CN109633147A - The detection method of fluoquinolone constituents in a kind of fresh royal jelly - Google Patents
The detection method of fluoquinolone constituents in a kind of fresh royal jelly Download PDFInfo
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Abstract
The present invention relates to the detection methods of fluoquinolone constituents in a kind of fresh royal jelly of royal jelly detection technique field, including sample pre-treatments: extraction-purification-supernatant processing；Sample detection: it is detected using the detection kit of fluorine promise quinoline ketone drug.The application is intended to provide a kind of detection method of fluoquinolone constituents that pre-treatment is easy to operate.
Description
Technical field
The present invention relates to a kind of inspections of fluoquinolone constituents in royal jelly detection technique field more particularly to fresh royal jelly
Survey method.
Background technique
Honey is the necessary all kinds of nutriments of human body to be rich in, such as glucose, fructose, ammonia from honeybee acquisition nectar
Base acid, enzyme, mineral and antioxidant etc., are important wholefood.Royal jelly is the lingual gland and mandibular gland point by worker bee
The compounding substances secreted have extraordinary health care and medical value.However, in actual production honeybee be easy by bacterium, fungi,
Viral and external parasitic infecting for mite class and cause disease.Controlling and treat these disease usual ways is Direct-fed veterinary drug
Or veterinary drug is sprayed in honeycomb, it is thus possible to lead to the residue of veterinary drug problem in the bee products such as honey and royal jelly, to consumption
Person causes potential threat.
Quinolone drugs is each commonly used to prevention and treatment and treatment in beekeeping as a kind of common antibiotics class veterinary drug
Class bacterial disease can especially effectively prevent bee numbers caused by American foul brood and reduce and honey output drop
It is low.
Currently, reporting the detection method of quinolones medicament relict in honey or royal jelly in some documents, mainly have
Liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry, because Liquid Chromatography-Tandem Mass Spectrometry detection limit is low, sensitivity and accuracy
High and be widely used, it is also the prescriptive procedure in China's honey and royal jelly.Pre-treating method mostly uses solid phase extraction techniques,
Because it is required respectively by some column processes such as purification, concentration, complicated for operation, time-consuming and laborious, sensitivity and poor repeatability.Mesh
Before, it is few can simple, quick sample-pretreating method for detecting quinolones medicament relict in royal jelly.
Summary of the invention
The purpose of the present invention is to provide a kind of detection methods of fluoquinolone constituents in fresh royal jelly, before having sample
Handle simple, quick advantage.
Above-mentioned purpose of the invention has the technical scheme that
The detection method of fluoquinolone constituents in a kind of fresh royal jelly, including following detecting step,
(1) sample pre-treatments:
Step 1: extracting, weigh 2g fresh royal jelly sample into 50ml centrifuge tube, 5.0ml acetic acid zinc solution is added in centrifuge tube
5min sample dissolution is shaken, 10ml ethyl acetate is added into sample solution, after 10min is extracted in concussion, 1.0g sodium chloride is added
With 4.0g anhydrous zinc sulfate, after whirling motion 1min, 5min is shaken, is centrifuged 5min under the conditions of 8000-10000rpm；
Step 2: purification accurately pipettes supernatant 5.0ml in the 15 mL centrifuge tubes equipped with 500 mg PSA, oscillation 5
Min, 6000rpm are centrifuged 5 min, and 1.0 mL of supernatant is taken to be dried with nitrogen at 30 DEG C in 2 mL centrifuge tubes；
Step 3: after supernatant is dried with nitrogen, 500 μ L sodium carbonate liquor (0.1 mM, pH=10.5) whirlpools being added into centrifuge tube
Inula is molten, adds 500 μ L dansyl Cls-acetone soln (1.0 mg/mL) and is vortexed at once mixing；In 60 DEG C of 15 min of heating；
After derivatization, it is placed in 4 DEG C of 10 min of refrigerator cold-storage；Final sample solution crosses 0.2 μm of filter membrane；
Sample detection: it is detected using the detection kit of fluorine promise quinoline ketone drug.
Implement above-mentioned technical proposal, royal jelly sample mesostroma is complicated, contains protein, fat, lactose, organic acid and ammonia
The chaff interferents such as base acid, effective degreasing, deproteinization directly determine the efficiency extracted.And in the process of dissolution royal jelly sample
In, the effect of pure water and acetic acid zinc solution dissolved matrix is compared, is occurred more when instrument analysis results show pure water as lytic agent
More impurity Interference Peaks, influence the accurate quantitative analysis of target compound；And when using acetic acid zinc solution as lytic agent, because it is mentioned
The high concentrated acid radical ion of confession can seize the spray layer of protein surface, protein micelle " dehydration ", and then condense and precipitate
It is precipitated, on the other hand, protein is minimum in the solubility of isoelectric point, and acid pH provided by zinc acetate buffer solution is close to bee
The isoelectric point of most of protein plays the effect of decontamination substrate so that also accelerating protein is precipitated precipitating in royal jelly.
In addition, sodium chloride and anhydrous zinc sulfate are added into solution can play salting out, ionic strength is improved, is deepened
Sediment and supernatant separation, and increase the extraction efficiency to target compound.
The PSA of addition can remove organic acid, fatty acid, certain pigments and carbohydrate isopolarity matrix in sample in extract
Ingredient selects PSA as scavenging material, easy to operate, and for organic acid, fatty acid, certain pigments and carbohydrate isopolarity base
The processing of matter is more thorough.
And the sulfonyl of dansyl Cl can and the phenolic hydroxyl group of quinolones hormone react and generate the easily combination that ionizes
Object increases the sensitivity of detection.
The detection of fluoquinolone constituents in the detection kit detection fresh royal jelly of fluorine promise quinoline ketone drug is finally selected,
It is simple to operate.
Test sample is just directly obtained by extraction-purification-supernatant processing during whole operation, compared with traditional benefit
It is simpler with the operating method of solid-phase extraction column.
Further, the detection method of the detection kit of the fluoroquinolones includes the following steps,
S1. fluoroquinolones kit is returned and is warmed to room temperature；
S2. it takes out and needs the microwell plate of quantity to get all the ready and be inserted on micropore frame, record sample and standard items correspond to micropore and by suitable
Sequence number, each sample and gauge orifice do 2 hole parallel tests；
S3. plus standard items/sample, 50 μ L Norfloxacin standard solution of absorption draw 50 μ L sample solution into corresponding micropore
In remaining micropore, draws 75 μ L Norfloxacin monoclonal antibodies and thyroid peroxidase marks the mixed of sheep anti mouse antiantibody
Closing liquid, gently oscillation mixes in each micropore, reacts 30min in cover board membrane cover plate 25 DEG C of light protected environments of postposition；
S4. board-washing, open cover board film, liquid in hole is dried, with 250 hole μ L/ of wash operating solution, sufficiently washing three times, every time
It is spaced 10s, is patted dry with blotting paper；
S5. it develops the color: substrate colour developing 50 hole μ l/ of A liquid is added, substrate colour developing 50 hole μ l/ of B liquid, gently oscillation mixes, with cover board membrane cover
15min is reacted in 25 DEG C of light protected environments of plate postposition；
S6. it measures, 50 hole μ L/ of terminate liquid is added, gently concussion mixes, and sets microplate reader at 450nm, measures every hole OD value.
Implement above-mentioned technical proposal, is detected using the detection method of the detection kit of fluoroquinolones, most
OD value is determined eventually, and the content of fluorine quino ketone in royal jelly is obtained eventually by conversion.
Further, the H of the terminate liquid selection 2mol/L2SO4。
Implement above-mentioned technical proposal, terminate liquid selection sulfuric acid can effectively inhibit the activity of enzyme, avoid the biological enzyme of survival
It is influenced caused by testing result.
Further, the substrate developing solution A liquid is the citrate-phosphate two of the carbamide peroxide containing 0.4~0.6mmol/L
Hydrogen sodium buffer solution, substrate develop the color B liquid for tetramethyl biphenyl amine aqueous solution.
Implement above-mentioned technical proposal, the substrate developing solution A and substrate developing solution B of selection, which can cooperate, increases reaction
Sensitivity.
Further, the wash operating solution includes the PBS solution containing 0.05% Tween-20.
Implement above-mentioned technical proposal, Tween-20 has the part of oleophylic as surfactant, existing hydrophilic part,
It is able to ascend clean level.
Further, the Norfloxacin standard solution is the storage for selecting ethyl acetate to be configured to 100ng/mL as solvent
Liquid saves under the conditions of -20 DEG C.
Further, the preparation of the fluoroquinolones monoclonal antibody:
Animal immune: 8~10 week old Balb/c mouse are immunized in fluoroquinolones haptens and carrier protein couplet object；
Cell fusion and cloning: the mice spleen cell after being immunized is taken, with SP2/0 myeloma cell in fusion agent Macrogol 4000
Under the action of merge, screening obtain can stably excreting monoclonal antibody hybridoma cell strain；
The monoclonal hybridoma strain of fluoroquinolones is obtained through screening;The Monoclonal hybridomas of fluoroquinolones
Cell strain can an unbounded quantity of generation fluoroquinolones specific antibody, which is for fluoroquinolones medicine
Object, sensitivity reaches 0.1 μ g/L；
Hybridoma: being made the cell suspension of 1 × 109/ml by cell cryopreservation and recovery of frozen stock solution, long-term in liquid nitrogen
It saves；
Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, moves into training in culture bottle
It supports.
Implement above-mentioned technical proposal, the monoclonal hybridoma strain of fluoroquinolones can an unbounded quantity of generation fluorine
Quinolone drugs specific antibody, for the antibody specificity for fluoroquinolones, sensitivity reaches 0.1 μ g/L.
Entire preparation exploitativeness is strong.
Further, the preparation process of the thyroid peroxidase label sheep anti mouse antiantibody includes: by marker enzyme first
Shape gland peroxidase and sheep anti mouse antiantibody are coupled to obtain thyroid peroxidase label using Euplotes woodruffi
Sheep anti mouse antiantibody.
Implement above-mentioned technical proposal, Euplotes woodruffi is a kind of common homotype bi-functional cross-linking agent, passes through its two
A aldehyde radical in conjunction with the amino of HRP and antibody protein, forms HRP- glutaraldehyde-Ab protein conjugates respectively.Prepare thyroid gland mistake
Oxide enzyme marks the preparation process of sheep anti mouse antiantibody simple.
In conclusion the invention has the following advantages:
One, it selects zinc acetate as buffer, can speed up the wash-off precipitating of protein, decontamination substrate effect is obvious;
Two, using the operating method of the application, the process of pre-treatment is easy to operate.
Specific embodiment
1, the preparation of fluoroquinolones monoclonal antibody:
Animal immune: 8~10 week old Balb/c mouse are immunized in fluoroquinolones haptens and carrier protein couplet object；
Cell fusion and cloning: the mice spleen cell after being immunized is taken, with SP2/0 myeloma cell in fusion agent Macrogol 4000
Under the action of merge, screening obtain can stably excreting monoclonal antibody hybridoma cell strain；
The monoclonal hybridoma strain of fluoroquinolones is obtained through screening;
Cell cryopreservation and recovery: hybridoma is made 1 × 10 with frozen stock solution9The cell suspension of a/ml, it is long-term in liquid nitrogen
It saves；
Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, moves into training in culture bottle
It supports.
2, the preparation process of thyroid peroxidase label sheep anti mouse antiantibody includes: by marker enzyme thyroid gland peroxidating
Object enzyme and sheep anti mouse antiantibody are coupled to obtain thyroid peroxidase label sheep anti mouse anti-using Euplotes woodruffi
Body.
3, the concentration of Norfloxacin standard solution is respectively set to 0ng/mL, 0.125ng/mL, 0.5 ng/mL, 2.5ng/
mL、5ng/mL、12.5ng/mL。
Embodiment one
(1) fresh royal jelly sample pre-treatments:
Step 1: extracting, weigh 2g fresh royal jelly sample into 50ml centrifuge tube, 5.0ml acetic acid zinc solution is added in centrifuge tube
5min sample dissolution is shaken, 10ml ethyl acetate is added into sample solution, after 10min is extracted in concussion, 1.0g sodium chloride is added
With 4.0g anhydrous zinc sulfate, after whirling motion 1min, 5min is shaken, is centrifuged 5min under the conditions of 8000rpm；
Step 2: purification accurately pipettes supernatant 5.0ml in the 15mL centrifuge tube equipped with 500mg PSA, vibrates 5min,
6000rpm is centrifuged 5min, and supernatant 1.0mL is taken to be dried with nitrogen at 30 DEG C in 2mL centrifuge tube；
Step 3: after supernatant is dried with nitrogen, 500 μ L sodium carbonate liquors (0.1 mM, pH=10.5) vortex being added into centrifuge tube
It redissolves, adds 500 μ L dansyl Cls-acetone soln (1.0 mg/mL) and be vortexed at once mixing；In 60 DEG C of heating 15min；Spread out
After biochemistry, it is placed in 4 DEG C of refrigerator cold-storage 10min；Final sample solution crosses 0.2 μm of filter membrane；
(2) it sample detection: is detected using the detection kit of fluorine promise quinoline ketone drug；
S1. fluoroquinolones kit is returned and is warmed to room temperature；
S2. it takes out and needs the microwell plate of quantity to get all the ready and be inserted on micropore frame, record sample and standard items correspond to micropore and by suitable
Sequence number, each sample and gauge orifice do 2 hole parallel tests；
S3. plus standard items/sample, 50 μ L Norfloxacin standard solution of absorption draw 50 μ L sample solution into corresponding micropore
In remaining micropore, draws 75 μ L Norfloxacin monoclonal antibodies and thyroid peroxidase marks the mixed of sheep anti mouse antiantibody
Closing liquid, gently oscillation mixes in each micropore, reacts 30min in cover board membrane cover plate 25 DEG C of light protected environments of postposition；
S4. board-washing, open cover board film, liquid in hole is dried, with 250 hole μ L/ of wash operating solution, sufficiently washing three times, every time
It is spaced 10s, is patted dry with blotting paper；
S5. it develops the color: substrate colour developing 50 hole μ l/ of A liquid is added, substrate colour developing 50 hole μ l/ of B liquid, gently oscillation mixes, with cover board membrane cover
15min is reacted in 25 DEG C of light protected environments of plate postposition；
S6. it measures, 50 hole μ L/ of terminate liquid is added, gently concussion mixes, and sets microplate reader at 450nm, measures every hole OD value.
Analysis of test results: the percentage absorptance of standard items or sample is equal to being averaged for the absorbance value of standard items or sample
Value obtains the percentage absorbance of standard items or sample multiplied by 100% divided by the average value of the absorbance value of first standard items
Value.Using standard items percentage absorptance as ordinate, using the logarithm of fluoroquinolones standard concentration (μ g/L) as abscissa,
Draw canonical plotting.The percentage absorptance of sample is substituted into standard curve, is read corresponding to sample from standard curve
Concentration is the actual concentrations of fluoroquinolones in sample multiplied by its corresponding extension rate.
Embodiment 2
Embodiment 2 is the difference from embodiment 1 is that centrifugal speed is 9000rpm/ in extraction process in the step 1 of sample pre-treatments
min。
Embodiment 3
Embodiment 2 is the difference from embodiment 1 is that centrifugal speed is in extraction process in the step 1 of sample pre-treatments
10000rpm/min。
Experiment Data Records
Standard curve obtained in embodiment 1 is y=- 0.341x+0.998
The calculation formula of the residual quantity of fluoquinolone
The residual quantity of fluoquinolone in X- sample, unit are ng/kg (μ g/kg)；
C- blood concentration norfloxacin from the sample obtained on standard working curve, unit are nanograms per milliliter (ng/ml)；
The final constant volume of V- sample solution, unit are milliliter (mL)；
Final sample mass representated by m- sample solution, unit are gram (g).
The testing result of the residual quantity of 1 embodiment 1-3 fluoquinolone of table.
| Project | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| Lower limit of measurement μ g/kg | 2.5 | 2. 5 | 2.5 |
Pitch-based sphere is 2.5 μ g/kg, and fluoquinolone rate of recovery range is 70%~93%;
Pitch-based sphere is 5.0 μ g/kg, and fluoquinolone rate of recovery range is 75%~97%;
Pitch-based sphere is 10.0 μ g/kg, and fluoquinolone rate of recovery range is 74%~90%；
On 2.5 μ g/kg, 5.0 μ g/kg and 10.0 μ g/kg pitch-based spheres, relative standard deviation (RSD) is 4.0%~6.0%.
Claims (8)
1. the detection method of fluoquinolone constituents in a kind of fresh royal jelly, which is characterized in that including following detecting step,
(1) sample pre-treatments:
Step 1: extracting, weigh 2g fresh royal jelly sample into 50ml centrifuge tube, 5.0ml acetic acid zinc solution is added in centrifuge tube
5min sample dissolution is shaken, 10ml ethyl acetate is added into sample solution, after 10min is extracted in concussion, 1.0g sodium chloride is added
With 4.0g anhydrous zinc sulfate, after whirling motion 1min, 5min is shaken, is centrifuged 5min under the conditions of 8000-10000rpm；
Step 2: purification accurately pipettes supernatant 5.0ml in the 15 mL centrifuge tubes equipped with 500mg PSA, vibrates 5min,
6000rpm is centrifuged 5min, and 1.0 mL of supernatant is taken to be dried with nitrogen at 30 DEG C in 2 mL centrifuge tubes；
Step 3: after supernatant is dried with nitrogen, 500 μ L sodium carbonate liquors being added into centrifuge tube and are vortexed and redissolve, add 500 μ L
Dansyl Cl-acetone soln is vortexed mixing at once；In 60 DEG C of heating 15min；After derivatization, it is placed in 4 DEG C of refrigerator cold-storages
10min；Final sample solution crosses 0.2 μm of filter membrane；
(2) it sample detection: is detected using the detection kit of fluorine promise quinoline ketone drug.
2. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
The detection method for stating the detection kit of fluoroquinolones includes the following steps,
S1. fluoroquinolones kit is returned and is warmed to room temperature；
S2. it takes out and needs the microwell plate of quantity to get all the ready and be inserted on micropore frame, record sample and standard items correspond to micropore and by suitable
Sequence number, each sample and gauge orifice do 2 hole parallel tests；
S3. plus standard items/sample, 50 μ L Norfloxacin standard solution of absorption draw 50 μ L sample solution into corresponding micropore
In remaining micropore, draws 75 μ L Norfloxacin monoclonal antibodies and thyroid peroxidase marks the mixed of sheep anti mouse antiantibody
Closing liquid, gently oscillation mixes in each micropore, reacts 30min in cover board membrane cover plate 25 DEG C of light protected environments of postposition；
S4. board-washing, open cover board film, liquid in hole is dried, with 250 hole μ L/ of wash operating solution, sufficiently washing three times, every time
It is spaced 10s, is patted dry with blotting paper；
S5. it develops the color: substrate colour developing 50 hole μ l/ of A liquid is added, substrate colour developing 50 hole μ l/ of B liquid, gently oscillation mixes, with cover board membrane cover
15min is reacted in 25 DEG C of light protected environments of plate postposition；
S6. it measures, 50 hole μ L/ of terminate liquid is added, gently concussion mixes, and sets microplate reader at 450nm, measures every hole OD value.
3. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
State the H of terminate liquid selection 2mol/L2SO4。
4. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
State the citrate-phosphate sodium dihydrogen buffer solution that substrate developing solution A liquid is the carbamide peroxide containing 0.4~0.6mmol/L, substrate
Colour developing B liquid is tetramethyl biphenyl amine aqueous solution.
5. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
Stating wash operating solution includes the PBS solution containing 0.05% Tween-20.
6. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
Stating Norfloxacin standard solution is the storing liquid for selecting ethyl acetate to be configured to 100ng/mL as solvent, under the conditions of -20 DEG C
It saves.
7. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
State the preparation of fluoroquinolones monoclonal antibody:
Animal immune: 8~10 week old Balb/c mouse are immunized in fluoroquinolones haptens and carrier protein couplet object；
Cell fusion and cloning: the mice spleen cell after being immunized is taken, with SP2/0 myeloma cell in fusion agent Macrogol 4000
Under the action of merge, screening obtain can stably excreting monoclonal antibody hybridoma cell strain；
The monoclonal hybridoma strain of fluoroquinolones is obtained through screening;
Hybridoma: being made the cell suspension of 1 × 109/ml by cell cryopreservation and recovery of frozen stock solution, long-term in liquid nitrogen
It saves；
Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, moves into training in culture bottle
It supports.
8. the detection method of fluoquinolone constituents in a kind of fresh royal jelly according to claim 1, which is characterized in that institute
The preparation process for stating thyroid peroxidase label sheep anti mouse antiantibody includes: by marker enzyme thyroid peroxidase and sheep
Anti- mouse antiantibody is coupled to obtain thyroid peroxidase label sheep anti mouse antiantibody using Euplotes woodruffi.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766632A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting quinolones in animal derived food |
| CN102766212A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones |
| CN103257233A (en) * | 2013-05-31 | 2013-08-21 | 南京祥中生物科技有限公司 | Biochip and method capable of simultaneously visually detecting multiple antibiotics, illegal addition agents and biotoxins |
| CN103323594A (en) * | 2012-03-22 | 2013-09-25 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application |
| US20150219678A1 (en) * | 2012-07-04 | 2015-08-06 | Beijing Kwinbon Biotechnology Co., Ltd | Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof |
| CN104977406A (en) * | 2014-04-03 | 2015-10-14 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit |
| CN106645728A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for fluoroquinolones drugs in foods |
-
2018
- 2018-12-07 CN CN201811496858.6A patent/CN109633147A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766632A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting quinolones in animal derived food |
| CN103323594A (en) * | 2012-03-22 | 2013-09-25 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application |
| CN102766212A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting residues of fluoroquinolones |
| US20150219678A1 (en) * | 2012-07-04 | 2015-08-06 | Beijing Kwinbon Biotechnology Co., Ltd | Enzyme-linked immunosorbent assay kit for detecting dinitolmide and use thereof |
| CN103257233A (en) * | 2013-05-31 | 2013-08-21 | 南京祥中生物科技有限公司 | Biochip and method capable of simultaneously visually detecting multiple antibiotics, illegal addition agents and biotoxins |
| CN104977406A (en) * | 2014-04-03 | 2015-10-14 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit |
| CN106645728A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for fluoroquinolones drugs in foods |
Non-Patent Citations (2)
| Title |
|---|
| 任军主编: "《安徽省突发公共卫生事件处置技术方案》", 31 January 2008, 合肥：安徽科学技术出版社 * |
| 张文文: "蜂王浆中类固醇激素化合物检测方法的开发和应用", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
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