CN101191796A - Broad spectrum ELISA kit and preparation method - Google Patents
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Abstract
The invention relates to a broad-band chloramphenicol ELISA kit and preparing method thereof. The broad-band chloramphenicol ELISA kit consists of a coated micro-perforated plate, chloramphenicol series standard solution, antibody solution, concentrated cleaning solution, working buffer solution, visualization reagent and stop solution. The kit in the invention has a broad detection range (the standard curve range is 0.025ng/ml-100ng/ml) and higher sensitivity (the lowest limit of detection is 0.025ng/ml). The invention also relates to a method for preparing the chloramphenicol ELISA kit. The kit in the invention adopts 4-paramater (Y=(A-D)(1+(X/C)^B)+D) regression curve for data processing, thereby simplifying data processing and widening the range of data processing.
Description
Technical field
The present invention relates to a kind of broad spectrum ELISA kit and preparation method.
Background technology
Chloromycetin (CAP) is a kind of microbiotic that extracts from Venezuela's streptococcus culture fluid, have characteristics such as broad-spectrum high efficacy cheapness, can act on Grain-positive and negative bacteria, can prevent honeybee Pafeng disease, fish vibriosis and tail etc., once be widely used in apiculture and culture fishery, but chloromycetin can suppress human bone marrow's hematopoiesis function, cause human body granular white blood cells shortage, irreversible alpastic anemia and grey baby's syndrome etc., more seriously the low concentration medicament residue can bring out the drug resistance of germ, and human beings'health is constituted potential threat.Therefore the U.S., Canada and EU countries government propose to ban use of in the animal derived food in succession, but chloromycetin is also added in some countries and regions in the consumption animal feeding, cause the residual of chloromycetin in the relevant animal derived food like this, therefore many countries are all regularly detected residual chloromycetin.Raising along with detection sensitivity, international detection lower bound has been brought up to 100ng/kg, and with this as the standard (Guan Rong that judges that chloromycetin is whether residual, use the research [J] of residual chloromycetin in the Enzyme-multiplied immune technique detection animal derived food, the inspection and quarantine science, 2002,12 (4): 5-7; 10).
Chloromycetin detection technique method and the related assays lower bound of announcing is respectively at present: cylinder plate method (50000ng/kg), radioimmunology (100ng/kg), euzymelinked immunosorbent assay (ELISA) (100ng/kg) (Shen Meifang, Zhao Wenya, Fei Zhiliang, Deng, measure the residual quantity of chloromycetin in the aquatic products with euzymelinked immunosorbent assay (ELISA), Nanjing Normal University's journal (engineering version) 2003,3 (2): 1-4), vapor-phase chromatography (100ng/kg) (Li Rong, Ai Xiaohui, Zou Shiping, Deng. in the gas chromatography determination fishes and shrimps musculature residual [J] of chloromycetin, fresh water fishery, 2003,33 (6): 17-19), liquid phase chromatography (1500ng/kg) and liquid chromatography tandem mass spectrum (LC-MS/MS) coupling method (10ng/kg) (Zhu Weixia, Yang Jizhou, Lu Kui waits the research [J] of residual chloromycetin in the high performance liquid chromatography tandem mass spectrometry detection food, He'nan University of Technology's journal (natural science edition), 2005,26 (5): 61-64) etc.The detectability of cylinder plate method and liquid phase chromatography has been difficult to adapt to present detection requirement, radioimmunology, vapor-phase chromatography and liquid chromatography tandem mass spectrum coupling method, must be equipped with expensive instrument and equipment, the sample pre-treatments complexity, workload is big, and round of visits is long, and particularly radioimmunology also needs to be equipped with radioactive source, certain danger is arranged, be difficult in grass-roots unit and the extensive residual monitoring generaI investigation and use.And euzymelinked immunosorbent assay (ELISA) ELISA (Enzyme-Linked Immuno Sorbent Assay) continuous improvement and development technically, its specificity and sensitivity all improve a lot, euzymelinked immunosorbent assay (ELISA) is not very high to experimental requirements simultaneously, just can carry out this work in ordinary laboratory, and be the approval of multinational government department, AOAC is recommended as rapid screening method with this technology.
The at present kit and the detection method of chlorine detection mycin, employing be the competitiveness enzyme linked immunosorbent assay, but because the material of bag quilt is different and different.A kind of is that bag is by the antibody of goat anti-rabbit igg (two is anti-), when adding chloromycetin enzyme labeling thing, free chlorine mycin (chloromycetin standard items or sample) and the chloramphenicol antibody (antibody of rabbit chloramphenicol resistance, be rabbit igg) after, free chlorine mycin and chloromycetin enzyme labeling thing are competed a limited number of chloramphenicol antibody binding sites, the quantity inverse ratio that chloromycetin enzyme labeling thing combines with the chloramphenicol antibody site and the quantity of free chlorine mycin, chloromycetin enzyme labeling thing and compound after chloramphenicol antibody combines again be coated on microwell plate on goat anti-rabbit igg combine, do not have the material of combined chloromycetin enzyme labeling thing in washing procedure, to be removed.The substrate and the developer (TMB-hydrogen peroxide) that add enzyme, can be according to the quantity of chloromycetin enzyme labeling thing, demonstrate the different concentration of the depth, adding stop buffer can be with blue flavescence, following detection micropore solution absorbency at the 450nm wavelength, chloramphenicol concentration and absorbance in the sample are inversely proportional to, according to the typical curve measuring fixed amount (Guan Rong that formulates, use the research [J] of residual chloromycetin in the Enzyme-multiplied immune technique detection animal derived food, the inspection and quarantine science, 2002,12 (4): 5-7; 10).Be limited to 50ng/kg under the detection of this method, sensing range is 0.05ng/ml～4.05ng/ml.Second method be with chloramphenicol antibody (chloromycetin monoclonal antibody) direct coated on microwell plate, add chloromycetin enzyme labeling thing, chloramphenicol antibody on free chlorine mycin (chloromycetin standard items or sample) the direct competitive microwell plate, the substrate and the developer (TMB-hydrogen peroxide) that add enzyme then, can be according to the quantity of chloromycetin enzyme labeling thing, demonstrate the different concentration of the depth, adding stop buffer can be with blue flavescence, following detection micropore solution absorbency at the 450nm wavelength, chloramphenicol concentration and absorbance in the sample are inversely proportional to, according to the typical curve measuring fixed amount of formulating.Be limited to 50ng/kg under the detection of this method, sensing range is 0.05ng/ml～4.05ng/ml.Also having a kind of method is that chloromycetin protein conjugates (generally being the bond of chloromycetin and oralbumin (BSA)) is coated on the microwell plate, add the competition of free chlorine mycin (chloromycetin standard items or sample) and chloramphenicol antibody and be coated on chloromycetin one BSA on the microwell plate, adding the anti-mouse ELIAS secondary antibody of rabbit then combines with the chloramphenicol antibody bond, the substrate and the developer (o-phenylenediamine-hydrogen peroxide) that add enzyme again, can be according to the quantity of chloromycetin enzyme labeling thing, demonstrate the different concentration of the depth, add stop buffer, following detection micropore solution absorbency at the 490nm wavelength, chloramphenicol concentration and absorbance in the sample are inversely proportional to, according to the typical curve measuring fixed amount (Shi Deshi that formulates, Zhou Bin, Qin Yali, Deng, the foundation of chloromycetin indirect competitive ELISA (ciELISA) detection method, Chinese animal doctor's journal, 2002,22 (1): 77-79).The detection of this method is limited to 0.1 μ g/l, and the suitableeest sensing range is 1～100 μ g/l, because the Color Appearance System that adopts is o-phenylenediamine-hydrogen peroxide, human body is had certain toxic action simultaneously.Above method is adopt according to logit (B/B0%) or B/B0% on data processing more, and log (chloramphenicol concentration) is carried out regretional analysis, obtains a regression straight line, then according to the curve calculation sample concentration, calculates more miscellaneous.
Summary of the invention
The present invention utilizes following proposal to prepare a kind of broad spectrum ELISA kit, and this kit has very wide sensing range and higher detection sensitivity, has simplified data processing simultaneously.
One, the enzyme-linked immunologic detecting kit of the present invention's preparation
The kit of the present invention's preparation comprises: the microwell plate, chloromycetin series standard product solution, antibody-solutions, thickening and washing solution, working buffer solution, developer, the stop buffer that have wrapped quilt.
Two, the preparation of the related solution that relates in the kit among the present invention
Wide range chloromycetin standard items series, thickening and washing damping fluid and the working buffer formula of liquid that relates among the present invention is very big to the sensitivity influence that kit of the present invention detects.
The principal ingredient of thickening and washing damping fluid and working buffer liquid is a phosphate, can use sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, or dipotassium hydrogen phosphate-potassium phosphate buffer, also can use sodium hydrogen phosphate-potassium dihydrogen phosphate.
Phosphate buffered solution is particularly added the damping fluid of bovine serum albumin(BSA) or calf serum, if preserve the improper breed bacteria that is easy to, has added the thimerosal antiseptic in the buffer solution in preparation process.
1, chloromycetin standard solution: preparation wide range chloromycetin series standard product solution: 0,0.025ng/ml, 0.2ng/ml, 1.5ng/ml, 12ng/ml, 100ng/ml or 0,0.04ng/ml, 0.3ng/ml, 2ng/ml, 15ng/ml, 100ng/ml.
2, concentrated cleaning solution: compound concentration is the phosphate buffer of 0.1mol/l～0.15mol/l, and its pH value is 7.0～7.6.Contain 3%～6% Tween-20 and 0.05%～0.15% thimerosal antiseptic in this concentrated cleaning solution, all the other are phosphate buffer.This concentrated cleaning solution cycles of concentration is 10～15 times, in use dilution.Be used to wash microwell plate after the dilution.
3, working buffer liquid: compound concentration is the phosphate buffer of 0.01mol/l～0.015mol/l, and its pH value is 7.0～7.6.Contain 0.5%～1.5% bovine serum albumin(BSA) (BSA), 0.3%～0.6% Tween-20 and 0.005%～0.015% thimerosal antiseptic in this working buffer liquid, all the other are phosphate buffer.This working buffer liquid can be used as first antibody (anti-hereinafter to be referred as) solution, second antibody (anti-hereinafter to be referred as two) solution, confining liquid and sample diluting liquid and uses.
Bovine serum albumin(BSA) in the working buffer solution that relates among the present invention is replaceable to be calf serum, rabbit anteserum or gelatin.
Three, the preparation of microwell plate among the present invention
The microwell plate of bag quilt among the present invention is to adopt chloromycetin-BSA (bovine serum albumin(BSA)) in the coating buffer that is fit to, with suitable concentration, at room temperature, translation reaction overnight bag quilt on the shaking table.
What the present invention adopted is that pH is sodium carbonate-sodium bicarbonate buffer liquid of 9.6.Among the present invention in the microwell plate chloromycetin-BSA of the quilt that wraps under alkaline environment, can finely be combined on the microwell plate frosting, the coating protein concentration of employing is 50～400ng/ml.
Bag is more stable by the character of the chloromycetin-BSA of usefulness, does not need to wrap quilt under the distinct temperature, and bag of the present invention is at room temperature, and translation is spent the night on the shaking table.Bag can be stood repeatedly to wash plate by good microwell plate like this.
Bag can be closed with containing the working buffer fluid-tight by good microwell plate.Inert protein can be BSA in the confining liquid, also can be other inert protein such as calf serum, rabbit anteserum, gelatin.
Four, the preparation of antibody-solutions
One anti-among the present invention, two anti-concentration are key factors of ELISA kit sensing range and sensitivity among decision the present invention.
An anti-solution that relates among the present invention can be 20～100ng/ml solution with the concentration of work damping fluid preparation.
The two anti-solution that relate among the present invention can be the concentrated solution of 2000～10000ng/ml with the concentration of work damping fluid preparation, press 80～100 times of concentration dilutions with work buffer solution during use.
(standard curve range can reach 0.025ng/ml～100ng/ml) and good sensitivity (0.025ng/ml) can to reach very wide sensing range by the kit of above-mentioned one anti-concentration and two anti-prepared at concentrations.
Five, the preparation of developer and stop buffer
The present invention adopts horseradish peroxidase-labeled substrate Color Appearance System, mainly is 3,3 ', 5,5 '-tetramethyl benzidine (TMB)-hydrogen peroxide system and o-phenylenediamine (OPD)-hydrogen peroxide system.OPD solution has photosensitivity, and is unstable relatively, and have the mutagenesis characteristic.The background value of tmb substrate Color Appearance System is low, and is solution-stabilized, do not have the mutagenesis characteristic, relatively safety.Developer adopts unique prescription among the present invention, with the sensitivity of guarantee reagent box.
The substrate developer that relates among the present invention comprises developer A and developer B two parts: a part is developer A (a TMB solution), and another part is developer B (phosphoric acid-citrate buffer solution-hydrogen peroxide).Developer A mixes in 1: 7～1: 10 ratio with developer B during use, and fully use the vibration back.The prescription of developer can improve the sensitivity of kit among the present invention, and developing time was controlled at 10～15 minutes, reduces detection time.
Developer A can use organic solvent dissolution TMB, and the concentration that makes TMB solution is 1g/l～3g/l, and all the other are dimethyl sulfoxide (DMSO)-ethanolic solution; Developer B can adopt phosphoric acid-citrate buffer solution-hydrogen peroxide, and pH value of buffer solution can be 4.0～6.0, and the concentration of hydrogen peroxide can be 0.01%～0.03%.
Among the present invention among the developer A TMB can replace with OPD.Hydrogen peroxide can replace with urea peroxide among the developer B.Phosphoric acid-citrate buffer solution can replace with phosphoric acid-acetate buffer solution among the developer B.
Stop buffer can adopt 0.05mol/l～2mol/l sulfuric acid solution.
Six, the data analysis of wide range chloromycetin kit
The data processing of traditional competitive ELISA kit is used logit (B/B0%) or B/B0% more, and log (concentration) is carried out regretional analysis, obtains a regression straight line, and data processing is miscellaneous.
The present invention relates to a kind of wide range chloromycetin and detect data analysing method.(Y=(A-D) (1+ (X/C) ^B)+D) regression curve is drawn to adopt 4-Paramater in data handling procedure, be that ordinate is represented the average light absorption value of standard items, horizontal ordinate is represented the logarithm of each standard items chloramphenicol concentration (ng/ml), can handle by reduced data.(typical curve drawn of Y=(A-D) (1+ (X/C) ^B)+D) regression curve is a curve, and is more more accurate than general straight-line regression, can enlarge the recurrence scope simultaneously, satisfies the needs that wide range detects to adopt 4-Paramater.
Description of drawings,
The typical curve of Fig. 1 ELISA kit, the composition of Fig. 2 ELISA kit.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The preparation that embodiment 1 kit is formed
1, bag is by the hole microwell plate
It is that protein concentration is 50～400ng/ml in sodium carbonate-sodium bicarbonate buffer liquid of 9.6 that chloromycetin-BSA is dissolved in pH.Add 100ul in every hole and add microwell plate, build and seal film and place shaking table, the room temperature shaken over night.Wash plate machine (lavation buffer solution) and wash plate, every hole lavation buffer solution 300ul washes plate and repeats 4 times.Pat dry, every hole adds 250ul sealing damping fluid, seals film well and places shaking table, and room temperature is shaken 1h.Wash plate machine (lavation buffer solution) and wash plate, every hole lavation buffer solution 300ul washes plate and repeats 4 times.Pat dry, seal 4 ℃ and preserve down.
2, thickening and washing damping fluid
The preparation working concentration is the 0.1mol/l phosphate buffer (the pH value is 7.0～7.6) that contains 3%～6%Tween-20 and 0.05%～0.15% thimerosal antiseptic.Its cycles of concentration is 10 *.
3, working buffer liquid
Preparation contains the PBST (contain the 0.01mol/l～0.03mol/l phosphate buffer of 0.3%～0.6%Tween-20 and 0.005%～0.015% thimerosal antiseptic, its pH value is 7.0～7.6) of 0.5%～1.5% bovine serum albumin(BSA) (BSA).
4, colour developing liquid
The concentration of developer A (3,3 ', 5,5 '-tetramethyl benzidine (TMB) solution) TMB is 1g/l～3g/l, and all the other are dimethyl sulfoxide (DMSO)-ethanolic solution; Developer B (0.05mol/l phosphoric acid-citrate buffer solution-hydrogen peroxide), the pH value is 4.0～6.0, the concentration of hydrogen peroxide is 0.01%～0.03%.A liquid and B liquid mix in 1: 7～1: 10 ratio during use.
5, stop buffer
Compound concentration is 0.05mol/l～1mol/lH
2SO
4As stop buffer.
6, first antibody solution
With work damping fluid dissolving first antibody, being mixed with protein concentration is the solution use of 20～100ng/ml.
7, second antibody concentrate
With work damping fluid dissolving second antibody, be mixed with the solution that protein concentration is 2000～10000ng/ml, dilute 100 times of uses with the work damping fluid during use.
8, chloromycetin standard solution
Be respectively with chloromycetin standard items preparations chloromycetin series standard product concentration: 0,0.025ng/ml, 0.2ng/ml, 1.5ng/ml, 12ng/ml, 100ng/ml (or 0,0.04ng/ml, 0.3ng/ml, 2ng/ml, 15ng/ml, 100ng/ml).
The establishment of embodiment 2 ELISA kits
Set up ELISA kit, its composition comprises:
1, wrapped by good micro-reaction plate (Coating Plate): 1.
2, chloromycetin standard items (Standard): 6 bottles (1ml～1.2ml/ bottle), content is respectively: CM100 (100ng/ml), CM12 (12ng/ml), CM1.5 (1.5ng/ml), CM0.2 (0.2ng/ml), CM0.025 (0.025ng/ml), B0 (0ng/ml).
3, one resists one bottle (6～8ml).
4, two is anti-: 1 bottle (100 *, 0.12～0.15ml).
5, developer A (Chromogen SolutionA): 1 bottle (1.2～1.5ml).
6, developer B (Chromogen Solution B): 1 bottle (12～15ml).
7, stop buffer (Stop Reagent): 1 bottle (12～15ml).
8, sample dilution (Assay Buffer): 1 bottle (1 *, 30～60ml).Be used for the dilution of horseradish peroxidase bond and sample.
9, concentrated cleaning solution (Dilution Wash Buffer): 1 bottle (10 *, 30～50ml).Be used for the flushing of micro-reaction plate.
10, the cover plate film is 1.
11, data analysis software (CD)
The establishment of embodiment 3 ELISA kits
Set up ELISA kit, its composition comprises:
1, wrapped by good micro-reaction plate (Coating Plate): 1.
2, chloromycetin standard items (Standard): 6 bottles (1ml～1.2ml/ bottle), content is respectively: CM100 (100ng/ml), CM15 (15ng/ml), CM2 (2ng/ml), CM0.3 (0.3ng/ml), CM0.04 (0.04ng/ml), B0 (0ng/ml).
3, one resists one bottle (6～8ml).
4, two is anti-: 1 bottle (100 *, 0.12～0.15ml).
5, developer A (Chromogen Solution A): 1 bottle (1.2～1.5ml).
6, developer B (Chromogen Solution B): 1 bottle (12～15ml).
7, stop buffer (Stop Reagent): 1 bottle (12～15ml).
8, sample dilution (Assay Buffer): 1 bottle (1 *, 30～60ml).Be used for the dilution of horseradish peroxidase bond and sample.
9, concentrated cleaning solution (Dilution Wash Buffer): 1 bottle (10 *, 30～50ml).Be used for the flushing of micro-reaction plate.
10, the cover plate film is 1.
11, data analysis software (CD)
Claims (14)
1. a broad spectrum ELISA kit is characterized in that it comprises microwell plate, chloromycetin series standard product solution, antibody-solutions, thickening and washing solution, working buffer solution, developer, the stop buffer that wraps quilt.
2. according to kit described in the claim 1, it is characterized in that: its maximum detection range is 0.025ng/ml～100ng/ml.
3. according to kit described in claim 1 or 2, it is characterized in that: described chloromycetin series standard product solution is 0 respectively, 0.025ng/ml, 0.2ng/ml, 1.5ng/ml, 12ng/ml, 100ng/ml, or 0,0.04ng/ml, 0.3ng/ml, 2ng/ml, 15ng/ml, 100ng/ml.
4. according to kit described in the claim 1, it is characterized in that: described concentrated cleaning solution is to comprise that the pH value is 7.0～7.6, concentration is the phosphate buffer of 0.1mol/l～0.15mol/l, the thimerosal antiseptic of the Tween-20 of percentage by weight 3%～6% and percentage by weight 0.05%～0.15%.
5. according to kit described in the claim 1, it is characterized in that: described working buffer solution is to comprise that the pH value is 7.0～7.6, concentration is the phosphoric acid of 0.01mol/l～0.015mol/l, the Tween-20 of percentage by weight 0.5%～1.5% bovine serum albumin(BSA) (BSA), percentage by weight 0.3%～0.6% and the thimerosal antiseptic of percentage by weight 0.005%～0.015%.
6. according to kit in the claim 5, it is characterized in that: the bovine serum albumin(BSA) in the described working buffer solution is replaceable to be one of calf serum, rabbit anteserum or gelatin.
7. according to the described kit of claim 1, it is characterized in that: described developer comprises developer A and developer B.
8. according to described in the claim 7, it is characterized in that: developer A comprises 3,3 ', 5 of concentration 1g/l～3g/l, 5 '-tetramethyl benzidine or o-phenylenediamine.
9. according to described in the claim 7, it is characterized in that: developer B is for one of being selected from 0.03mol/l～0.06mol/l phosphoric acid-citrate buffer solution-hydrogen peroxide, 0.03mol/l～0.06mol/l phosphoric acid-acetate buffer solution-hydrogen peroxide, the 0.03mol/l～0.06mol/l phosphoric acid-acetate buffer solution-urea peroxide.
10. according to described in the claim 9, it is characterized in that: the pH value of developer B is 4.0～6.0, and wherein the concentration of hydrogen peroxide is 0.01%～0.03%.
11. described in each, it is characterized in that: developer A and developer B proportioning in use are 1: 7～1: 10 according to claim 7-10.
12. the preparation method of a broad spectrum ELISA kit comprises that bag is by the preparation of microwell plate and sealing, antibody-solutions preparation, concentrated cleaning solution preparation, the preparation of working buffer liquid, the preparation of colour developing liquid, the preparation of stop buffer.
13. the preparation method according to the described kit of claim 12 is characterized in that: bag is 50～400ng/ml by the microwell plate protein concentration, and antibody-solutions concentration is 20～100ng/ml.
14. the data analysing method of a wide range chloromycetin enzyme linked immunosorbent detection is characterized in that: ((1+ (X/C) ^B)+D) regression curve carries out data processing to Y=(A-D) to adopt 4-Paramater.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788560A (en) * | 2010-03-17 | 2010-07-28 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN102288761A (en) * | 2011-05-09 | 2011-12-21 | 广州万孚生物技术有限公司 | Chloramphenicol testing kit and preparation method of same |
| CN105319369A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting chloramphenicol, and detection method thereof |
| CN106568933A (en) * | 2016-10-31 | 2017-04-19 | 深圳市安鑫宝科技发展有限公司 | Aquatic animal diagnosing instrument |
-
2006
- 2006-11-20 CN CNA2006101458262A patent/CN101191796A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101788560A (en) * | 2010-03-17 | 2010-07-28 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN101788560B (en) * | 2010-03-17 | 2013-01-16 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN102288761A (en) * | 2011-05-09 | 2011-12-21 | 广州万孚生物技术有限公司 | Chloramphenicol testing kit and preparation method of same |
| CN105319369A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting chloramphenicol, and detection method thereof |
| CN106568933A (en) * | 2016-10-31 | 2017-04-19 | 深圳市安鑫宝科技发展有限公司 | Aquatic animal diagnosing instrument |
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