CN101458252A - Dairy antibiotic rapid detection kit - Google Patents
Dairy antibiotic rapid detection kit Download PDFInfo
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- CN101458252A CN101458252A CNA2008101642043A CN200810164204A CN101458252A CN 101458252 A CN101458252 A CN 101458252A CN A2008101642043 A CNA2008101642043 A CN A2008101642043A CN 200810164204 A CN200810164204 A CN 200810164204A CN 101458252 A CN101458252 A CN 101458252A
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Abstract
The invention discloses a rapid detection kit for antibiotics in a dairy product. The kit comprises a detection kit which is provided with a chromatographic film, a cleaning solution, a developing solution and absorbent paper as well as a sample tube which contains an enzyme-labeled antigen, wherein, the chromatographic film is coated with emulsion of an antibiotic antibody and emulsion of a control antibody. The detection kit is formed by assembling a movable component and a base. As the detection kit is provided with the cleaning solution and the developing solution, the detection can be completed only by adding samples and then pressing the movable component without manually adding a reagent, thus greatly improving convenience and reliability of the detection and reducing error probability; reaction is performed in the kit by chromatography, which causes a reactant to be capable of fully contacting the antibody, thus being beneficial to improving the reaction efficiency and the detection sensitivity and shortening the detection time.
Description
Technical field
The present invention relates to field of biological detection, especially the food safety detection field.
Background technology
Milk cow is owing to the diseases such as mastitis very easily takes place of often milking, and injection of antibiotics is the most direct at present effective treatment means.And after giving sick beef injection medicine, have a large amount of antibiotic residues in the milk of secretion.Even after drug withdrawal, Determination of antibiotic in milk content still can continue to exceed standard in a period of time.If the milk that the long-term drinking microbiotic exceeds standard can make the passive acceptance of normal person, accumulation microbiotic, cause the Human Physiology disorder, microbiotic is produced drug resistance.European and American countries i.e. legislation is many years ago forbidden the milk listing of the antibiotic residue amount of transfiniting, and antibiotic content is up to standard to have become general international raw material milk acquisition criteria.
The detection method of antibiotic residue roughly is divided three classes in the dairy products at present: bioassay method, physico-chemical analysis method and immunization.Be described below respectively:
1. microorganism detection method
The microorganism detection method is to use method more widely, its measuring principle is to the physiological function of microorganism, the inhibiting effect of metabolism according to microbiotic, antimicrobial agents is residual in next qualitative or quantitatively definite sample, as Delvotest method, paper disk method (PD), TTC method, swab method (STOP) etc.Delvotest method and TTC method are two kinds of milk Chinese traditional medicine residue detection microorganism detection methods commonly used.
The Delvotest method: its principle is to add thermophilic brood cell bacterium in milk sample, can produce acid after cultivating 2.5～3 hours under 64 ℃ of conditions, and acid causes that indicator BCP (bromcresol purple) becomes yellow.If do not contain microbiotic in the milk sample, cultivate the back sample and be yellow; As containing microbiotic in the sample, thermophilic brood cell bacterium growth is suppressed and can't produces acid, and indicator is with nondiscolouring.
The TTC method: i.e. triphenyl tetrazolium chloride method (tripheye tetrazolium chloride) is the detection method (GB5409-85) of antibiotic residue in the inspection milk of stipulating in present China food hygienic standard.Its principle is that streptococcus thermophilus can be with colourless 2,3 when growing in milk, and (2,3,5-Triphenyltetrazolium Chloride TTC) is reduced to red triphen first a ceremonial jade-ladle, used in libation to 5-triphenyltetrazolium chloride.When having antibacterial materials such as microbiotic in the milk, can suppress the growth of streptococcus thermophilus, TTC then can't be reduced.
The advantage of microorganism detection method is, equipment, reagent cost are low; Can detect the microbiotic of wide spectrum.Shortcoming is: 1. the time long, usually need to wait for more than 3 hours; 2. the color status judgement is distinguished by naked eyes, easily produces error, can't make accurate judgement; 3. complicated operation requires operating personnel that the strict working specification of observing in certain professional knowledge and the experimentation need be arranged; 4. detect and disturbed easily, cause the result false positive or false negative to occur.
2. physics and chemistry detection method
The physics and chemistry detection method is to utilize special reaction that group had or character in the antibiotic molecule to measure its content, comprises high performance liquid chromatography, vapor-phase chromatography, mass spectroscopy or the like, and is wherein commonly used with high performance liquid chromatography (HPLC) method.
High performance liquid chromatography (HPLC): be a kind of physics and chemistry detection method of present widespread use, utilize the special reaction that group had in the antibiotic molecule to measure its content.It has introduced the gas chromatography theory, has adopted high-pressure pump technically, and efficient stationary phase and high sensitivity detector have realized that velocity of separation is fast, efficient is high and operation automation.Nearly all compound comprises high polarity/ion-type determinand and macromolecular substances, and all available HPLC measures.Testing process generally will be through the extraction of sample, take off steps such as albumen, centrifugal, chromatographic column purifications, derivatization, the concrete content of energy detection of antibiotics, and susceptibility is higher.
Other physics and chemistry method that is used for the dairy antibiotic residue detection also comprises vapor-phase chromatography (GC), high performance thin layer chromatography (HPTLC), supercritical fluid chromatography (SFC) and capillary zone electrophoresis method (CZE) etc.Although these methods are of little use,, can remedy weak point in the common method because of its distinctive separately performance in residue detection.The strong detecting device of many highly sensitive, versatilities or selectivity is arranged for selecting for use as GC, as hydrogen flame ionization detector (FID), chlorine P detector (NPD) etc., detectability can reach μ g/kg level; Quantitative, the qualitative and high efficiency separation material (Φ 3～10 μ m) of the former bit scan of the spot of HPTLC has changed the deficiency of conventional TLC aspect sensitivity and reappearance, but easy, the quick and big advantage of sample capacity that has kept TLC, can use positive or anti-phase plate, resolution is almost suitable with HPLC, is being widely used aspect rapid screening inspection 5 surveys of antibiotic residue; SFC can remedy the deficiency of GC and HPLC, connects the detecting device (MS, ECD etc.) of various sensitivities easily; CZE has been one of most active analytical technology of research since the nineties, high speed, high-resolution and the HPLC that has high-voltage power supply concurrently flexibly, advantage efficiently.
The advantage of physics and chemistry method is to carry out qualitative, quantitative and drug identification, and susceptibility is higher.Shortcoming is but the trace routine complexity that expense is higher, needs to buy expensive detection equipment, is not suitable for small-sized control laboratory.
3. immunization
Immunoassay (immunoassays, IAs) can be divided into euzymelinked immunosorbent assay (ELISA) (EIA), put the method for exempting from (RIA) etc., be to utilize between antibody and the antigen specific immune response to carry out qualitative or detection by quantitative.At the comparatively common immunodetection of dairy antibiotic detection range ELISA method and gold mark chromatography etc. are arranged at present.
The ELISA method: be coated with adding sample and enzyme-labelled antigen on 96 orifice plates of antibody, the microbiotic that contains in the sample (antigen) is competed limited antibody combining site on the plate with enzyme-labelled antigen.Antigenic content in the sample is high more, and is just many more with the amount of antibodies, and the binding capacity of enzyme-labelled antigen is just low more, and promptly antigenic content is inversely proportional to the enzyme labelled antibody content that combines in the sample.Add colour developing liquid, antigenic content is inversely proportional in the colour developing depth and the sample.The advantage of ELISA method is the detection sensitivity height, but accurate quantification.
Gold mark chromatography: add sample and gold mark antigen on the film of antibody being coated with, the microbiotic that contains in the sample (antigen) and enzyme-labelled antigen utilize capillary action by the antibody on the film and be hunted down.Antigenic content in the sample is high more, and is just many more with the amount of antibodies, and the binding capacity of gold mark antigen is just low more, and the colour developing of antibody location is just shallow more, and antigenic content is inversely proportional in the depth that promptly develops the color and the sample.The advantage of gold mark method is that reaction velocity is fast, and a few minutes can go out the result usually.But can only qualitatively judge usually, aspect sensitivity, have shortcoming simultaneously.
The advantage that immuno analytical method is the most outstanding is simple to operate, and speed is fast, analysis cost is low.Be example with the ELISA that uses 96 orifice plates, the immunoassay sampling amount is little, and pre-treatment is simple, capacity is big, and the instrumentation degree is low, and it is similar to GC/MS or GC/ECD to detect milk, can reach the ng/ml level easily, and analysis efficiency then is more than tens times of HPLC or GC.When being detection by quantitative, shortcoming need operating personnel to possess certain professional knowledge and skill; Need detection time simultaneously more than 1 hour, needs go out the needs of report immediately in the time of can not satisfying milk corporate buyout fresh milk fully; And gold mark chromatography can only qualitatively judge usually, and the result is stable inadequately, has shortcoming simultaneously aspect sensitivity.Present most of microbiotic has been set up immunoassay, as sulfamethazine, chloromycetin, streptomysin, tetracycline, coban etc.
Summary of the invention
Defective at prior art exists the purpose of this invention is to provide a kind of easy, quick, stable dairy antibiotic detection kit based on the enzyme linked immunological chromatographic technique.
Dairy antibiotic rapid detection kit of the present invention, comprise: have the detection box of chromatographic film, cleansing solution, colour developing liquid and thieving paper and the sample hose that includes enzyme-labelled antigen, said chromatographic film is coated with the latex of antibiotic antibody, the latex of control antibodies;
Wherein, the antibiotic antibody latex that is coated on the chromatographic film comprises: penicillins antibody, cynnematin antibody-like, aminoglycoside antibody-like, sulfamido antibody, Tetracyclines antibody, chloramphenicol antibody, aflatoxins antibody
Enzyme-labelled antigen comprises: penicillins antigen, cephalosporins antigen, aminoglycoside antigen, sulfamido antigen, Tetracyclines antigen, chloromycetin antigen, aflatoxins antigen
Colour developing liquid: 0.02%TMB, 1.5%PVP, 0.75%DMSO, 0.1% dextran sulfate, 0.015%H
2O
2, pH5.0.
Cleansing solution: 0.01MTris-HCl, 0.5%NaCl, 0.1%Tween-20, pH6.9
The preparation method of dairy antibiotic rapid detection kit comprises the steps:
1) get pure antibiotic marker horseradish peroxidase ,-80 ℃ of preservations are stand-by;
2) get above-mentioned steps 1) microbiotic-horseradish peroxidase solution of mark, be divided in the sample hose freeze-drying;
3) get antibiotic antibody, the control antibodies bag latex that becomes commercialized is sprayed on the check point and the control point position of chromatographic film with the latex of antibody sandwich respectively with point sample instrument, 37 ℃ of oven dry 1 hour;
4) get the blue dyes aqueous solution, be sprayed on chromatographic film with point sample instrument and be positioned at the position of detecting the box indicating window, oven dry;
5) preparation cleansing solution, colour developing liquid are respectively charged into the relevant position of detecting box.Wherein cleansing solution is 300ul, and colour developing liquid is 700ul.220 ℃ of sealings by fusing behind the covering aluminium foil;
6) the suction rod of in detecting the suction pin of box, packing into, the chromatographic film of in the movable component that detects box, packing into; Detecting the box base relevant position thieving paper of packing into.At last with movable component and base assembling.
Advantage of the present invention is summarized as follows:
1) adopts the euzymelinked immunosorbent assay (ELISA) principle, improve detection sensitivity;
2) adopt the chromatography mode to carry out, reactant (comprising the antigen in the sample, the substrate in the colour developing liquid etc.) can fully be contacted with antibody, improve reaction efficiency, further improve the sensitivity that detects, shorten detection time;
3) be provided with control point on the check point next door, come judged result, can realize fast detecting by the colour developing depth that compares both.
Prepackage cleansing solution and the colour developing liquid step that can simplify the operation in detecting box.The user need not manual liquid feeding when detecting, as long as press movable component, cleansing solution and colour developing liquid can be participated in reaction successively; Reduce the possibility of misoperation: avoid occurring leaking the possibility that adds or add solution by mistake.
Utilize the present invention to detect to comprise fresh milk, sour milk, water-soluble dairy products, other contains whey solution etc.
Description of drawings
Fig. 1 is the vertical view that detects box in the dairy antibiotic rapid detection kit;
1. well; 2. reading window; 3. control point; 4. check point; 5. indicating window; 6. button
Fig. 2 is the side view (original state) that detects box in the dairy antibiotic rapid detection kit;
1. well; 2. reading window; 3. control point; 4. check point; 5. indicating window; 6. button; 7. chromatographic film; 8. the suction rod 1; 9. the suction rod 2; 10. suction pin; 11. movable component; 12. base; 13. thieving paper; 14. cleansing solution; 15. colour developing liquid; 16. aluminium foil
Fig. 3 is the side view (color status) that detects box in the dairy antibiotic rapid detection kit.
Embodiment
Below further specify the present invention for example.
With reference to accompanying drawing, dairy antibiotic rapid detection kit, comprise: have chromatographic film 7, cleansing solution 14, colour developing liquid 15 and the detection box of thieving paper 13 and the sample hose that includes enzyme-labelled antigen, said chromatographic film is coated with the latex of antibiotic antibody, the latex of control antibodies;
Wherein, the antibiotic antibody latex that is coated on the chromatographic film comprises: penicillins antibody, cynnematin antibody-like, aminoglycoside antibody-like, sulfamido antibody, Tetracyclines antibody, chloramphenicol antibody, aflatoxins antibody
Enzyme-labelled antigen comprises: penicillins antigen, cephalosporins antigen, aminoglycoside antigen, sulfamido antigen, Tetracyclines antigen, chloromycetin antigen, aflatoxins antigen
Colour developing liquid: 0.02%TMB, 1.5%PVP, 0.75%DMSO, 0.1% dextran sulfate, 0.015%H
2O
2, pH5.0.
Cleansing solution: 0.01MTris-HCl, 0.5%NaCl, 0.1%Tween-20, pH6.9
The preparation of embodiment 1. dairy products beta-lactam quick detection kit
1. get pure penicillin 5mg, add 200ul0.01M PBS (pH7.2) dissolving.Add 4mg horseradish peroxidase (HRP) and 1.5mg cyclohexyl carbodiimide, 37 ℃ were reacted 2 hours.Reaction product to 0.01M PBS (pH7.2) dialysis 3 times, is removed free penicillin.-80 ℃ of preservations are stand-by;
2. get above-mentioned penicillin-HRP solution, add the 0.01M PBS (pH7.2) that contains 10% trehalose and be diluted to about 2ug/ml, add the mouse-anti BSA-HRP of some again, mix, be divided in the sample hose freeze-drying;
3. get the 100ul latex, the EDC that adds the 20mg/ml of 900ul0.1M pH6.2 NaH2PO4 solution, new preparation, add penicillin antibody and control antibodies and wrap quilt, bag is sprayed on the check point and the control point position of chromatographic film by good latex respectively with point sample instrument, 37 ℃ of oven dry 1 hour;
4. get 0.01% blue dyes aqueous solution, be sprayed on chromatographic film with point sample instrument and be positioned at the position of detecting the box indicating window, oven dry;
The preparation cleansing solution (0.01MTris-HCl, 0.5%NaCl, 0.1%Tween-20, pH6.9) and the colour developing liquid (contain 0.02%TMB, 1.5%PVP, 0.75%DMSO, 0.1% dextran sulfate, 0.015%H
2O
2, pH5.0), be respectively charged into the relevant position of detection kit.Wherein cleansing solution is 300ul, and colour developing liquid is 700ul.220 ℃ of sealings by fusing behind the covering aluminium foil;
6. the suction of packing in the suction pin that detects box is excellent, the chromatographic film of packing in the movable component that detects box; Detecting the box base relevant position thieving paper of packing into.At last with movable component and base assembling.
The present invention adopts chromatographic film as solid phase carrier, and the latex particle of specific antibody bag quilt is fixed on the chromatographic film surface.The enzyme labeling thing is kept in the sample hose with lyophilized form, the cleansing solution and the colour developing liquid that react required then have been contained in the detection box in advance, press the button that detects box one side, cleansing solution and colour developing liquid can flow through reaction zone automatically by capillary action, finish washing and development step.After the colour developing, the reading window 2 spots (check point and control point) occur and gets final product judged result by the depth that compares both colors.Both can also can utilize instrument to read the colour developing depth, obtain semiquantitative numerical value by the direct judged result of range estimation.
A feature of the present invention is to detect to have pre-installed cleansing solution and the colour developing liquid that reacts volume required in the box.During reaction, at first the determinand (as milk) with certain volume adds in the sample hose, and the wherein enzyme labeling thing of freeze-drying dissolving mixes with milk.Then biased sample is added in the well 1 of detection kit, sample infiltrates chromatographic film 7 and also utilizes capillary action to spread, the control point 3 by being coated with antibody and the detection zone of check point 4.Limited binding site on determined antigen in the milk and the enzyme mark microbiotic antigenic competition check point position antibody, the antibody (or antigen) on the control point is caught corresponding enzyme mark thing simultaneously.When sample arrives indicating window 5, press the button 6, movable component one side descends, and suction rod 8,9 punctures aluminium foil 16, and the chromatographic film of well one end contacts with thieving paper 13 simultaneously.Cleansing solution 14 that pre-install the aluminium foil below and colour developing liquid 15 arrive chromatographic film 7 by suction pin 10 successively and also finally are absorbed to thieving paper one end motion along chromatographic film.Cleansing solution will be not by the materials such as enzyme-labelled antigen of antibodies flush away from the film, the liquid that develops the color then reacts by detection zone and with the enzyme that is combined on control point 3 and the check point 4, the TMB in the colour developing liquid is oxidized to be blue.
1. add the 400ul milk sample to sample hose, shake up;
2. will contain the sample hose of milk and detect box place together 45 ℃ 5 minutes;
3. the milk in the sample hose is poured in the well that detects box;
4. the button of movable component one end appears pressing when mobile when solution diffusion to indicating window, dyestuff spot;
5. after waiting for 4 minutes, by range estimation or readout instrument judged result.
During range estimation, when the check point color was deeper than control point, the result was negative in prompting, and microbiotic does not exceed standard in the milk; When the check point color was shallower than control point, the result was positive in prompting, and microbiotic exceeds standard in the milk.When adopting readout instrument, when reading less than 1.05 the time, the result is negative in prompting, microbiotic does not exceed standard in the milk; When reading greater than 1.05 the time, the result is positive in prompting, microbiotic exceeds standard in the milk.
Claims (2)
1, dairy antibiotic rapid detection kit, it is characterized in that comprising: have the detection box of chromatographic film, cleansing solution, colour developing liquid and thieving paper and the sample hose that includes enzyme-labelled antigen, said chromatographic film is coated with the latex of antibiotic antibody, the latex of control antibodies;
Wherein, the antibiotic antibody latex that is coated on the chromatographic film comprises: penicillins antibody, cynnematin antibody-like, aminoglycoside antibody-like, sulfamido antibody, Tetracyclines antibody, chloramphenicol antibody, aflatoxins antibody
Enzyme-labelled antigen comprises: penicillins antigen, cephalosporins antigen, aminoglycoside antigen, sulfamido antigen, Tetracyclines antigen, chloromycetin antigen, aflatoxins antigen
Colour developing liquid: 0.02%TMB, 1.5%PVP, 0.75%DMSO, 0.1% dextran sulfate, 0.015%H
2O
2, pH5.0.
Cleansing solution: 0.01MTris-HCl, 0.5%NaCl, 0.1%Tween-20, pH6.9.
2, the preparation method of dairy antibiotic rapid detection kit according to claim 1 is characterized in that comprising the steps:
1) get pure antibiotic marker horseradish peroxidase ,-80 ℃ of preservations are stand-by;
2) get above-mentioned steps 1) microbiotic-horseradish peroxidase solution of mark, be divided in the sample hose freeze-drying;
3) get antibiotic antibody, the control antibodies bag latex that becomes commercialized is sprayed on the check point and the control point position of chromatographic film with the latex of antibody sandwich respectively with point sample instrument, 37 ℃ of oven dry 1 hour;
4) get the blue dyes aqueous solution, be sprayed on chromatographic film with point sample instrument and be positioned at the position of detecting the box indicating window, oven dry;
5) preparation cleansing solution, colour developing liquid are respectively charged into the relevant position of detecting box.Wherein cleansing solution is 300ul, and colour developing liquid is 700ul.220 ℃ of sealings by fusing behind the covering aluminium foil;
6) the suction rod of in detecting the suction pin of box, packing into, the chromatographic film of in the movable component that detects box, packing into; Detecting the box base relevant position thieving paper of packing into.At last with movable component and base assembling.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101642043A CN101458252A (en) | 2008-12-31 | 2008-12-31 | Dairy antibiotic rapid detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101642043A CN101458252A (en) | 2008-12-31 | 2008-12-31 | Dairy antibiotic rapid detection kit |
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| Publication Number | Publication Date |
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| CN101458252A true CN101458252A (en) | 2009-06-17 |
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| CNA2008101642043A Pending CN101458252A (en) | 2008-12-31 | 2008-12-31 | Dairy antibiotic rapid detection kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103399008A (en) * | 2013-07-30 | 2013-11-20 | 刘岩 | Rapid detection kit for gutter oil |
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN106885802A (en) * | 2015-12-16 | 2017-06-23 | 范石军 | The real-time monitoring system of the antibiotic residue of sewage discharge in a kind of breeding process |
| CN109085342A (en) * | 2018-09-14 | 2018-12-25 | 武汉伊莱瑞特生物科技股份有限公司 | A kind of ELISA Sample dilution and preparation method improving detection sensitivity |
| CN110286110A (en) * | 2019-07-29 | 2019-09-27 | 邓鑫 | A kind of hepatocarcinoma early diagnosis kit |
| CN110333345A (en) * | 2019-06-28 | 2019-10-15 | 浙江大学山东工业技术研究院 | A kind of magnetic focusing rapid detection system with elution function |
-
2008
- 2008-12-31 CN CNA2008101642043A patent/CN101458252A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN103524427B (en) * | 2012-07-03 | 2017-02-08 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN103399008A (en) * | 2013-07-30 | 2013-11-20 | 刘岩 | Rapid detection kit for gutter oil |
| CN106885802A (en) * | 2015-12-16 | 2017-06-23 | 范石军 | The real-time monitoring system of the antibiotic residue of sewage discharge in a kind of breeding process |
| CN109085342A (en) * | 2018-09-14 | 2018-12-25 | 武汉伊莱瑞特生物科技股份有限公司 | A kind of ELISA Sample dilution and preparation method improving detection sensitivity |
| CN110333345A (en) * | 2019-06-28 | 2019-10-15 | 浙江大学山东工业技术研究院 | A kind of magnetic focusing rapid detection system with elution function |
| CN110286110A (en) * | 2019-07-29 | 2019-09-27 | 邓鑫 | A kind of hepatocarcinoma early diagnosis kit |
| CN110286110B (en) * | 2019-07-29 | 2024-03-19 | 邓鑫 | Early diagnosis kit for liver cancer |
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Effective date of registration: 20090508 Address after: Hangzhou City, Zhejiang province high tech Industrial Development Zone Waterfront Road 1192 Applicant after: Hangzhou Bo sun Technology Co., Ltd. Co-applicant after: Zhejiang Institute of Quality Inspection and Technical Research Address before: Hangzhou City, Zhejiang province high tech Industrial Development Zone Waterfront Road 1192 Applicant before: Hangzhou BIOER Technology Co., Ltd. |
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Application publication date: 20090617 |