CN106596955A - Rapid detection kit for pork, beef and mutton and preparation method of kit - Google Patents
Rapid detection kit for pork, beef and mutton and preparation method of kit Download PDFInfo
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- CN106596955A CN106596955A CN201611146453.0A CN201611146453A CN106596955A CN 106596955 A CN106596955 A CN 106596955A CN 201611146453 A CN201611146453 A CN 201611146453A CN 106596955 A CN106596955 A CN 106596955A
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- sheep
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a rapid detection kit for pork, beef and mutton and a preparation method of the kit. The rapid detection kit can rapidly identify types of the pork, the beef and the mutton. The rapid detection kit is characterized in that a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorbing pad are sequentially pasted on a test paper bottom plate, colloidal labeling special monoclonal antibodies 1 of a pig, a cattle and a sheep are adhered on the colloidal gold pad, and detection lines and quality control lines of the special monoclonal antibodies 2 of the pig, the cattle and the sheep wrap on the nitrocellulose membrane. The rapid detection kit has the advantages of simplicity in operation, rapidness, high sensitivity, high specificity and the like, and the kit is suitable for on-site rapid detection and identification of meat types.
Description
Technical field
The present invention is a kind of pork, beef and mutton quick detection kit and preparation method, belongs to immuno-chromatographic assay technology neck
Domain, it particularly relates to meat detection technique field.
Background technology
Food quality and safety is closely related with the existence of the mankind and health, is that people reach certain quality of life level
Basic demand, resident improves constantly the demand to nutrition and health needs high-quality meat as guarantee.Along with meat city
Drastically expansion, on the market the event of meat adulteration occur again and again, adulterated content becomes increasingly complex, and mode is more and more,
Economic problems are not only produced, What is more the serious consequence such as may damage to consumer.It now appear that, adulterated means
Mainly adulterate, mix the spurious with the genuine.Illegal enterprise adopts the meat raw materials such as relatively inexpensive Carnis Sus domestica, Carnis Gallus domesticus, by various sides
The deep processing of formula, pretends to be beef, Carnis caprae seu ovis product to be sold to try to gain illegal profit, the legal power of this consumer that constituted a serious infringement
Benefit.In China, the cattle and sheep roulade of personation is exposed by the media again and again.The shortage of effectively identification meat adulteration technology is puzzlement meat productss
One of difficult problem of security control.Traditional method carries out discriminating and depends on morphology and organoleptic examination to the introduces a collection of meat.Sense organ
Inspection limitation is big, accuracy is low, and finished meat ball etc. cannot be differentiated substantially.Therefore one is urgently needed currently on the market
Plant the high discrimination method of cheap, easily operated, accuracy to improve the accuracy of detection and the speed of inspection.Use technology
Means provide scientific basis for supervision department, hit the meat adulteration infringement being becoming increasingly rampant.Therefore this research is intended to set up one
Plant the method for being adapted to vast basic unit reviewer scene quick discriminating meat kind.
At present both at home and abroad main meat differentiates that modern technologies method is the PCR methods for determining DNA sequence, proteomics,
Spectrographic method, biomarker method, Electromagnetic Wave Method also has been reported that, but only PCR methods are widely used.Needed for above-mentioned every technology
Technical requirements height, expensive equipment, detection time high to personnel requirement it is long, be not appropriate for carrying out basic unit's inspection.At present in China
Most widely used method or sense organ discriminating, fully rely on the experience of trier.The error that sense organ differentiates is larger, easily by mistake
Sentence, therefore currently on the market in the urgent need to a kind of high discrimination method of cheap, easily operated, accuracy improving detection
Accuracy and inspection speed, provides scientific basis for supervision department with technological means, hit the meat adulteration being becoming increasingly rampant separated
Judicial act.
Immunochromatography technique is widely used in the multiple fields such as disease marker analyte detection, food inspection, scientific research.Using exempting from
Epidemic disease Chromatographic techniques differentiate meat species rarely seen research both at home and abroad, by extracting the specific proteinses and system that separate various meats
Standby polyclonal antibody, monoclonal antibody, prepare the technical characteristic that rapid immune detecting kit is this patent.
The content of the invention
Goal of the invention：A kind of pork, beef and mutton quick detection kit is provided, for the species of quick discriminating meat.
Technical scheme：
A kind of pork, beef and mutton quick detection kit, the test kit adopts immunochromatographic method, for quick discriminating pork, beef and mutton
Species, it is characterised in that：The test kit includes reagent paper base plate；The reagent paper base plate last time be pasted with sample pad, colloidal gold pad,
Nitrocellulose filter and adsorptive pads；
The colloidal gold pad adheres to pig, cattle, the respective monoclonal antibody specific 1 of sheep of colloidal label；
Detection line and the Quality Control of the respective monoclonal antibody specific 2 of pig, cattle, sheep are coated with described nitrocellulose filter
Line.
Described pork, beef and mutton quick detection kit, preferably：Described pig, cattle, the respective specific monoclonal of sheep
Antibody specific is to isolate and purify out that pig, beef or mutton are respective, be different from the specific proteinses of other two kinds of meat, then the list for preparing
Clonal antibody.
Described pork, beef and mutton quick detection kit, preferably：The pig of the colloidal label, cattle, sheep are respective special
Property monoclonal antibody 1 be specially gold colloidal difference labelling pig, cattle, the respective monoclonal antibody specific 1 of sheep, then be prepared into one
Colloidal gold pad.
Described pork, beef and mutton quick detection kit, preferably：The detection line be on same nitrocellulose filter,
Drawing film respectively successively has the monoclonal antibody 2 of pig, cattle, sheep.
The step of a kind of preparation method of pork, beef and mutton quick detection kit, preparation method is：
1）Colloidal gold solution is taken, pH value is adjusted as 7.6 with solution of potassium carbonate；
2）Pig, cattle, sheep monoclonal antibody 1, stirring is added to stand；
3）Deca BSA while stirring；
4）By gold labeling antibody low-temperature centrifugation obtained in above-mentioned steps, bulky grain is removed, remove the unmarked albumen in supernatant, used
Diluent redissolves；
5）The gold mark monoclonal antibody for preparing is sprayed on same polyester film with gold spraying instrument, is drying to obtain gold colloidal gold pad, it is standby
With；
6）Drawn successively on nitrocellulose filter with film instrument pig, cattle, sheep monoclonal antibody 2 is drawn, form three detections line；By rabbit-anti
Mus IgG is drawn on nitrocellulose filter, forms nature controlling line, is dried.
7）Gold colloidal gold pad, sample pad, nitrocellulose filter, adsorptive pads are adhered to successively on PVC backings；Nitric acid is fine
One end that the plain film of dimension draws detection line is connected with gold colloidal gold pad, and the gold colloidal gold pad other end is connected with sample pad, celluloid
Film is drawn one end of nature controlling line and is connected with absorbent paper, and test strips are cut into after the completion of stickup；The upper plastics of test strips pressure are got stuck, is loaded
In sealed plastic bag with desiccant, preparation is finished.
Described preparation method, preferably：In terms of 100ml colloidal gold solutions, pig, cattle, the sheep monoclonal anti of 0.5mg is added
Body 1；5% BSA of the mL of Deca 2, the diluent is the diluent of original volume 1/10.
Described preparation method, preferably：The step 5）In the gold mark monoclonal antibody spray of each 4x5mm gold colloidals gold pad
Measure as 400 ng.
Described preparation method, preferably：The step 6）In, by the pig of 1 mgmL -1, cattle, sheep monoclonal anti
Body 2 is drawn successively at the detection line of nitrocellulose filter with the amount of 1 μ Lcm -1；By the rabbit-anti Mus of 1 mgmL -1
IgG is drawn at the nature controlling line of nitrocellulose filter with the amount of 1 μ Lcm -1.
Advantage and effect：The art of this patent can make the identification and detection accuracy of meat greatly improve, improve detection efficiency,
Can be widely applied to on-site rapid inspection, for meat, roulade, meat ball, skewer Adulteration identification, strike is illegal adulterated, is
Supervision department provides technological means, and effective guarantee consumer's interests, social meaning is great.
The art of this patent can develop multiple meat immunity detection reagent, for the Rapid identification of various meats,
Food-safety problem serious today, have a extensive future.
Description of the drawings：
Fig. 1 is schematic diagram of the present invention；
Mark in figure：1 reagent paper base plate；2 sample pad；3 colloidal gold pads；4 nitrocellulose filters；5 adsorptive pads；6 detection lines；7 Quality Controls
Line.
Specific embodiment：
Every kind of animal flesh all contains multiple proteins, and because species are different, every kind of animal certainly exists and is different from other animals
Protein, i.e., containing its exclusive specific proteins.The fact that foundation, the specific proteinses of every kind of meat are found out, by mirror
Other this specific proteins, you can differentiate the species of meat.
By using SDS-PAGE electrophoresis, multiple specific proteinses of pig, cattle, sheep are found out.
By using being centrifuged, saltout, ultrafiltration, the technological means such as gel post separation, pig, cattle, sheep after purification is obtained respectively
The single albumen of specificity.
With specific proteinses, monoclonal antibody is prepared, respectively obtain pig, cattle, the sheep monoclonal antibody of pairing.
Gold colloidal difference labelling pig, cattle, sheep monoclonal antibody 1, are then mixed and made into gold pad.
On nitrocellulose filter, film pig, cattle, the detection line of sheep monoclonal antibody 2 are drawn successively respectively.
Assembling detection card.
As shown in the figure：
The test kit includes reagent paper base plate 1；It is pasted with sample pad 1, colloidal gold pad 3, cellulose nitrate on the reagent paper base plate 1 successively
Plain film 4 and adsorptive pads 5；
The colloidal gold pad 3 adheres to pig, cattle, the respective monoclonal antibody specific 1 of sheep of colloidal label；
The detection line 6 and matter of the respective monoclonal antibody specific 2 of pig, cattle, sheep are coated with described nitrocellulose filter
Control line 7.
Embodiment
With reference to embodiment, the present invention will be described in further detail, but protection scope of the present invention does not receive embodiment institute
Limit.
Embodiment
1st, colloid gold label pig, cattle, sheep monoclonal antibody：The colloidal gold solution 100ml for preparing is taken respectively adds beaker
In, determined by acidometer, adjust pH value as 7.6 with the solution of potassium carbonate of 0.2M, it is separately added into pig, cattle, the sheep monoclonal of 0.5mg
Antibody 1, magnetic agitation 30min.After stirring 30 min, 30 min are stood.The 5% of 2 mL are slowly added dropwise in stirring
BSA, stirs 30 min.By the min of 1000 rpm low-temperature centrifugations of gold labeling antibody 30, bulky grain is removed.12000 rpm are used again
1 h is centrifuged, the unmarked albumen in supernatant is removed.Finally the precipitation diluent of original volume 1/10 redissolves, and obtains final product immunity
Gold colloidal,
2nd, the preparation of gold pad：With gold spraying instrument by the gold mark monoclonal antibody for preparing according to every kind of each gold pad（4×5 mm）400
The amount of ng, is sprayed on same polyester film, is drying to obtain gold colloidal gold pad, standby.
3rd, the preparation of nitrocellulose filter：With stroke film instrument by the pig of 1 mgmL -1, cattle, sheep monoclonal antibody 2 with 1
The amount of μ Lcm -1 is drawn successively in nitrocellulose filter（NC films）T lines at, formed three T lines；By 1 mgmL -1
Rabbit anti-mouse igg with the amount of 1 μ Lcm -1 draw in nitrocellulose filter（NC films）C lines at, be dried after it is stand-by.
4th, test strips assembling
The gold pad handled well, sample pad, NC films, adsorptive pads are adhered to successively on PVC backings, NC films are drawn into the one of T lines
End is connected with gold pad, and the gold pad other end is connected with sample pad, and NC films are drawn one end of C lines and are connected with absorbent paper, after the completion of stickup
Cut into the test strips of 4 × 60 mm.The upper plastics of test strips pressure are got stuck, in being fitted into the sealed plastic bag with desiccant, i.e.,
.
Inspection checking
The each 1g of pig, beef or mutton is taken respectively, in putting centrifuge tube, is blended with hand mixer, it is each to add extracting solution 3ml, vibrate 1 point
Clock, it is static after take supernatant, instill well.Either it is individually added into or mixing is added, can obtains correct result.
Claims (8)
1. a kind of pork, beef and mutton quick detection kit, the test kit adopts immunochromatographic method, for quick discriminating pork, beef and mutton
Species, it is characterised in that：The test kit includes reagent paper base plate（1）；The reagent paper base plate（1）On be pasted with sample pad successively
（2）, colloidal gold pad（3）, nitrocellulose filter（4）And adsorptive pads（5）；
The colloidal gold pad（3）Pig, cattle, the respective monoclonal antibody specific 1 of sheep of attachment colloidal label；
The detection line of the respective monoclonal antibody specific 2 of pig, cattle, sheep is coated with described nitrocellulose filter（6）And
Nature controlling line（7）.
2. pork, beef and mutton quick detection kit according to claim 1, it is characterised in that：Described pig, cattle, sheep is each
Monoclonal antibody specific be specially isolate and purify out the specific protein that pig, beef or mutton are respective, be different from other two kinds of meat
In vain, then the monoclonal antibody for preparing.
3. pork, beef and mutton quick detection kit according to claim 1, it is characterised in that：The pig of the colloidal label,
The respective monoclonal antibody specific 1 of cattle, sheep is specially gold colloidal difference labelling pig, cattle, the respective specific monoclonal of sheep and resists
Body 1, then it is prepared into a colloidal gold pad.
4. pork, beef and mutton quick detection kit according to claim 1, it is characterised in that：The detection line（6）Be
On same nitrocellulose filter, drawing film respectively successively has the monoclonal antibody 2 of pig, cattle, sheep.
5. the preparation method of pork, beef and mutton quick detection kit as claimed in claim 1, it is characterised in that：The preparation method
The step of be：
1）Colloidal gold solution is taken, pH value is adjusted as 7.6 with solution of potassium carbonate；
2）Pig, cattle, sheep monoclonal antibody 1, stirring is added to stand；
3）Deca BSA while stirring；
4）By gold labeling antibody low-temperature centrifugation obtained in above-mentioned steps, bulky grain is removed, remove the unmarked albumen in supernatant, used
Diluent redissolves；
5）The gold mark monoclonal antibody for preparing is sprayed on same polyester film with gold spraying instrument, is drying to obtain gold colloidal gold pad, it is standby
With；
6）Drawn successively on nitrocellulose filter with film instrument pig, cattle, sheep monoclonal antibody 2 is drawn, form three detections line；By rabbit-anti
Mus IgG is drawn on nitrocellulose filter, forms nature controlling line, is dried；
7）Gold colloidal gold pad, sample pad, nitrocellulose filter, adsorptive pads are adhered to successively on PVC backings；By celluloid
Film is drawn one end of detection line and is connected with gold colloidal gold pad, and the gold colloidal gold pad other end is connected with sample pad, and nitrocellulose filter is drawn
One end of nature controlling line is connected with absorbent paper, and test strips are cut into after the completion of stickup；The upper plastics of test strips pressure are got stuck, loads band dry
In the sealed plastic bag of drying prescription, preparation is finished.
6. preparation method according to claim 5, it is characterised in that：In terms of 100ml colloidal gold solutions, add 0.5mg's
Pig, cattle, sheep monoclonal antibody 1；5% BSA of the mL of Deca 2, the diluent is the diluent of original volume 1/10.
7. preparation method according to claim 5, it is characterised in that：The step 5）In each 4x5mm gold colloidal gold pad
Gold mark monoclonal antibody discharge rate is 400 ng.
8. preparation method according to claim 5, it is characterised in that：The step 6）In, by 1 mgmL's -1
Pig, cattle, sheep monoclonal antibody 2 are drawn successively at the detection line of nitrocellulose filter with the amount of 1 μ Lcm -1；By 1
The rabbit anti-mouse igg of mgmL -1 is drawn at the nature controlling line of nitrocellulose filter with the amount of 1 μ Lcm -1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611146453.0A CN106596955A (en) | 2016-12-13 | 2016-12-13 | Rapid detection kit for pork, beef and mutton and preparation method of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611146453.0A CN106596955A (en) | 2016-12-13 | 2016-12-13 | Rapid detection kit for pork, beef and mutton and preparation method of kit |
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| Publication Number | Publication Date |
|---|---|
| CN106596955A true CN106596955A (en) | 2017-04-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611146453.0A Pending CN106596955A (en) | 2016-12-13 | 2016-12-13 | Rapid detection kit for pork, beef and mutton and preparation method of kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593935A (en) * | 2018-05-08 | 2018-09-28 | 湖南汉康生物医药有限公司 | A kind of inflammation quickly detects colloidal gold strip |
| CN110195112A (en) * | 2019-04-11 | 2019-09-03 | 中国农业大学 | Pig derived components rapid detection method and kit in a kind of food |
| CN111537723A (en) * | 2020-05-12 | 2020-08-14 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chicken doped in beef and mutton |
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| CN103487576A (en) * | 2013-09-27 | 2014-01-01 | 无锡市产品质量监督检验中心 | Quick test-paper detection method for porcine-derived material adulteration |
| CN203479792U (en) * | 2013-10-08 | 2014-03-12 | 北京六角体科技发展有限公司 | Meat identifying infiltration card |
| CN204241478U (en) * | 2014-12-11 | 2015-04-01 | 北京智峰博泰生物科技有限公司 | For detecting the immuno-chromatographic test paper strip whether being mixed with certain animal flesh in meat |
-
2016
- 2016-12-13 CN CN201611146453.0A patent/CN106596955A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103487576A (en) * | 2013-09-27 | 2014-01-01 | 无锡市产品质量监督检验中心 | Quick test-paper detection method for porcine-derived material adulteration |
| CN203479792U (en) * | 2013-10-08 | 2014-03-12 | 北京六角体科技发展有限公司 | Meat identifying infiltration card |
| CN204241478U (en) * | 2014-12-11 | 2015-04-01 | 北京智峰博泰生物科技有限公司 | For detecting the immuno-chromatographic test paper strip whether being mixed with certain animal flesh in meat |
Non-Patent Citations (1)
| Title |
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| 唐穗平 等: "广东省牛羊肉及其制品中掺杂掺假情况的调查分析", 《食品安全质量检测学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593935A (en) * | 2018-05-08 | 2018-09-28 | 湖南汉康生物医药有限公司 | A kind of inflammation quickly detects colloidal gold strip |
| CN110195112A (en) * | 2019-04-11 | 2019-09-03 | 中国农业大学 | Pig derived components rapid detection method and kit in a kind of food |
| CN111537723A (en) * | 2020-05-12 | 2020-08-14 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chicken doped in beef and mutton |
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Application publication date: 20170426 |