CN202939174U - Colloidal gold reagent plate for rapidly detecting zearalenone in feed - Google Patents
Colloidal gold reagent plate for rapidly detecting zearalenone in feed Download PDFInfo
- Publication number
- CN202939174U CN202939174U CN 201220702189 CN201220702189U CN202939174U CN 202939174 U CN202939174 U CN 202939174U CN 201220702189 CN201220702189 CN 201220702189 CN 201220702189 U CN201220702189 U CN 201220702189U CN 202939174 U CN202939174 U CN 202939174U
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- Prior art keywords
- zearalenone
- feed
- pad
- colloidal gold
- sample
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- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 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 title claims abstract description 43
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 18
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Images
Abstract
The utility model relates to a colloidal gold reagent plate for rapidly detecting zearalenone in feed, which can be used to rapidly detect the zearalenone in the feed. The colloidal gold reagent plate for rapidly detecting the zearalenone in the feed consists of an upper plastic template, a lower plastic template and a backing, wherein the backing is orderly and closely pasted with a sample pad, a colloidal gold combined pad, a nitrocellulose membrane and a water absorbent pad. The nitrocellulose membrane is orderly sprayed with a detection line and a quality control line from the direction from the sample pad to the water absorbent pad, and the detection result of the zearalenone can be judged through naked eyes. The colloidal gold reagent plate for rapidly detecting the zearalenone in the feed can be semi-quantitatively and intuitively detected, the whole operation process just needs 15minutes, any expensive auxiliary experimental facilities are not needed, the colloidal gold reagent plate for rapidly detecting the zearalenone in the feed is beneficial for large sample screening, and is suitable to rapidly detect the zearalenone in the seed in large scale for industrial and commercial departments, inspection and quarantine institutions, farms and feed production and processing enterprises.
Description
Technical field
The utility model relates to the colloidal gold reagent plate of zearalenone in a kind of fast detecting feed, is specifically related to a kind of immune colloid gold quick detection reagent plate that detects zearalenone.
Background technology
Zearalenone (zearalenone, ZEN) claims again the F-2 toxin, at first separates obtaining from the corn that head blight is arranged.Its toxigenic bacterium is mainly the bacterium dwarf of Fusarium (Fusarium), as Fusarium graminearum (F.graminearum) and fusarium tricinctum (F.tricinctum).The thermotolerance of zearalenone is stronger, processes 1h under 110 ℃ and is just destroyed fully.Zearalenone mainly pollutes the cereal such as corn, wheat, rice, barley, millet and oat, and wherein the positive rate of corn is 45%, and the highest toxic amount can reach 2909mg/kg; The recall rate of wheat is 20%, and toxic amount is 0.364～11.05mg/kg.Zearalenone has estrogen action, and Main Function can make domestic animal, poultry and experiment mice produce the hyperfunction disease of female hormone in reproductive system.The edible food that contains zearalenone of gravidic animal (comprising the people) can cause miscarriage, stillborn foetus and monster.Edible contain the various wheaten food that Gibberella saubinetii flour makes and also can cause the toxicity symptom of central nervous system, as feel sick, feel cold, headache, mind depression and incoordination etc.
The harm of zearalenone has caused the attention of a lot of countries, and Countries has been formulated the limit standard of zearalenone in cereal.As the allowance of zearalenone in Australia regulation ox feed≤150 μ g/kg; Allowance in Russia's regulation cereal, vegetable oil, nut≤1000 μ g/kg; Allowance in France's regulation cereal, vegetable oil≤200 μ g/kg; Allowance in Brazil's regulation corn≤200 μ g/kg; Allowance in China GB13078.2-2006 " allowance of ochratoxin A and zearalenone in the forage health standard feed " regulation mixed feed, corn≤500 μ g/kg.
At present, generally take the methods such as thin layer chromatography, liquid and gas chromatography, euzymelinked immunosorbent assay (ELISA) that zearalenone is measured.The thin layer chromatography method is simple, but its sensitivity is lower; Although and the sensitivity of the detection methods such as liquid and gas chromatogram is very high, method is comparatively complicated, and is also very high to the requirement of instrument.In addition, also can analyze from molecular biological angle, the scope of mensuration is at 5～100 μ g/mL.Said method respectively has its advantage, but still can not satisfy for outdoor, Site Detection, therefore need to be by means of the immune colloidal gold chromatography technology.
Colloidal gold immunity chromatography is the immunology detection technology of a kind of simple and fast of setting up on the basis of immunity percolation technology, except the plurality of advantages that possesses euzymelinked immunosorbent assay (ELISA), has also overcome the some shortcomings of euzymelinked immunosorbent assay (ELISA).Whole or the major part that the method will be reacted needed raw material all has been incorporated in reagent, and reaction only needs several minutes to tens of minutes usually, and testing result is brought interpretation with macroscopic colour developing bar.This method has easy quick, simple to operate, high specificity, does not need the advantages such as extras, now has been widely used in the numerous areas of biotechnology.
The utility model content
The utility model conducts a research for the zearalenone in feed is residual, and a kind of immune colloid gold quick detection reagent plate for the positive feed sample of on-the-spot rapid screening zearalenone is provided.The utlity model has the characteristics such as simple, that quick, sensitivity is high, need not coordinate mensuration by instrument and equipment.Testing process is not subjected to site limitation, both can carry out in the laboratory, can carry out on-site measurement yet, and 15min can complete the left and right quantitative and semi-quantitative of zearalenone in sample is measured.
This agent plate is comprised of two of up and down plastic formwork and backing, is closely pasting successively sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on backing.Have in sample pad, collaurum pad, place that nitrocellulose filter is adjacent with adsorptive pads that 1～2mm's is overlapping, in order to guarantee that the chromatography effect is from sample pad carrying out smoothly to the adsorptive pads position on the one hand, in order to make sample solution and sample be lined with sufficient reaction time on the other hand, buffer system on sample pad can in and the potential of hydrogen of sample solution, guarantee that effective constituent in sample solution and the golden labeling antibody on the collaurum pad react smoothly.
Be coated with the conjugate of anti-ZEN monoclonal antibody and collaurum on the collaurum pad; Be coated with successively ZEN-OVA conjugate and sheep anti-mouse igg from sample pad to the adsorptive pads direction on nitrocellulose filter, respectively as detection line and nature controlling line.
The utility model agent plate is used chromatography type antibody mediated immunity competition principle, and by antigen and golden labeling antibody reaction solution, the zearalenone in the specific detection feed is residual.If sample solution contains residual, antibody response on zearalenone elder generation and colloid gold particle, therefore when colloid gold particle diffused to detection line with sample solution, on colloid gold particle, the avtive spot of antibody can't be combined by the zearalenone specific antigen on detection line because being occupied by the zearalenone in sample solution; When the zearalenone content in sample surpassed the agent plate detection limit, the detection line colour developing on agent plate was shallow even without colour developing than nature controlling line, is judged to be the positive.On the contrary, if in sample, zearalenone content is below the agent plate detection limit or during noresidue, the detection line colour developing on agent plate is close with nature controlling line or partially dark, is judged to be feminine gender.
Each several part constituent and the function of the utility model agent plate are as follows:
Plastic formwork plays fixedly test strips and referential function district (well, detection zone, Quality Control district).
Backing scribbles the toughness material that does not absorb water of adhesive sticker for one side, as the PVC plate, play fixing other ingredients of test paper of supporting.
Sample pad is made by glass fibre, works to absorb sample solution and buffering sample solution pH value.
The collaurum pad is made by polyester film, is marked with the conjugate of anti-zearalenone monoclonal antibody and collaurum on it, is the place that effective constituent in sample solution and golden labeling antibody react.
Cellulose nitrate membrane portions Main Function is with reaction result with macroscopic characterization out.
Adsorptive pads is filter paper, and its effect is that the mobile unnecessary solution that comes up is absorbed as the suction part.
The utility model agent plate has following beneficial effect:
(1) specificity is good, highly sensitive, detection limit is adjustable
The utility model agent plate is 100% to the cross reacting rate of ZEN, comprises that with the mycotoxin of other kinds aflatoxin, vomitoxin, volt horse mycin, ochracin etc. all do not have cross reaction.
The lowest detectable limit of the utility model agent plate can reach 100 μ g/kg, can satisfy national residue limits requirement fully.Demands different from the user, agent plate can be selected different detection limits by regulating the sample solution dilution ratio.
(2) easy and simple to handle, quick, little to the dependence of experimental facilities
The whole testing process of the utility model agent plate only needs 15min.Sample pre-treatments is simple, drips after sample in 3～5min with the naked eye reading result.
The utility model agent plate needs to use compact centrifuge in sample pretreatment process, does not relate to other instrument and equipments, is convenient to field and execute-in-place.
(3) cost is low, and is profitable
The utility model agent plate production technology is simple, and production cost is low, both can detect single sample, also is applicable to batch samples and detects, and greatly reduces testing cost.
Description of drawings
Fig. 1 is zearalenone immune colloid gold quick detection reagent plate structure schematic diagram, and wherein 1 is sample pad, and 2 is the collaurum pad, and 3 is nitrocellulose filter, and 4 is detection line, and 5 is nature controlling line, and 6 is adsorptive pads, and 7 is adhesive sticker, and 8 is the PVC plate.
Fig. 2 is zearalenone immune colloid gold quick detection reagent plate operation chart, and wherein S is well, and T is detection zone, and C is the Quality Control district.
Fig. 3,4,5 really judges schematic diagram for zearalenone immune colloid gold quick detection reagent hardens, and wherein T is detection zone, and C is the Quality Control district; The negative result of Fig. 3, the positive result of Fig. 4, Fig. 5 is invalid.
Embodiment
The preparation of the utility model agent plate comprises preparation, the preparation of colloidal gold solution, the preparation of the anti-ZEN monoclonal antibody of colloid gold label and the assembling of zearalenone immune colloid gold quick detection reagent plate of the preparation of zearalenone-carrier protein couplet thing, anti-ZEN monoclonal antibody.
1, the preparation of zearalenone-carrier protein couplet thing
1.1 the preparation of zearalenone oxime (ZENO)
2mg ZEN is dissolved in 200 μ L anhydrous pyridines, adds 4mg ethyloic azanol half hydrochloride, oscillating reactions 24h under room temperature, vacuum drying.Residue is dissolved in 100 μ L distilled water, transfer pH to 8.0 with 1mol/L NaOH, unreacted ZEN removes with the benzene extraction, add 0.1mol/L HCI to transfer the reaction product of pH to 3.0 precipitation aqueous phase, and with ethyl acetate extraction 5 times, collect the ethyl acetate after extracting, vacuumize drying, obtain the crude extract of ZENO.Above-mentioned residue is dissolved in 1mL methyl alcohol, and take ether: ethyl acetate (V: V=60: 40) as developping agent, carry out thin-layer chromatography and launch.Collection contains the silica gel of ZENO component, contains the silica gel of ZENO with methanol-eluted fractions, collects methyl alcohol and vacuumize drying to obtain ZENO.1.2ZEN-BSA the preparation with ZEN-OVA
Get 0.5mgZEN and be dissolved in 50 μ L pyridines, add 20 μ L 37% formalins, mixing.Get 3.6mgBSA and be dissolved in 200 μ L 0.5mol/LNaAc solution, ZEN solution slowly is added dropwise in BSA solution, the limit edged stirs, and under room temperature, stirring reaction spends the night.Coupled product 4 ℃ of dialysis 72h of distilled water, freeze drying ,-20 ℃ save backup.The ZEN-BSA that obtains is immunogene.
Adopting uses the same method carries out coupling with OVA and ZEN, and the ZEN-OVA that obtains is coating antigen.
2, the preparation of anti-ZEN monoclonal antibody
2.1 animal immune
Get the about female BALB/c mouse of 18～20g of 6～8 week body weight in age, first immunisation is with 100 μ g ZEN-BSA, with the complete freund adjuvant mixing of equivalent, lumbar injection.After 2 weeks, with 50 μ g ZEN-BSA, after the incomplete freund adjuvant mixing of equivalent, lumbar injection.After this all with 50 μ g ZEN-BSA, with the incomplete freund adjuvant mixing of equivalent, lumbar injection every 2.Immunity in the spleen after 8 weeks, 50 μ g ZEN-BSA are as booster immunization, and after 3d, extracting spleen cell merges.
2.2 Fusion of Cells
Immune mouse spleen cell mixes with 10: 1 volume ratios with murine myeloma cell (SP2/0), (PEG) makes fusion agent with 50% polyglycol, fused cell is suspended from the selection nutrient culture media that contains 20% calf serum, divide to plant and do to put 5%CO in trophoblastic 96 porocyte culture plates in being added with the BALB/c mouse peritoneal exudate cells
2, cultivate in 37 ℃ of incubators, when microscopy hybridoma clonal growth reaches 1/3～1/2 visual field, get supernatant and screen.
2.3 hybridoma screening and antibody test
Take ZEN-HSA as envelope antigen, with the cell hole of the anti-zearalenone antibody of indirect non-competing ELISA method screening secretion, with the checking of indirect competition inhibition ELISA method.With the positive contrast of the serum of immune mouse, the supernatant of cultivating take the SP2/0 cell is as blank, with the negative contrast of the cells and supernatant that does not grow the clone in Tissue Culture Plate, the criterion in positive cell hole is (A test-A is blank)/(A contrast-A blank) 〉=2.1.
2.4 monoclonal antibody production
Induce ascites in body: to the injection of the mouse peritoneal after lumbar injection whiteruss 10d cloning cell line 10
7Individual cell extracted ascites after 7 days.
3, the preparation of colloidal gold solution
Add 1mL 1% trisodium citrate in the 100mL deionized water, boil rear 1mL 1% gold chloride that adds rapidly, continue to boil 10min, cooling after, save backup under 4 ℃.Through experiment sieving, the particle diameter of measuring this colloid gold particle is about 40nm.
4, the preparation of the anti-ZEN monoclonal antibody of colloid gold label
Pipette the colloidal gold solution 100mL that has prepared, transfer pH to 8.0 with the 0.1mol/L solution of potassium carbonate.Add while stirring the anti-ZEN monoclonal antibody of 1mg, stir 20min, dropwise add 1mL 25mol/L PEG 20000 (PEG 20000), stir 15min.The centrifugal 15min of 20000r/min abandons supernatant, adds the PBS damping fluid (containing 0.4mol/L PEG) of 10mL pH 7.4 to clean 2 times.Precipitation is contained PBS damping fluid (pH 7.4) dissolving of 2%BSA with 10mL, after sterilizing filter filtered, 4 ℃ saved backup.
5, the assembling of zearalenone immune colloid gold quick detection reagent plate
With a film machine, the ZEN-OVA conjugate of debita spissitudo and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and nature controlling line, 37 ℃ of oven drying 8h.In kind, the anti-ZEN monoclonal antibody of the colloid gold label for preparing is coated on the collaurum pad.Detecting reagent set becomes a PVC backing, is stained with in order sample pad, collaurum pad, nitrocellulose filter and adsorptive pads thereon.After the test strips assembling is complete, oven dry in drying box.Then with cutting machine, the kilocalorie that posts is cut into the wide bar of 3.84mm, make the detection agent plate in the plastic formwork of packing into, then put into the aluminium foil bag sealed storage with drying agent.
6, zearalenone immune colloid gold quick detection reagent plate detects the implementation and operation method
6.1 determination
Get the 1g sample and be placed in the 5mL centrifuge tube, add the 4mL extraction agent, concuss 3min, the centrifugal 5min of 4000r/min.Get the upper solution of corresponding amount according to detectability in following table, then draw the special-purpose diluted of corresponding amount, after piping and druming evenly, to be checked.
| Detection limit (μ g/kg) | Upper solution (mL) | Special-purpose dilution (mL) |
| 100 | 0.1 | 0.1 |
| 200 | 0.1 | 0.3 |
| 250 | 0.1 | 0.4 |
| 500 | 0.05 | 1.0 |
| 750 | 0.02 | 0.6 |
| 1000 | 0.02 | 0.8 |
6.2 detecting step
The horizontal positioned check-out console is drawn sample solution 100 μ L to be checked and is added drop-wise in well, begins timing after application of sample; Result should read at 3～5min, and the other times interpretation is invalid.
6.3 result judgement
During reading result, the agent plate level is placed in the observer front.
Negative (-): T line colour developing (detection line is near well one end) than C line (control line, i.e. nature controlling line) deeply or equally dark, in the expression sample, the zearalenone residual content is lower than detectability or not contain zearalenone residual.
Positive (+): the colour developing of T line is more shallow than C line, or the T line is without colour developing, and in the expression sample, the zearalenone residual content is equal to or higher than detectability, and the colour developing of T line is more shallow than C line, and in the expression sample, zearalenone content is higher.
Invalid: the C line not occurring, may be that misoperation or agent plate lost efficacy.Should again read instructions, and retest with new agent plate.
Claims (4)
1. the colloidal gold reagent plate of zearalenone in a fast detecting feed, agent plate is comprised of two of up and down plastic formwork and backing, closely pasting successively sample pad, glue gold pad, nitrocellulose filter and adsorptive pads on backing, it is characterized in that having between sample pad, collaurum pad, nitrocellulose filter and adsorptive pads each several part that 1～2mm's is overlapping.
2. agent plate according to claim 1, it is characterized in that being sprayed with on the collaurum pad bond of anti-zearalenone monoclonal antibody and collaurum, be sprayed with successively ZEN-OVA conjugate and sheep anti-mouse igg from sample pad to the adsorptive pads direction on nitrocellulose filter, respectively as detection line and nature controlling line.
3. agent plate according to claim 1, is characterized in that backing is the PVC plate that one side scribbles adhesive sticker, and sample pad is made by glass fibre, and the collaurum pad is made by polyester film, and adsorptive pads is filter paper.
4. agent plate according to claim 2, is characterized in that the particle size of colloid gold particle is about 40nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220702189 CN202939174U (en) | 2012-11-30 | 2012-11-30 | Colloidal gold reagent plate for rapidly detecting zearalenone in feed |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220702189 CN202939174U (en) | 2012-11-30 | 2012-11-30 | Colloidal gold reagent plate for rapidly detecting zearalenone in feed |
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| CN202939174U true CN202939174U (en) | 2013-05-15 |
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| CN 201220702189 Expired - Lifetime CN202939174U (en) | 2012-11-30 | 2012-11-30 | Colloidal gold reagent plate for rapidly detecting zearalenone in feed |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849461A (en) * | 2014-02-14 | 2015-08-19 | 北京勤邦生物技术有限公司 | Test paper strip for detecting zearalenone and application of test paper strip |
| CN106397597A (en) * | 2016-09-14 | 2017-02-15 | 湖南沙博安科技有限责任公司 | Preparation method of F2 resisting egg yolk antibody |
| CN108226487A (en) * | 2016-12-22 | 2018-06-29 | 中粮集团有限公司 | Zearalenone detection card and the zearalenone detection method blocked using the detection |
-
2012
- 2012-11-30 CN CN 201220702189 patent/CN202939174U/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849461A (en) * | 2014-02-14 | 2015-08-19 | 北京勤邦生物技术有限公司 | Test paper strip for detecting zearalenone and application of test paper strip |
| CN106397597A (en) * | 2016-09-14 | 2017-02-15 | 湖南沙博安科技有限责任公司 | Preparation method of F2 resisting egg yolk antibody |
| CN108226487A (en) * | 2016-12-22 | 2018-06-29 | 中粮集团有限公司 | Zearalenone detection card and the zearalenone detection method blocked using the detection |
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| Date | Code | Title | Description |
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| C14 | Grant of patent or utility model | ||
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| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of utility model: Colloidal gold reagent plate for rapid detection of zearalenone in feed Effective date of registration: 20211026 Granted publication date: 20130515 Pledgee: Xiaoshan sub branch of Hangzhou United Rural Commercial Bank Co.,Ltd. Pledgor: HANGZHOU NANKAI BIOTECH Co.,Ltd. Registration number: Y2021330002066 |
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