CN106526175B - Tetracyclines detection method and detection card in a kind of poultry - Google Patents
Tetracyclines detection method and detection card in a kind of poultry Download PDFInfo
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- CN106526175B CN106526175B CN201611078778.XA CN201611078778A CN106526175B CN 106526175 B CN106526175 B CN 106526175B CN 201611078778 A CN201611078778 A CN 201611078778A CN 106526175 B CN106526175 B CN 106526175B
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- tetracyclines
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- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 239000004098 Tetracycline Substances 0.000 title claims abstract description 33
- 235000019364 tetracycline Nutrition 0.000 title claims abstract description 33
- 150000003522 tetracyclines Chemical class 0.000 title claims abstract description 31
- 244000144977 poultry Species 0.000 title claims abstract description 23
- 229940040944 tetracyclines Drugs 0.000 title claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims description 35
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 18
- 235000013372 meat Nutrition 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 14
- BEGBSFPALGFMJI-UHFFFAOYSA-N ethene;sodium Chemical group [Na].C=C BEGBSFPALGFMJI-UHFFFAOYSA-N 0.000 claims description 13
- 239000003480 eluent Substances 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims description 6
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 235000006408 oxalic acid Nutrition 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 6
- 238000005341 cation exchange Methods 0.000 claims description 5
- 241001411320 Eriogonum inflatum Species 0.000 claims 1
- 230000009514 concussion Effects 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 238000003908 quality control method Methods 0.000 abstract description 19
- 238000010521 absorption reaction Methods 0.000 abstract description 18
- 229960002180 tetracycline Drugs 0.000 abstract description 17
- 229930101283 tetracycline Natural products 0.000 abstract description 17
- 238000006243 chemical reaction Methods 0.000 abstract description 14
- 230000001681 protective effect Effects 0.000 abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 3
- 229920002678 cellulose Polymers 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 8
- 239000003814 drug Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 5
- 229960001484 edetic acid Drugs 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 229910052708 sodium Inorganic materials 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- -1 tetracycline compound Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000007705 chemical test Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses Tetracyclines detection method in a kind of poultry and detection cards; including micropore reagent and test paper; the test paper includes reaction film, sample absorption pad, water absorption pad, protective film and bottom plate; the lower surface of the sample absorption pad is fixedly connected with reaction film; the lower surface of the reaction film is fixedly connected with water absorption pad; the lower surface of the water absorption pad is fixedly connected with protective film, and the lower surface of the protective film is fixedly connected with bottom plate, has detection zone and quality control region on the reaction film.The present invention provides Tetracyclines detection cards in a kind of poultry, high specificity of the present invention, sensibility is high, meets the limit detection requirement of China's tetracycline, realizes the quick detection of tetracycline residue, detection time is greatly saved, testing result is can determine that in 5min for Site Detection, it is easy to be quick, it can quickly detect the content of tetracycline in poultry, it ensures food safety, reduces harm of the metopycide to human body.
Description
Technical field
The present invention relates to Tetracyclines detection method and detections in poultry detection technique field more particularly to a kind of poultry
Card.
Background technology
Tetracycline medication has effects that the sterilization antibiosis of broad-spectrum high efficacy, can be mixed with most drugs or feed addictive
With being widely used in Production of Livestock and Poultry.But it is excessively used and inevitably causes serious medicament residue, become serious
Endanger one of the food safety hazards of human health.Countries in the world detection remaining to tetracycline medication thus is extremely paid close attention to.
Tentatively provide that the total amount of the tetracycline compound in muscle and milk must not exceed 100 μ g/ in the 675/92nd command of European Union
Kg must not exceed 600 μ g/kg, 300 μ g/kg and 200 μ g/kg respectively in kidney, liver and egg.Japanese Positive List System
The total amount that metric determines tetracycline compound in milk and egg must not exceed 100ng/mL and 400ng/mg respectively.
In the remaining detection method of tetracycline medication Physico-chemical tests mainly have thin-layered chromatography capillary electrophoresis, efficiently
Liquid chromatography, chemoluminescence method and liquid chromatography-mass spectrometry etc..Biological detection method is broadly divided into microbial method
With immunoassay etc..But the instrument and equipment of these method some need costlinesses, needs skilled practitioner to operate, operated
Journey is complicated, needs the time longer, limits its scope of application, it is difficult to be promoted and applied in produce reality.Immune test paper method has
Sxemiquantitative and certain quantitation capabilities, can provide the preliminary information of determinand, and the method high sensitivity, analytic process is simple, is
The detection technique first developed is needed, in order to detect the content of tetracycline in poultry.
Invention content
Technical problems based on background technology, the present invention propose Tetracyclines detection method and inspection in a kind of poultry
Card is surveyed, has the characteristics that analytic process simple, high sensitivity and easy to operate, the content for solving to detect tetracycline in poultry wants ripe
Experienced professional's operation, operating process is complicated, needs time longer problem.
The present invention provides the following technical solutions：Tetracyclines detection card in a kind of poultry, including micropore reagent and test paper, institute
It includes reaction film, sample absorption pad, water absorption pad, protective film and bottom plate to state test paper, and the lower surface of the sample absorption pad, which is fixed, to be connected
It is connected to reaction film, the lower surface of the reaction film is fixedly connected with water absorption pad, and the lower surface of the water absorption pad is fixedly connected with guarantor
The lower surface of cuticula, the protective film is fixedly connected with bottom plate, has detection zone and quality control region on the reaction film, and detect position
In the side of quality control region, detection zone is coated with tetracycline hapten-carrier protein conjugate, and quality control region is coated with antiantibody, institute
Stating freeze-drying in micropore reagent has tetracycline medication specific antibody-colloid gold label object.
Preferably, the bottom plate is PVC board, and the sample absorption pad is suction strainer paper, and the water absorption pad is blotting paper, described
Reaction film is nitrocellulose filter, and the protective film is PE material protective films.
Preferably, it is printed on MAX mark lines on the protective film.
Tetracyclines detection method in a kind of poultry, includes the following steps：
S1, birds meat is cleaned, after mincing, weighs 5-10g birds meats and be placed in the centrifuge tube of 50mL, 2- is added
Protein in the trichloroacetic acid removal meat of the 0.01mol/L of 4mL, on liquid blending device after vortex mixed 1-2min, crosses 0.2 μm
Filter membrane.
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle
Corking tightening seal vibrates 8-10min with oscillator.
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and ethylenediamine tetra-acetic acid
The pH value of sodium buffer solution is 4-6, centrifuge tube is placed in vortex mixed 1-2min on liquid blending device, then vibrate 8- with oscillator
10min centrifuges 10-12min, and by supernatant in 100mL volumetric flasks, sodium ethylene diamine tetracetate buffer solution 20mL is added in residue,
It repeats to extract once, then is loaded into 100mL volumetric flasks.
S4, it is taken in 0.6mL supernatants to small glass with suction pipe, small glass is put into 30-45 DEG C of insulating box, it will
Moisture in supernatant is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid.
S5, detection liquid is exchanged with the flow velocity of 3-5mL/min by activated carboxylic acid type cation under reduced pressure
Column washes column with 5mL methanol, discards whole effluxes after eluent all outflow, under the negative pressure of 65kPa, decompressing and extracting 5-
8min, then eluted with 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors, eluent is collected in conical flask.
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX lines are printed on
End downward, is allowed to be sufficiently submerged in solution, after being incubated 5min at 20-25 DEG C of room temperature, takes out test strips, judges result.
Preferably, in S3, in centrifugal process, the power of centrifugation is 1000r/min.
Preferably, in S2 and S3, during earthquake, the power of oscillator is 800-1000W.
The present invention provides metopycide detection method and kits in a kind of poultry, have following beneficial to effect
Fruit：
The present invention provides Tetracyclines detection cards in a kind of poultry, and high specificity of the present invention, sensibility is high, meets China
The limit detection requirement of tetracycline, realizes the quick detection of tetracycline residue, detection time is greatly saved, for scene inspection
It surveys and can determine that testing result in 5min, it is easy quickly quickly to detect the content of tetracycline in poultry, ensure food peace
Entirely, harm of the metopycide to human body is reduced.
Specific implementation mode
Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
The present invention provides a kind of technical solution：Tetracyclines detection card in a kind of poultry, including micropore reagent and test paper, examination
Paper includes reaction film, sample absorption pad, water absorption pad, protective film and bottom plate, and bottom plate is PVC board, and sample absorption pad is suction strainer paper, is inhaled
Water cushion is blotting paper, and reaction film is nitrocellulose filter, and protective film is PE material protective films, is printed on MAX mark lines on protective film,
The lower surface of sample absorption pad is fixedly connected with reaction film, and the lower surface of reaction film is fixedly connected with water absorption pad, under water absorption pad
Surface is fixedly connected with protective film, and the lower surface of protective film is fixedly connected with bottom plate, there is detection zone and quality control region on reaction film, and
Detection zone is located at the side of quality control region, and detection zone is coated with tetracycline hapten-carrier protein conjugate, and quality control region is coated with anti-
Antibody, in micropore reagent freeze-drying have tetracycline medication specific antibody-colloid gold label object.
In the following, technical scheme of the present invention is described in detail by specific embodiment.
Embodiment 1
Tetracyclines detection method in a kind of poultry, includes the following steps：
S1, birds meat is cleaned, after mincing, weighs 5g birds meats and be placed in the centrifuge tube of 50mL, be added 2mL's
Protein in the trichloroacetic acid removal meat of 0.01mol/L, on liquid blending device after vortex mixed 1min, crosses 0.2 μm of filter membrane.
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle
Corking tightening seal vibrates 8min with oscillator, and during earthquake, the power of oscillator is 800-1000W.
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and ethylenediamine tetra-acetic acid
The pH value of sodium buffer solution is 4, centrifuge tube is placed in vortex mixed 1min on liquid blending device, then vibrate 8-10min with oscillator,
During earthquake, the power of oscillator is 800-1000W, centrifuges 10min, in centrifugal process, the power of centrifugation is
1000r/min, by supernatant in 100mL volumetric flasks, in residue plus sodium ethylene diamine tetracetate buffer solution 20mL, repetition extract one
It is secondary, then be loaded into 100mL volumetric flasks.
S4, it is taken in 0.6mL supernatants to small glass, small glass is put into 30 DEG C of insulating box with suction pipe, it will be upper
Moisture in clear liquid is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid.
S5, will detection liquid under reduced pressure with the flow velocity of 3mL/min by activated carboxylic acid type cation exchange column,
After eluent all outflow, column is washed with 5mL methanol, discards whole effluxes, under the negative pressure of 65kPa, decompressing and extracting 5min,
It uses 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors to elute again, collects eluent in conical flask.
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX lines are printed on
End downward, is allowed to be sufficiently submerged in solution, after being incubated 5min at 20 DEG C of room temperature, takes out test strips, judgement is as a result, when quality control region is aobvious
It showing that band, detection zone are not shown, is judged to the positive, Fourth Ring cellulose content is not exceeded, when quality control region and detection zone all show band,
It is judged to feminine gender, Fourth Ring cellulose content is exceeded, when quality control region does not show band, test paper failure.
Embodiment 2
Tetracyclines detection method in a kind of poultry, includes the following steps：
S1, birds meat is cleaned, after mincing, weighs 10g birds meats and be placed in the centrifuge tube of 50mL, 4mL is added
0.01mol/L trichloroacetic acid removal meat in protein, on liquid blending device after vortex mixed 2min, cross 0.2 μm of filter membrane.
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle
Corking tightening seal vibrates 10min with oscillator, and during earthquake, the power of oscillator is 800-1000W.
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and ethylenediamine tetra-acetic acid
The pH value of sodium buffer solution is 6, centrifuge tube is placed in vortex mixed 2min on liquid blending device, then vibrate 8-10min with oscillator,
During earthquake, the power of oscillator is 800-1000W, centrifuges 12min, in centrifugal process, the power of centrifugation is
1000r/min, by supernatant in 100mL volumetric flasks, in residue plus sodium ethylene diamine tetracetate buffer solution 20mL, repetition extract one
It is secondary, then be loaded into 100mL volumetric flasks.
S4, it is taken in 0.6mL supernatants to small glass, small glass is put into 45 DEG C of insulating box with suction pipe, it will be upper
Moisture in clear liquid is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid.
S5, will detection liquid under reduced pressure with the flow velocity of 5mL/min by activated carboxylic acid type cation exchange column,
After eluent all outflow, column is washed with 5mL methanol, discards whole effluxes, under the negative pressure of 65kPa, decompressing and extracting 8min,
It uses 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors to elute again, collects eluent in conical flask.
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX lines are printed on
End downward, is allowed to be sufficiently submerged in solution, after being incubated 5min at 25 DEG C of room temperature, takes out test strips, judgement is as a result, when quality control region is aobvious
It showing that band, detection zone are not shown, is judged to the positive, Fourth Ring cellulose content is not exceeded, when quality control region and detection zone all show band,
It is judged to feminine gender, Fourth Ring cellulose content is exceeded, when quality control region does not show band, test paper failure.
Embodiment 3
Tetracyclines detection method in a kind of poultry, includes the following steps：
S1, birds meat is cleaned, after mincing, weighs 8g birds meats and be placed in the centrifuge tube of 50mL, be added 3mL's
Protein in the trichloroacetic acid removal meat of 0.01mol/L, on liquid blending device after vortex mixed 1min, crosses 0.2 μm of filter membrane.
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle
Corking tightening seal vibrates 9min with oscillator, and during earthquake, the power of oscillator is 800-1000W.
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and ethylenediamine tetra-acetic acid
The pH value of sodium buffer solution is 5, centrifuge tube is placed in vortex mixed 2min on liquid blending device, then vibrate 8-10min with oscillator,
During earthquake, the power of oscillator is 800-1000W, centrifuges 11min, in centrifugal process, the power of centrifugation is
1000r/min, by supernatant in 100mL volumetric flasks, in residue plus sodium ethylene diamine tetracetate buffer solution 20mL, repetition extract one
It is secondary, then be loaded into 100mL volumetric flasks.
S4, it is taken in 0.6mL supernatants to small glass, small glass is put into 40 DEG C of insulating box with suction pipe, it will be upper
Moisture in clear liquid is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid.
S5, will detection liquid under reduced pressure with the flow velocity of 4mL/min by activated carboxylic acid type cation exchange column,
After eluent all outflow, column is washed with 5mL methanol, discards whole effluxes, under the negative pressure of 65kPa, decompressing and extracting 6min,
It uses 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors to elute again, collects eluent in conical flask.
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX lines are printed on
End downward, is allowed to be sufficiently submerged in solution, after being incubated 5min at 22 DEG C of room temperature, takes out test strips, judgement is as a result, when quality control region is aobvious
It showing that band, detection zone are not shown, is judged to the positive, Fourth Ring cellulose content is not exceeded, when quality control region and detection zone all show band,
It is judged to feminine gender, Fourth Ring cellulose content is exceeded, when quality control region does not show band, test paper failure.
Embodiment 4
S1, birds meat is cleaned, after mincing, weighs 5g birds meats and be placed in the centrifuge tube of 50mL, be added 4mL's
Protein in the trichloroacetic acid removal meat of 0.01mol/L, on liquid blending device after vortex mixed 1min, crosses 0.2 μm of filter membrane.
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle
Corking tightening seal vibrates 10min with oscillator, and during earthquake, the power of oscillator is 800-1000W.
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and ethylenediamine tetra-acetic acid
The pH value of sodium buffer solution is 6, centrifuge tube is placed in vortex mixed 2min on liquid blending device, then vibrate 8-10min with oscillator,
During earthquake, the power of oscillator is 800-1000W, centrifuges 10min, in centrifugal process, the power of centrifugation is
1000r/min, by supernatant in 100mL volumetric flasks, in residue plus sodium ethylene diamine tetracetate buffer solution 20mL, repetition extract one
It is secondary, then be loaded into 100mL volumetric flasks.
S4, it is taken in 0.6mL supernatants to small glass, small glass is put into 40 DEG C of insulating box with suction pipe, it will be upper
Moisture in clear liquid is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid.
S5, will detection liquid under reduced pressure with the flow velocity of 3mL/min by activated carboxylic acid type cation exchange column,
After eluent all outflow, column is washed with 5mL methanol, discards whole effluxes, under the negative pressure of 65kPa, decompressing and extracting 5min,
It uses 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors to elute again, collects eluent in conical flask.
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX lines are printed on
End downward, is allowed to be sufficiently submerged in solution, after being incubated 5min at 20 DEG C of room temperature, takes out test strips, judgement is as a result, when quality control region is aobvious
It showing that band, detection zone are not shown, is judged to the positive, Fourth Ring cellulose content is not exceeded, when quality control region and detection zone all show band,
It is judged to feminine gender, Fourth Ring cellulose content is exceeded, when quality control region does not show band, test paper failure.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (3)
1. Tetracyclines detection method in a kind of poultry, which is characterized in that include the following steps：
S1, birds meat is cleaned, after mincing, weighs 5-10g birds meats and be placed in the centrifuge tube of 50mL, 2-4mL is added
0.01mol/L trichloroacetic acid removal meat in protein, on liquid blending device after vortex mixed 1-2min, cross 0.2 μm of filter
Film；
S2, filter residue is placed in 50mL centrifuge tubes, then pure water and each 2mL of ethyl acetate is added into centrifuge tube, by bottle stopper
Tightening seal vibrates 8-10min with oscillator；
S3, be added into centrifuge tube 30mL 0.1mol/L sodium ethylene diamine tetracetate buffer solution, and sodium ethylene diamine tetracetate is slow
The pH value of fliud flushing is 4-6, centrifuge tube is placed in vortex mixed 1-2min on liquid blending device, then vibrate 8-10min with oscillator,
10-12min is centrifuged, by supernatant in 100mL volumetric flasks, in residue plus sodium ethylene diamine tetracetate buffer solution 20mL, repetition carry
It takes once, then is loaded into 100mL volumetric flasks；
S4, it is taken in 0.6mL supernatants to small glass with suction pipe, small glass is put into 30-45 DEG C of insulating box, by supernatant
Moisture in liquid is evaporated, and then 0.3mL volume dilution liquid is used to redissolve bottom of a cup solid, obtains detection liquid；
S5, liquid will be detected under reduced pressure with the flow velocity of 3-5mL/min by activated carboxylic acid type cation exchange column, waited for
Eluent all after outflow, washes column with 5mL methanol, discards whole effluxes, under the negative pressure of 65kPa, decompressing and extracting 5-8min,
It uses 4mL mobile phase acetonitrile-methanol 0.01mol/ml oxalic acid mixed liquors to elute again, collects eluent in conical flask；
S6,200 μ l are pipetted with micropipettor in micropore, the test strips marked are inserted into micropore, MAX line ends court is printed on
Under, it is allowed to be sufficiently submerged in solution, after being incubated 5min at 20-25 DEG C of room temperature, takes out test strips, judge result.
2. Tetracyclines detection method in a kind of poultry according to claim 1, which is characterized in that in S3, centrifuging
In the process, the power of centrifugation is 1000r/min.
3. Tetracyclines detection method in a kind of poultry according to claim 1, which is characterized in that in S2 and S3,
During concussion, the power of oscillator is 800-1000W.
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