CN107703130A - Suitable for the acetes chinensis method for the beta lactamase for detecting Residues in Milk - Google Patents
Suitable for the acetes chinensis method for the beta lactamase for detecting Residues in Milk Download PDFInfo
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- CN107703130A CN107703130A CN201710846108.6A CN201710846108A CN107703130A CN 107703130 A CN107703130 A CN 107703130A CN 201710846108 A CN201710846108 A CN 201710846108A CN 107703130 A CN107703130 A CN 107703130A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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Abstract
The invention discloses the acetes chinensis method of the beta lactamase suitable for detecting Residues in Milk, utilize colorimeter detection method, certain density milk soln is prepared by raw material of nonreactive milk powder, different amounts of beta-lactam enzyme stock solution and enough substrate nitrocefins are separately added into after quantitative absorption, after water-bath for a period of time, generation color change, and determined with colour difference meter, the standard curve of various concentrations beta lactamase is drawn, the beta lactamase content in testing sample is determined further according to calibration curve method；Chromogenic reaction of the present invention, discoloration is obvious, high sensitivity, and time-consuming short, compare other detection methods, can reduce the minimum detectability of beta lactamase, suitable for the present situation of the commercially available milk detection in current China.
Description
Technical field
The invention discloses a kind of milk detection field, is especially adapted for use in the beta-lactamase of detection Residues in Milk
Acetes chinensis method.
Background technology
With the rapid development of economy, the living standard of people improves therewith, the also more and more higher of the pursuit to quality of the life.
Milk, it has also become daily life drink enters in the life of people.But the beta-lactamase of residual can be contained in milk, due to
It is both domestic and external for Journal of Sex Research it is fewer, or even connect temperature to the influence degree of beta-lactam enzymatic activity, beta-lactamase and its
The edible safety of metabolite is indefinite, so beta-lactamase is not allowed using addition in food service industry.
In order to realize the detection to beta-lactamase antibiotic in commercially available milk, the present invention is using according to kit detection method
On the basis of add colour difference meter method and detected, can quickly, economic, the accurate residual antibiotic detected in milk, expire
The current China market demand of foot.
The content of the invention
In order to realize the detection to commercially available milk beta-lactamase residues, while also lack economy to solve current China,
Accurately, the problem of quick beta-lactamase residues detection, the purpose for allowing consumer to trust is reached, the present invention proposes one kind and existed
Colour difference meter method is added on the basis of kit detection method makes the detection method that its final minimum detectability substantially reduces, its technical side
Case is as follows：
Suitable for the acetes chinensis method for the beta-lactamase for detecting Residues in Milk, it is characterised in that：Utilize colour difference meter
The colourity of beta-lactamase and substrate after Nitrocefin colour developing in sample is detected, and is determined by calibration curve method in sample
The content of beta-lactamase, concrete operation step are as follows：
(1) 10mL milk testing samples are drawn to insert in water white transparency test tube；
(2) the Nitrocefin solution and 1-5U/ μ l for being 500mg/ml by addition concentration in the test tube of above-mentioned steps (1)
The concussion of beta-lactam enzyme stock solution is uniform；
(3) the test tube mouth of pipe in above-mentioned steps (2) is sealed with preservative film, is placed in reacting in water-bath, water-bath is completed
After carry out reduction enzyme activity；
(4) it is uniform that the test tube after above-mentioned steps (3) enzyme deactivation is taken out rapidly to concussion, and carries out colour difference meter measure, record is surveyed
Fixed number evidence, including but not limited to：Lightness (L), a passages (scope of carmetta to green), the b passages (model of yellow to blueness
Enclose), aberration (Δ E)；
(5) according to the aberration Value Data determined in above-mentioned steps (4), the content of beta-lactamase in sample is calculated.
Further, totally six groups of test tubes, one of which are blank control group for setting in the step (1).
Further, the minimum detectability of the beta-lactamase is in 1U/mL.
Further, milk testing sample in the step (1), using nonreactive milk powder and 50 DEG C of -100 DEG C of warm water, with 1:
6—1:10 ratio configure stand-by.
Further, water-bath reaction temperature is 30 DEG C in the step (3), water bath time 3min.
Further, enzyme activity is reduced using ice-water bath in the step (3), the reduction enzyme activity reaction time is 5s-20s.
Further, in the step (2) beta-lactam enzyme stock solution dosage be respectively 0 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l,
10μl。
Further, the Nitrocefin solution usage is 30 μ of μ l -50 l.
The present invention compared with prior art, has the following advantages：
The present invention determines the content of the beta-lactamase of Residues in Milk using colour difference meter measure value of chromatism, with existing head
Spore nitre thiophene test paper detection method, the minimum detectability of beta-lactamase is greatly reduced, examined suitable for the commercially available milk in current China
The present situation of survey；The present invention utilizes orthogonal test simultaneously, determines reaction temperature, Nitrocefin amount of solution, reaction time for whole
The influence of precursor reactant, and then standard curve is drawn, and then optimum reaction conditionses are determined, then premised on optimum reaction conditionses,
Influence of the different beta-Lactamase concentrations to Nitrocefin colour developing degree is carried out using colour difference meter measure value of chromatism；Present invention tool
Coloured change is clear, and high sensitivity is simple to operate, takes the characteristics of short, can fast and efficiently detect residual in milk
The beta-lactamase stayed, financial cost is low, and social benefit is high.
Brief description of the drawings
Fig. 1 is the graph of a relation of reaction time and test index.
Fig. 2 is the graph of a relation of reaction temperature and test index.
Fig. 3 is cephalo addition and the graph of a relation of test index.
Fig. 4 is enzyme concentration and contrastive colours difference standard curve schematic diagram.
Embodiment
With reference to the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Ground describes, it is clear that a described embodiment only well-behaved embodiment of the invention, rather than whole embodiments.Based on this
Embodiment in invention, those of ordinary skill in the art are obtained every other on the premise of creative work is not made
Embodiment, belong to protection scope of the present invention.
In order to reach the purpose of the present invention, the present invention proposes to be applied to the aberration of the beta-lactamase of detection Residues in Milk
Detection method, the colourity of beta-lactamase and substrate after Nitrocefin colour developing in sample is detected using colour difference meter, and passes through mark
Directrix curve method determines the content of beta-lactamase in sample, and concrete operation step is as follows：
(1) milk testing sample is prepared, using nonreactive milk powder and 50 DEG C of -100 DEG C of warm water, with 1:6—1:10 ratio is entered
Row configuration；Draw 10mL milk testing samples to insert in water white transparency test tube, be provided with 6 groups of test tubes, and set one group as sky
White control group；
(2) will in six groups of test tubes of above-mentioned steps (1) add be separately added into concentration be 500mg/ml Nitrocefin it is molten
Liquid dosage is the 30 μ l of μ l -50,1-5U/ μ l μ l of beta-lactam enzyme stock solution 0,2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l, and concussion is equal
It is even；
(3) the test tube mouth of pipe in above-mentioned steps (2) is sealed with preservative film, is placed in temperature to be reacted in 30 DEG C of water-baths
3min, enzyme activity 5s-20s is reduced using ice-water bath after the completion of water-bath；
(4) it is uniform that the test tube after above-mentioned steps (3) enzyme deactivation is taken out rapidly to concussion, and carries out colour difference meter measure, record is surveyed
Fixed number evidence, including but not limited to：Lightness (L), a passages (scope of carmetta to green), the b passages (model of yellow to blueness
Enclose), aberration (Δ E)；
(5) according to the aberration Value Data determined in above-mentioned steps (4), the content of beta-lactamase in sample is calculated.
Embodiment 1：
(1) the colorimeter measurement method that the present invention designs, is determined with reference to orthogonal test, and specific data parameters are following just
Experiment gauge outfit one, orthogonal test gauge outfit two are handed over, wherein, A represents enzyme concentration (U/mL), and B represents the reaction time (min), and C represents anti-
Temperature (DEG C) is answered, D represents cephalo addition (μ l).
Orthogonal test table is first
Orthogonal test designs table first two
According to orthogonal test table above is first, orthogonal test gauge outfit two adds beta-lactamase and Nitrocefin solution
Amount, control reaction time and reaction temperature, then determine the color after this nine groups of reactions with colour difference meter, do again in addition
One group of blank, also write down data with colour difference meter measure.Every group has three data, takes its average value to be designated as final data.
Colorimeter measurement orthogonal experiment data table
L represents lightness wherein in colorimeter measurement orthogonal experiment data table, a represents the passage (model of carmetta to green
Enclose), b represents the passage scope of blueness (yellow to), E represents aberration.In order to determine minimum detectability, so the concentration of enzyme can be with
Do not know first, enzyme concentration gradient can be controlled, reduce the overall density of enzyme, draw minimum detectability.Painted according to orthogonal test
The curve map that luminosity changes under the change condition of each different factors is made, so as to draw most suitable factor.Enzyme is not known first
Concentration, other most suitable factors are first determined, enzyme concentration is then changed so as to draw minimum detectability according to most appropriate factor.
Orthogonal test is analyzed by intiutive analysis method, can be intuitively by colorimeter measurement orthogonal experiment data table
Find out optimal level and be combined as No. 4, i.e. A2B1C2D3, index of test 86.56, i.e. reaction time are 3min, and reaction temperature is
30 DEG C, cephalo addition is 50 μ l；Secondly it is No. 1, i.e. A1B1C1D1, index of test 86.51.Now by intuitively analyzing
Verified：
Calculate the K of each rowijValue.By taking the 1st row A factors as an example, then
1st horizontal sum K11=86.51+86.45+86.48=259.44
2nd horizontal sum K21=86.56+86.33+86.44=259.33
3rd horizontal sum K31=86.30+86.40+86.41=259.11
Remaining factor computational methods is identical, then calculates the average value of each row same levelConcrete numerical value is as follows：
Each factor level sum and average value
Calculate the extreme difference R of each factor rowj：
A factors：Rj=64.86-64.78=0.08；
B factors：Rj=64.84-64.80=0.04；
C factors：Rj=64.86-64.78=0.08；
D factors：Rj=64.86-64.80=0.06
Find out that A factors and C factors are more important by extreme difference value, next to that D factors, are finally B factors.But A factors are enzyme
Concentration, variable can be voluntarily controlled, so determining C factors, D factors and B factors in sequence.Each factor is made with experiment to refer to
Target graph of a relation.The relation of the graph of a relation of 1 reaction time and test index, the reaction temperature of accompanying drawing 2 and test index with reference to the accompanying drawings
The graph of a relation of figure, the cephalo addition of accompanying drawing 3 and test index can be seen that B, C, and D is key factor, according to each factor most
Good level is chosen for B1C2D3, i.e. the reaction time is 3min, and reaction temperature is 30 DEG C, and Nitrocefin solution addition is 50 μ l,
This be combined as Nitrocefin and β-LactamsThe optimum combination of enzyme reaction.Next make these conditions certain, changing enzyme concentration is
Can, measure minimum detectability.
(2) according to above-mentioned test data, the minimum detectability that colour difference meter method detects milk lactamase is carried out：
With nonreactive milk powder, 80 DEG C of warm water press 1:10 proportional arrangement is respectively charged into numbering 0-5 into milk after being cooled to room temperature
Test tube in, every bottle of 10ml；No. 0 is used as blank control group, and 2,4,6,8,10 microlitres of concentration are sequentially added into 1-5 test tubes
For 5U/ μ l beta-lactam enzyme stock solution, it is respectively 1U/ml, 2U/ml, 3U/ml, 4U/ml, 5U/ml to make its concentration；To No. 1-5
50 microlitres of Nitrocefin solution is separately added into test tube, concussion is uniform；Sealed with preservative film, be placed in 30 DEG C of water-bath reclaimed waters
Bathe 3min；Taken out immediately after 3min reduces enzyme activity with ice-water bath 15s；Rapid take out carries out colour difference meter measure after ice-water bath；Record
Chromatism data is determined, standard curve is drawn, as shown in Figure 4, determines enzyme minimum detectability 1U/mL.
The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all be contained
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (8)
1. the acetes chinensis method of the beta-lactamase suitable for detection Residues in Milk, it is characterised in that：Examined using colour difference meter
In test sample product after the colour developing of beta-lactamase and Nitrocefin substrate colourity, and by calibration curve method determine β in sample-
The content of lactamase, concrete operation step are as follows：
(1) 10mL milk testing samples are drawn to insert in water white transparency test tube；
(2) β-interior of Nitrocefin solution that concentration is 500mg/ml and 1-5U/ μ l will be added in the test tube of above-mentioned steps (1)
The concussion of acid amides enzyme stock solution is uniform；
(3) the test tube mouth of pipe in above-mentioned steps (2) is sealed with preservative film, is placed in reacting in water-bath, water-bath is completed laggard
Row reduces enzyme activity；
(4) it is uniform that the test tube after above-mentioned steps (3) enzyme deactivation is taken out rapidly to concussion, and carries out colour difference meter measure, record measure number
According to including but not limited to：Lightness (L), a passages scope of green (carmetta to), b passages (scope of yellow to blueness),
Aberration (Δ E)；
(5) according to the aberration Value Data determined in above-mentioned steps (4), the content of beta-lactamase in sample is calculated.
2. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：Totally six groups of test tubes, one of which are blank control group for setting in the step (1).
3. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：The minimum detectability of beta-lactamase is in 1U/mL.
4. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：Milk testing sample in the step (1), using nonreactive milk powder and 50 DEG C of -100 DEG C of warm water, with 1:6—1:10 ratio
Example configure stand-by.
5. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：Water-bath reaction temperature is 30 DEG C in the step (3), water bath time 3min.
6. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：Enzyme activity is reduced using ice-water bath in the step (3), the reduction enzyme activity reaction time is 5s-20s.
7. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：Beta-lactam enzyme stock solution dosage is respectively 0 μ l, 2 μ l, 4 μ l, 6 μ l, 8 μ l, 10 μ l in the step (2).
8. the acetes chinensis method of the beta-lactamase according to claim 1 for being applied to detection Residues in Milk, it is special
Sign is：The Nitrocefin solution usage is 30 μ of μ l -50 l.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710846108.6A CN107703130A (en) | 2017-09-19 | 2017-09-19 | Suitable for the acetes chinensis method for the beta lactamase for detecting Residues in Milk |
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| CN201710846108.6A CN107703130A (en) | 2017-09-19 | 2017-09-19 | Suitable for the acetes chinensis method for the beta lactamase for detecting Residues in Milk |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112683893A (en) * | 2020-12-21 | 2021-04-20 | 南京康容健康科技有限公司 | High-sensitivity helicobacter pylori detection kit |
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| CN112683893A (en) * | 2020-12-21 | 2021-04-20 | 南京康容健康科技有限公司 | High-sensitivity helicobacter pylori detection kit |
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