CN102252986B - Kit for detecting beta-lactamase in raw milk and dairy products and detection method thereof - Google Patents
Kit for detecting beta-lactamase in raw milk and dairy products and detection method thereof Download PDFInfo
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- CN102252986B CN102252986B CN 201110162366 CN201110162366A CN102252986B CN 102252986 B CN102252986 B CN 102252986B CN 201110162366 CN201110162366 CN 201110162366 CN 201110162366 A CN201110162366 A CN 201110162366A CN 102252986 B CN102252986 B CN 102252986B
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Abstract
The invention discloses a kit for detecting beta-lactamase in raw milk and dairy products and a detection method thereof, and reagents of the kit for detecting the beta-lactamase comprise a beta-lactamase standard product, a benzylpenicillin potassium standard liquid product, a protein precipitator, namely trichloroacetic acid (TCA), a color developing agent, namely 2,2'-biquinoline ethanol solution and a complexing agent, namely CuSO4. The detection method comprises the following steps: firstly setting parameters of a detecting instrument, adding distilled water, standard liquid and a sample into a blank tube, a standard tube and a sample tube respectively, simultaneously adding the protein precipitator TCA, fully oscillating and mixing, then placing into a centrifuge for centrifugation at 4000rpm for 10 minutes, taking supernatant liquid, adding the 2,2'-biquinoline ethanol solution, fully uniformly shaking, standing at room temperature for 5 minutes, then performing colorimetric determination through the instrument and indicating the content of the beta-lactamase according to color development reaction. The kit and the detection method thereof are simple and easy to operate, the detection cost is low, the detection result is accurate and the beta-lactamase in the raw milk and the dairy products can be fast detected.
Description
Technical field
The present invention relates to for detection of with the semiquantitative determination dairy products in the kit and preparation method thereof of beta-lactamase, belong to the food safety detection field.
Background technology
Beta-lactamase (β-lactamase), be commonly called as fresh cow's milk microbiotic distintegrant, have another name called and separate anti-dose or magnificent blue enzyme preparation, it is produced and secretion by gram-positive bacterium, residual beta-lactam antibiotic in the alternative decomposing milk, conventional detection method such as microbial method, euzymelinked immunosorbent assay (ELISA) was lost efficacy, thereby can't correctly judge whether to be " antibiotic-free milk ", cover the fact that microbiotic exceeds standard.
Beta-lactam antibiotic is most widely used microbiotic in milk is produced, be used for the treatment of garget and other bacterial infection diseases, if in strict accordance with diseases such as predetermined treatment cow's milk inflammation, abuse of antibiotics will not cause the microbiotic in the cow's milk to exceed standard.Because antibiotic existence, being difficult on the one hand becomes the qualified product of safety, on the other hand, also can influence the processing of dairy produces such as sour milk, cheese.(1985) such as Korycka-Dahl M. utilize residual microbiotic in the beta-lactamase degraded milk, can be reduced to the level that the conventional sense method can't detect.Some lawless persons as antibiotic distintegrant in the milk, to cover the fact that its antibiotic residue exceeds standard, become beta-lactamase into qualified milk thereby microbiotic is exceeded standard suckle, and this has just caused the illegal interpolation of exogenous beta-lactamase.
The beta-lactam endonuclease capable makes the structural failure of penicillin lactams and loses activity, and causes antibiotics drug resistances such as penicillin, cynnematin to increase, thereby greatly reduces the ability that people resist infectious disease, and that gives the consumer healthyly brings harm.Penicillin under the beta-lactamase effect, in the enzyme nucleophilic group to the beta-lactam nucleus attack, but decarboxylation reset to generate the intermediate penicilloic acid after the open loop.The beta-lactam antibiotic allergic reaction that with penicillin is representative is very general, and the base substance that causes allergic reaction has two kinds, and a kind of is that the penicillin cracking generates some penicilloic acids, forms antigen and sensitization with protein bound.Another kind of anaphylactogen is some superpolymer, and for self-polymerization after the beta-lactam nucleus open loop forms, extent of polymerization is more high, and allergic reaction is more strong.Though cause the case of penicillin anaphylaxis not to be studied proof owing to add beta-lactamase, but the irritated mechanism according to penicillin, and the interpolation of beta-lactamase may cause the chemical sproof reason of beta-lactam antibiotic, really should enhance your vigilance to adding excessively of beta-lactamase, strictly management.Though some microorganism can produce beta-lactamase, but because component, sterilization method and the packing and storing method etc. of adopting milk mode, milk are unsuitable for producing the existence of beta-lactamase microorganism, be the beta-lactamase that can't detect microorganisms under the condition of non-artificial interpolation beta-lactamase.
On February 4th, 2009, according to nine departments such as the Ministry of Public Health regulation of " about carrying out the emergency notice that the non-edible material from soybeans of illegal interpolation and abuse food additives focus efforts on special areas are hit in the whole nation ", hit carrying out in a deep going way of the non-edible material from soybeans of illegal interpolation and abuse food additives sole rectification for cooperating the whole nation, the focus efforts on special areas Committee of Experts proposes " the non-edible material from soybeans list of the illegal interpolation of possibility in the food ", wherein just comprises beta-lactamase.Exogenous beta-lactamase is that China does not allow the chemical substance used in food, and the fact of residues of antibiotics in the milk has been covered in its use.
Healthy for consumers in general safeguarded the competitive environment that dairy market is good, sets up quick detection kit and the detection method thereof of beta-lactamase in a kind of simple, convenient, economic dairy products, has been a problem of needing solution badly.
Summary of the invention
A kind of kit for detection of beta-lactamase in the dairy products comprises beta-lactamase standard items, benzylpenicillin potassium titer product, protein precipitant trichloroacetic acid (TCA), developer 2,2'-diquinoline ethanolic solution and complexing agent CuSO
4Wherein said reagent beta-lactamase standard items enzyme activity is 2000U/mL, and benzylpenicillin potassium titer product concentration is 50000U/mL, and protein precipitant TCA concentration is 10% ~ 15%, and 2,2'-diquinoline ethanolic solution concentration is 0.05% ~ 0.1%, complexing agent CuSO
4Concentration is 20 ~ 40mmol/L.
Use the mentioned reagent box to detect the detection method of beta-lactamase in the dairy products, concrete steps are as follows:
(1) according to reaction principle and instrument performance specification test parameter;
(2) in blank pipe, add distilled water, in sample hose, add the dairy products sample; Add penicillin thiazole acid reaction liquid in standard pipe, this penicillin thiazole acid reaction liquid is prepared as follows and obtains: the benzylpenicillin potassium concentration of substrate is 50000U/mL, adds the 2000U/mL beta-lactamase, 37 ℃ of water-bath 60min; High speed refrigerated centrifuge 10min, rotating speed 10000rpm, supernatant are the accurate reactant liquor of penicillin thiazole acidity scale;
(3) blank pipe, standard pipe and sample hose in the step (2) are added protein precipitant 10% ~ 15%TCA simultaneously, the volume ratio of TCA and distilled water, dairy products sample and penicillin thiazole acid reaction liquid is 1:1; Abundant vibration 5min, the centrifugal 10min of 4000rpm gets supernatant 100 ~ 200 μ L after centrifugal, adds 2.5 ~ 3.5mL 2,2'-diquinoline ethanol, 0.2 ~ 0.4mL, 20 ~ 40mmol/L CuSO
4Solution fully shakes up, and after room temperature leaves standstill the chromogenic reaction of 5 min, by the spectrophotometric determination of absorbance method, draws the absorbance result.
Described absorbance method adopts single wavelength to detect, and measures absorbance under the single wavelength condition of 547nm.
Positive beneficial effect: the principle of invention, the decomposition product that beta-lactamase decomposes benzylpenicillin potassium is penicilloic acid, penicilloic acid has reductibility, can be with Cu
2+Be reduced to Cu
+, and cuproine 2,2 '-diquinoline and Cu
+Coupling generates the aubergine complex compound, utilizes visible spectrophotometer to measure complex compound, at the 547nm place maximum absorption band is arranged, and color is along with the beta-lactamase concentration becomes the positive correlation (see figure 1).
Description of drawings
Fig. 1 reaction principle figure;
Fig. 2 benzylpenicillin potassium standard items liquid chromatogram;
The liquid chromatogram of Fig. 3 penicillin thiazole acid;
The linear relationship chart of Fig. 4 absorbance and beta-lactamase.
Embodiment
A kind of kit for detection of beta-lactamase in the dairy products comprises beta-lactamase standard items, benzylpenicillin potassium titer product, protein precipitant trichloroacetic acid (TCA), developer 2,2'-diquinoline ethanolic solution and complexing agent CuSO
4Wherein said reagent beta-lactamase standard items enzyme activity is 2000U/mL, benzylpenicillin potassium titer product concentration is 50000U/mL, and protein precipitant TCA concentration is 10% ~ 15%, 2,2'-diquinoline ethanolic solution concentration is 0.05% ~ 0.1%, and complexing agent CuSO4 concentration is 20 ~ 40mmol/L.
Below in conjunction with the drawings and specific embodiments, the present invention is further illustrated:
A kind of kit for detection of beta-lactamase in the dairy products is characterized in that comprising beta-lactamase standard items, benzylpenicillin potassium titer product, protein precipitant trichloroacetic acid (TCA), developer 2,2'-diquinoline ethanolic solution and complexing agent CuSO
4Wherein said reagent beta-lactamase standard items enzyme activity is 2000U/mL, and benzylpenicillin potassium titer product concentration is 50000U/mL, and protein precipitant TCA concentration is 10% ~ 15%, and 2,2'-diquinoline ethanolic solution concentration is 0.05% ~ 0.1%, complexing agent CuSO
4Concentration is 20 ~ 40mmol/L.
Described penicillin thiazole acid reaction liquid is prepared as follows and obtains:
The benzylpenicillin potassium concentration of substrate is 50000U/mL, adds the 2000U/mL beta-lactamase, 37 ℃ of water-bath 60min; High speed refrigerated centrifuge (10000rpm) 10min, supernatant is penicillin thiazole acid solution.The standard items of benzylpenicillin potassium with the efficient liquid phase collection of illustrative plates of penicillin thiazole acid shown in Fig. 2 and 3.
A kind of kit for detection of beta-lactamase in the dairy products, it is for detection of the detection method of beta-lactamase in the dairy products, and concrete steps are as follows:
(1) according to reaction principle and instrument performance specification test parameter;
(2) in blank pipe, add distilled water, in standard pipe, add titer, in sample hose, add raw milk or dairy products sample;
(3) blank pipe, standard pipe and sample hose in the step (2) are added protein precipitant 10% ~ 15% TCA simultaneously, 5min fully vibrates, the centrifugal 10min of 4000rpm, get supernatant 100 ~ 200 μ L after centrifugal, add 2.5 ~ 3.5mL 2,2'-diquinoline ethanol, 0.2 ~ 0.4mL 20mmol/mL CuSO
4Solution fully shakes up, and after room temperature left standstill the chromogenic reaction of 5 min, the spectrophotometric determination by the inspection absorbance method drew the absorbance result.
Described inspection absorbance method adopts single wavelength to detect, and measures absorbance under the single wavelength condition of 547nm.
Embodiment 1
The invention provides a kind of kit that detects beta-lactamase, comprise beta-lactamase standard items, benzylpenicillin potassium titer product, developer 0.05% 2,2'-diquinoline ethanolic solution, protein precipitant 10%TCA and 20mmol/L CuSO
4
(1) penicillin thiazole acid reaction liquid is prepared as follows and obtains:
The benzylpenicillin potassium concentration of substrate is 50000U/mL, adds the 2000U/mL beta-lactamase, 37 ℃ of water-bath 60min; High speed refrigerated centrifuge (10000rpm) 10min, supernatant are penicillin thiazole acid reaction liquid.
(2) configuration of kit reagent
The configuration of 10%TCA: the 10g trichloroacetic acid is dissolved in the 100mL water;
0.05% 2,2'-diquinoline ethanolic solution: 0.05g 2, and the 2'-diquinoline is dissolved in the 100mL ethanol;
20mmol/L CuSO
4: the anhydrous CuSO of 0.5g
4Be dissolved in 100mL distilled water.
Embodiment 2
A kind of kit that detects beta-lactamase in the dairy products is when carrying out the beta-lactamase detection, and the concrete steps of this method are as follows:
(1) gets the penicillin thiazole acid reaction liquid of 0 μ L, 40 μ L, 120 μ L, 250 μ L, 500 μ L, 1000 μ L, 1500 μ L, 2000 μ L respectively, be settled to 10mL, be mixed with the gradient concentration that liquid is used in the acid of penicillin thiazole.
(2) the instrument detected parameters is set, detects wavelength 547nm;
(3) get distilled water, dairy products sample and penicillin thiazole acid reaction liquid 5mL, add protein precipitant 15%TCA 5mL respectively, pour the centrifugal 10min of 4000rpm in the centrifuge tube into;
(4) get supernatant 100 μ L, insert test tube, add 0.1% 2,2'-diquinoline ethanolic solution 2.5mL and 40mmol/L CuSO4 0.1mL and mix, room temperature by the spectrophotometric determination of absorbance method, is obtained a result after the chromogenic reaction of 5min.
Embodiment 3
The test of kit minimum detectability
(1) prepares the sample that contains 0U/mL, 4U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, 150U/mL, 200U/mL, 250U/mL beta-lactamase respectively with milk, the benzylpenicillin potassium concentration of substrate is the solution of 5000U/mL, mixes back 37 ℃ of water-bath 60min;
(2) take out the back and add 15% TCA solution 5 mL in the 1:1 ratio, fully put into hydro-extractor after the vibration, the centrifugal 10min of 4000rpm adds 0.05% 2,2'-diquinoline ethanolic solution 3.5mL and 30mmol/L CuSO4 0.2mL mix, and room temperature is through the chromogenic reaction of 5min;
(3) utilize visible spectrophotometer to measure absorbance at 547nm, obtain a result.
(4) drawing standard curve: beta-lactamase (U/ mL) standard solution with variable concentrations is marked as vertical X, with absorbance as the Y value, drawing standard curve (as shown in Figure 4).The test result of its empty pipe is as shown in table 1, and its theory detects and is limited to 3.44U/mL, and actual detecting is limited to 4 U/mL.
The absorbance at table 1 blank test revision test 547nm place
The theory that can calculate this method by table 1 detects and is limited to:
CL=3S
b/b=3×0.058/0.0144=3.44U/mL
Embodiment 4
The contrast of kit method of testing and additive method
Iodimetric titration, high performance liquid chromatography, visible spectrophotometry and kit are compared in detectability, correlativity, operability, detection time, the simple and easy degree of operation, as shown in table 2 below.
The contrast of table 2 kit method of testing and additive method
The principle of invention, the decomposition product that beta-lactamase decomposes benzylpenicillin potassium is penicilloic acid, penicilloic acid has reductibility, can be with Cu
2+Be reduced to Cu
+, and cuproine 2,2 '-diquinoline and Cu
+Coupling generates the aubergine complex compound, utilizes visible spectrophotometer to measure complex compound, at the 547nm place maximum absorption band is arranged, and color is along with the beta-lactamase concentration becomes positive correlation.Kit and detection method thereof among the present invention are easy to operation, and it is low to detect cost, and testing result is accurate, can realize the fast detecting to beta-lactamase in the dairy products.
Above embodiment only is used for explanation preferred implementation of the present invention; but the present invention is not limited to above-mentioned embodiment; in the ken that described field those of ordinary skill possesses; any modification of doing within the spirit and principles in the present invention, be equal to and substitute and improvement etc., it all should be encompassed within the technical scheme scope that the present invention asks for protection.
Claims (3)
1. kit for detection of beta-lactamase in the dairy products, it is characterized in that: comprise beta-lactamase standard items, benzylpenicillin potassium titer product, protein precipitant trichloroacetic acid (TCA), developer 2,2'-diquinoline ethanolic solution and complexing agent CuSO
4Wherein said reagent beta-lactamase standard items enzyme activity is 2000U/mL, and benzylpenicillin potassium titer product concentration is 50000U/mL, and protein precipitant TCA concentration is 10% ~ 15%, and 2,2'-diquinoline ethanolic solution concentration is 0.05% ~ 0.1%, complexing agent CuSO
4Concentration is 20 ~ 40mmol/L.
2. a right to use requires 1 described kit to detect the detection method of beta-lactamase in the dairy products, and it is characterized in that: concrete steps are as follows:
(1) according to reaction principle and instrument performance specification test parameter;
(2) in blank pipe, add distilled water, in sample hose, add the dairy products sample; Add penicillin thiazole acid reaction liquid in standard pipe, this penicillin thiazole acid reaction liquid is prepared as follows and obtains: the benzylpenicillin potassium concentration of substrate is 50000U/mL, adds the 2000U/mL beta-lactamase, 37 ℃ of water-bath 60min; High speed refrigerated centrifuge 10min, rotating speed 10000rpm, supernatant are the accurate reactant liquor of penicillin thiazole acidity scale;
(3) blank pipe, standard pipe and sample hose in the step (2) are added protein precipitant 10% ~ 15%TCA simultaneously, the volume ratio of TCA and distilled water, dairy products sample and penicillin thiazole acid reaction liquid is 1:1; Abundant vibration 5min, the centrifugal 10min of 4000rpm gets supernatant 100 ~ 200 μ L after centrifugal, adds 2.5 ~ 3.5mL 2,2'-diquinoline ethanol, 0.2 ~ 0.4mL, 20 ~ 40mmol/L CuSO
4Solution fully shakes up, and after room temperature leaves standstill the chromogenic reaction of 5 min, by the spectrophotometric determination of absorbance method, draws the absorbance result.
3. right to use according to claim 2 requires 1 described kit to detect the detection method of beta-lactamase in the dairy products, it is characterized in that: described absorbance method adopts single wavelength to detect, and measures absorbance under the single wavelength condition of 547nm.
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| CN 201110162366 CN102252986B (en) | 2011-06-16 | 2011-06-16 | Kit for detecting beta-lactamase in raw milk and dairy products and detection method thereof |
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| CN 201110162366 CN102252986B (en) | 2011-06-16 | 2011-06-16 | Kit for detecting beta-lactamase in raw milk and dairy products and detection method thereof |
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| CN103344769A (en) * | 2013-07-12 | 2013-10-09 | 上海市动物疫病预防控制中心 | Kit for quantitatively detecting beta-lactamase residue in milk |
| CN103940865B (en) * | 2014-04-10 | 2016-01-13 | 山东理工大学 | A kind of belt-lactam antibiotics residues fast detector |
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2011
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| CN102095724A (en) * | 2010-11-24 | 2011-06-15 | 青岛佳明测控仪器有限公司 | Method for determining copper content in water by biquinolyl spectrophotometry |
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