label antibody 0.5mL.
4. ochratoxin A content kit in the qualitative detection food according to claim 1, it is characterized in that: the point sample amount of described ochratoxin A and ovalbumin connector is 0.5-2 μ g, the test strips specification is 1.0-2.2cm * 4.5-9.0cm, the width in A, two zones of B equates, be all 3-5mm, two zones of A, B are at a distance of 10-15cm.
5. detection method of using ochratoxin A content kit in the qualitative detection food as claimed in claim 1: may further comprise the steps:
(1) sample pre-treatments;
(2) get colloidal gold mark test paper, golden labelled antibody and sample diluting liquid were mixed in 1: 5 by volume, after competitive reaction 8-12 minute, draw 100 these reaction mixtures of μ L and join and be coated with haptenic zone;
(3) centre between the zone of the detection line on the control stripes bar and effective control line zone drips the blank mixed liquor of 100 μ L, in test strip with control stripes bar validity control zone redness all occurs and surveyed area occurs under the situation of red lines, if the comparison of test strip surveyed area color is shallow according to test strips, contain ochratoxin A in the interpret sample.
6. the detection method of ochratoxin A content kit in the qualitative detection food according to claim 5, it is characterized in that: described sample pre-treatments is for powdered with sample powder essence, 40-60 ℃ of oven dry in drying box, sample thief adds dilution, ultrasonic extraction 20min, centrifugal, get supernatant.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100565581A CN101231290A (en) | 2007-01-25 | 2007-01-25 | Kit and method for quantitative determination of ochratoxin A content in food product |
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| CNA2007100565581A CN101231290A (en) | 2007-01-25 | 2007-01-25 | Kit and method for quantitative determination of ochratoxin A content in food product |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN103575886A (en) * | 2012-07-20 | 2014-02-12 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof |
| CN103728451A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN104459062A (en) * | 2014-12-08 | 2015-03-25 | 北京市理化分析测试中心 | Magnetic immunochromatographic kit for detecting ochratoxin A and preparation method of magnetic immunochromatographic kit |
| CN104569399A (en) * | 2013-10-22 | 2015-04-29 | 北京勤邦生物技术有限公司 | Test strip for testing ochratoxin A and application of test strip |
| CN105793707A (en) * | 2013-11-29 | 2016-07-20 | 积水医疗株式会社 | Immunochromatography-assisted detection method |
| CN106353493A (en) * | 2016-09-21 | 2017-01-25 | 成都测迪森生物科技有限公司 | Ochratoxin test rod |
| CN106771255A (en) * | 2017-01-23 | 2017-05-31 | 四川迈克生物科技股份有限公司 | Immuno-chromatographic test paper strip and detection kit for detecting CRP concentration |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN101881770B (en) * | 2009-05-08 | 2013-07-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN103575886A (en) * | 2012-07-20 | 2014-02-12 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof |
| CN104569399A (en) * | 2013-10-22 | 2015-04-29 | 北京勤邦生物技术有限公司 | Test strip for testing ochratoxin A and application of test strip |
| CN104569399B (en) * | 2013-10-22 | 2016-09-21 | 北京勤邦生物技术有限公司 | A kind of test strips detecting ochratoxin A and application thereof |
| CN105793707A (en) * | 2013-11-29 | 2016-07-20 | 积水医疗株式会社 | Immunochromatography-assisted detection method |
| CN103728451A (en) * | 2014-01-23 | 2014-04-16 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN103728451B (en) * | 2014-01-23 | 2015-06-24 | 湖南大学 | Colloidal gold immunoassay kit for detecting ochracin A and preparation method of kit |
| CN104459062A (en) * | 2014-12-08 | 2015-03-25 | 北京市理化分析测试中心 | Magnetic immunochromatographic kit for detecting ochratoxin A and preparation method of magnetic immunochromatographic kit |
| CN106353493A (en) * | 2016-09-21 | 2017-01-25 | 成都测迪森生物科技有限公司 | Ochratoxin test rod |
| CN106771255A (en) * | 2017-01-23 | 2017-05-31 | 四川迈克生物科技股份有限公司 | Immuno-chromatographic test paper strip and detection kit for detecting CRP concentration |
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Application publication date: 20080730 |