CN101928748A - Quick detection method for beta-lactamase in dairy food - Google Patents
Quick detection method for beta-lactamase in dairy food Download PDFInfo
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- CN101928748A CN101928748A CN2009100628522A CN200910062852A CN101928748A CN 101928748 A CN101928748 A CN 101928748A CN 2009100628522 A CN2009100628522 A CN 2009100628522A CN 200910062852 A CN200910062852 A CN 200910062852A CN 101928748 A CN101928748 A CN 101928748A
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Abstract
The invention discloses a quick determination method for beta-lactamase, which comprises the following steps: mixing a sample to be determined, such as the beta-lactamase contained in milk and a substrate such as penicillin in a mode of two groups of independent flowing phases in a reactor and carrying out catalytic reaction; converting heat generated by biological catalysis into change of temperature; and determining the components, concentration or activity and the like of the sample to be determined by measuring the relative change of temperature. The determinator of the beta-lactamase is an enzyme thermistor which can quantitatively and qualitatively analyze the content and activity of the beta-lactamase in the liquid sample. The method of the invention is suitable for quick detection (5 minutes/sample) of the beta-lactamase in analysis of food, especially dairy products, beverage and the like, and has low detection price (about ** yuan RMB/sample), simple operation, no need of pretreatment of the sample and high sensitivity and accuracy (activity below 0.5 active unit/mL can be measured).
Description
Technical field
The present invention relates to bioassay technique, more specifically relate to the method for quick of β-Nei Xiananmei in a kind of dairy products, be applicable to the fast check and analysis of the β-Nei Xiananmei in enzyme or enzyme substrates, the especially dairy products in all kinds of liquid-foods.
Background technology
At present, the method that directly detects β-Nei Xiananmei mainly is based on the detection kit of antigen-antibody reaction.Testing cost height (hundred yuan/every sample), and the specificity of antibody is bad, is subjected to the interference of other composition easily, and precision is not enough.Another kind method is to use the activity of the indirect measurement β-Nei Xiananmei of chromatogram, this method measuring accuracy height, but testing expense is expensive more, and test period is longer relatively, can't on-the-spotly detect.All existing methods all can not be fast and on-the-spot (as adopting milking station) test because they all need the milk sample is carried out pre-treatment.
The method for quick of β-Nei Xiananmei is based on the hot biological analyser technology of enzyme (Enzyme Thermistor) in the dairy products involved in the present invention.It is integrated zymotechnic, microfluid analysis technology, low-grade fever sensing technology etc.It does not need to sample carry out pre-treatment, have very high measuring accuracy, fast, advantage such as testing cost is low.This detector is made of two big parts: hot biological respinse of enzyme and transmitter and signal amplify and treater.
The hot biological analyser of enzyme is based on this principle, that is, specific enzymic catalytic reaction (as, beta-lactam enzyme catalysis penicillin in the milk) and a kind of general mobile calorimetric analysis are combined, thereby realize unique, omnipotent bioanalysis and measurement.
Summary of the invention
The object of the present invention is to provide the method for quick of β-Nei Xiananmei in a kind of dairy products, be applicable to, especially to the fast check and analysis of the β-Nei Xiananmei in the dairy products enzyme or enzyme substrates in all kinds of liquid-foods.
Main contents of the present invention are: the rapid assay methods that has developed a kind of β-Nei Xiananmei.This method is with testing sample, as the β-Nei Xiananmei that contains in the liquid of suckling and its substrate, as penicillin with two groups independently moving phase mix and carry out catalyzed reaction at a reactor, the heat that its biocatalysis is produced changes into variation of temperature, and determines composition, concentration or the activity etc. of its determinand by the relative variation of measuring its temperature.This β-Nei Xiananmei determinator is the hot biological analyser of enzyme, β-Nei Xiananmei content and activity in energy quantitative analysis and the qualitative analysis liquid sample.Be suitable in the food analysis rapid detection of the β-Nei Xiananmei in especially milk-product, the beverage etc., and have the price of detection low, easy and simple to handle, need not sample is carried out pre-treatment and very high sensitivity and precision.
The method for quick of β-Nei Xiananmei in a kind of dairy products, its step is as follows:
B. with SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4) 2-6g, Sodium phosphate dibasic (Na
2HPO
4) 17-24g (purchasing the company in sigma) mixing, deionized water is settled to 1000mL and obtains the 50-200mM phosphate solution then, and pH is 7-7.4.
C. be the penicillinase stoste (penicillin stoste is the mixed solution of penicillinase and 100mM phosphate solution) of 1000U/mL with the 100mM phosphate solution configuration concentration for preparing.Penicillinase Penicillinase (360U/mg) purchases the company in sigma.Use 100mM phosphorus concentration acid salt solution weaker concn 1000U/mL penicillinase stoste again, obtaining penicillinase concentration is the penicillinase stoste of 10U/mL.Continue to use the 100mM phosphate solution, by the same procedure gradient dilution, preparation penicillinase concentration is 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the penicillinase reference liquid of 0.25U/mL.
D. use penicillinase reference liquid (10U/mL through gradient dilution, 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, 0U/mL) mix (volume ratio 1: 1) with milk (lipid content 0.5-3.3%), obtain penicillinase concentration and be respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the milk sample of 0.25U/mL and 0U/mL (blank).
E. prepare substrate with phosphate solution, concentration is the penicillin solution (penicillin P enicillin G purchases the company in sigma) of 100mM, is diluted to required concentration of substrate 1mM.
F. assembling, regulate instrument (the hot biological analyser Enzyme of the enzyme Thermistor that adopts Ou Mike biotechnology (Wuhan) company limited to produce): peristaltic pump is calibrated to required flow velocity (0.25-1.0mL/min), insert sampling valve (sample size is 100-500 μ L), and instrument signal is amplified knob transfer to 50-200 (signal amplify 50-200 doubly), then the current meter pointer is transferred to 0 position of central authorities.At last registering instrument (or general chromatographic working station of market purchase) is connected signal output part, the voltage signal that record detects.
G. after treating baseline stability, injection contains penicillinase concentration and is respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the milk sample of 0.25U/mL and 0U/mL respectively in sampling valve.Then sampling valve is changeed to cut to " sample introduction " by " application of sample " end and hold.Detect a sample and take 5～10 minutes.When the reaction peak of a sample falls after rise to baseline, sampling valve is recalled to " application of sample " end, inject next sample.
H. when detection penicillinase concentration is the milk sample of 0U/mL, because small non-signal peak can appear in the relation of mixture heat and milk viscosity.When the processing detection contains the data of penicillinase milk sample, need deduct the peak value of blank sample with the peak value of its sample, thereby obtain correction data, the data of correction data for trying to achieve.Correction data is the linear ratio relation according to the concentration of test sample.
I. detect after the end, pump into 70～75% concentration spirituous solutions, clean instrument and sample loop (about 30 minutes).
The method for quick of β-Nei Xiananmei in a kind of dairy products, it may further comprise the steps:
The present invention is contained the scope and the analytic target that detect and analyze and is comprised:
β-Nei Xiananmei in A, all milk-product.All milk-product are defined as herein: comprise raw dairy and finished product breast, and liquid milk and solid-state breast, absolutely composition breast or part contain milk-content finished product, Chinese or foreign milk product;
B, the present invention can extend to other non-liquid-food that contains milk-content, beverage, medicine, makeup, chemicals, fermentation preparation, waste water etc.;
The detection of C, β-Nei Xiananmei is defined as: the activity of β-Nei Xiananmei, content, qualitative analysis or quantitative analysis, successive analysis or discontinuous analysis, Braking Analysis or manual analyzing, direct analysis or indirect analysis, pre-treatment or undressed sample analysis etc.
Detection of the present invention and analytical procedure comprise:
D, based on its substrate of biological enzyme (test analyte) the principle that changes of the heat content that produces;
E, measurement are detection signal by the temperature variation that this heat content changes the reactive system that is produced.This temperature signal can difference or non-differential mode measure;
F, the analysis of flow injection sample introduction combine with the hot bioanalysis of enzyme;
G, testing sample (as milk liquid) that contains β-Nei Xiananmei and the damping fluid (seeing for details as follows) that contains its substrate (as penicillin) respectively two independently flow cavity enter reactive system, mix the back at reactor and produce catalyzed reaction;
H, this enzyme-substrate catalyzed reaction can comprise: single enzyme to the catalysis of single substrate, single enzyme simultaneously to the catalysis of many substrates, multienzyme to the catalysis of single substrate, multienzyme simultaneously to catalysis of many substrates etc.
The right of beta-lactam enzyme analyser of the present invention comprises:
I, two-pass flow Controlling System and mixing tank, reactor and detector;
J, detecting sensor are temperature sensor, comprise thermistor, thermopair, thermopile, temperature-sensitive transistor etc.;
K, the hot analyzing electrode of enzyme comprise: flow system, reaction column and temperature sensor.Reactor is replaceable and not replaceable two kinds, and can fill and non-filling dual mode;
L, measurement system can be single measurement enzyme thermode and measure enzyme thermode mode more;
M, measurement system can be full-automatic type or type hand, integrated or non-integrated flow control, signal alarm or non-warning;
N, electronic measurement are comparison bridge, difference temperature survey mode.Its detection sensitivity can reach 10
-5℃.
Detection method and step:
O, two independently flow cavity by peristaltic pump simultaneously with phosphate buffered saline buffer (as 0.1M, pH7.0-7.4) and the same buffer that contains penicillin (as 1mM Penicillin G) with as, 0.5mL/ minute flow velocity be injected into the input terminus of the hot biological analyser of enzyme (ENZYME THERMISTOR INSTRUMENT) respectively, and in the reactor of this analyser, mix and flow out output terminal through heat sensitive sensor;
P, testing sample (as containing β-Nei Xiananmei milk liquid) by the sample feeding valve will as, 350 μ L sample sizes are sneaked into the only flow cavity of phosphoric acid salt buffer, and mix at reactor with its phosphate buffered saline buffer that contains penicillin, the heat that its catalyzed reaction produces passes through temperature sensor measurement.
Compared with prior art, the present invention has following advantage:
Directly the β-Nei Xiananmei in the dairy products is analyzed; Method is simple, instrumentation is convenient.Milk sample to be measured need not special pre-treatment, but direct injection analysis; Measure (about 5 minutes/every sample) fast; Detect cost low (several yuans approximately/every sample); Highly sensitive (be lower than 0.5U enzymic activity/mL sample of suckling, or be lower than 2ppm enzyme content); The milk sample is fit to the detection of the β-Nei Xiananmei in raw material milk, milk preparation and other liquid-foods, also can be used for the detection of other enzyme or the analysis of enzyme substrates.
Fig. 1 is the record diagram of specimen signal peak shown in the registering instrument.
The hot biological analyser of enzyme (Enzyme Thermistor) that is to use that shows among the figure detects active β-Nei Xiananmei sample detection response for 0U/mL, 0.25U/mL, 0.5U/mL, 1.0U/mL, 2.0U/mL and 5.0U/mL in the milk respectively.Twice of every kind of sample duplicate test.By this detection, can learn that this method detection method speed is fast, detection is stable and precision is high
Embodiment
Embodiment 1:
The method for quick of β-Nei Xiananmei the steps include: in a kind of dairy products
One. material is prepared
Sodium phosphate dibasic (Na
2HPO
4) (buying from sigma), SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4) (buying from sigma), penicillinase (Penicillinase from bacillus cereus, 360U/mg buys from sigma), penicillin (Penicillin G potassium salt, purchase is from sigma), milk (lipid content 3%), 4 ℃ of refrigerator storages.
Two. the configuration sample
1. prepare 100mM phosphate solution (pH=7.4): weighing NaH
2PO
42～6g, Na
2HPO
417-24g is settled to 1000mL with deionized water;
2. be the penicillinase stoste (penicillin stoste is the mixed solution of penicillinase and 100mM phosphate solution) of 1000U/mL with the 100mM phosphate solution configuration concentration for preparing.4 ℃ of refrigerator storage.(note, the easy inactivation of enzyme solution, dried frozen aquatic products can be stablized a week for 2～8 ℃.)；
3. use 100mM phosphorus concentration acid salt solution weaker concn 1000U/mL penicillinase stoste again, obtaining penicillinase concentration is the penicillinase stoste of 10U/mL;
4. continue to use the 100mM phosphate solution, by the same procedure gradient dilution, preparation penicillinase concentration is 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the penicillinase stoste of 0.25U/mL;
With the penicillinase original liquid concentration be respectively 10U/mL, 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, 0U/mL mix (volume ratio 1: 1) with milk (lipid content 3%), obtain penicillinase concentration and be respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the milk sample of 0.25U/mL and 0U/mL (blank).
6. prepare substrate with phosphate solution, concentration is the penicillin solution (penicillin P enicillin G potassium salt buys from sigma) of 100mM, and being diluted to required concentration of substrate is 1mM.
7. be that penicillin solution and the 100mM phosphate solution gradient dilution of 100mM is concentration 10mM with concentration, 5mM, the penicillin solution of 1mM.
Three. operation steps:
The method for quick of β-Nei Xiananmei the steps include: in a kind of dairy products
1. assembling instrument (the hot biological analyser Enzyme of the enzyme Thermistor that Ou Mike biotechnology company produces) is calibrated to 0.5mL/min with peristaltic pump.
2. 350 μ L sample loops (sample loop is that the general non-hydrophilic transparent plastics tubing in market is made) are installed.
3. instrument signal is amplified knob and transfer to 100 (signal amplifies 100 times), then the current meter pointer is transferred to 0 position of central authorities.
4. registering instrument (universal registering instrument is bought in market) voltage is transferred to 1 volt, recording paper rolling speed 10cm/hour.
5. after treating baseline stability, in sampling valve, inject penicillinase concentration respectively and be respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the milk sample of 0.25U/mL and 0U/mL.Then, sampling valve is transferred to " sample introduction " shelves by " application of sample " shelves.Detect a sample and take 5～10 minutes.When the response peak of a sample falls after rise to baseline, sampling valve is recalled to " sample introduction " shelves, inject next sample.
6. when detection penicillinase concentration is the milk sample of 0U/mL, because non-signal peak can appear in the relation of mixture heat and milk viscosity.When the processing detection contains the data of penicillinase milk sample, need deduct the milk sample peak value that penicillinase concentration is 0U/mL with the peak value of the milk sample that contains penicillinase, thereby obtain correction data, the data of correction data for trying to achieve.Correction data is the linear ratio relation according to the concentration of test sample.
7. detect after the end, pump into 70～75% concentration spirituous solutions, reach the purpose of cleaning instrument and sample valve.
Be the numerical value (every sample detection secondary) of test in the following table 1, can reach a conclusion by test: this method can detect the activity of the penicillinase in the sample comparatively accurately.
Claims (5)
1. the fast detection method of β-Nei Xiananmei in the dairy products the steps include:
A, SODIUM PHOSPHATE, MONOBASIC 2-6g, Sodium phosphate dibasic 17-24g are mixed, deionized water is settled to 1000mL and obtains the 100mM phosphate solution then, and pH is 7-8.6;
The 100mM phosphate solution configuration concentration that B, usefulness prepare is the penicillinase stoste of 1000U/mL, use 100mM phosphorus concentration acid salt solution weaker concn 1000U/mL penicillinase stoste again, obtaining penicillinase concentration is the penicillinase stoste of 10U/mL, continue to use the 100mM phosphate solution, by the same procedure gradient dilution, preparation penicillinase concentration is 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the penicillinase stoste of 0.25U/mL;
C, be 10U/mL through the penicillinase stoste of gradient dilution, 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, 0U/mL mixes with milk, obtains penicillinase concentration and is respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, the milk sample of 0.25U/mL and 0U/mL;
D, prepare substrate with phosphate solution, concentration is the penicillin solution of 100mM, and being diluted to required concentration of substrate is 1mM;
E, peristaltic pump is calibrated to required flow velocity is 0.5mL/min, sample loop is installed, and instrument signal is amplified knob transfers to 100 times, then the current meter pointer is transferred to 0 position of central authorities, at last registering instrument voltage is transferred to 1 volt, recording paper rolling speed 10cm/hour;
F, treat baseline stability after, in introduction valve, inject penicillinase concentration respectively and be respectively 5U/mL, 2U/mL, 1U/mL, 0.5U/mL, 0.25U/mL and the milk sample of 0U/mL, then introduction valve is transferred to the sample shelves by the injection shelves, test sample 5～7 minutes is when the peak of sample falls after rise to baseline, introduction valve is recalled to the injection shelves, inject next sample;
G, when detecting penicillinase concentration and be the milk sample of 0U/mL, registering instrument record peak signal detects when containing the data of penicillinase milk sample handling, and deducts the peak value of blank sample with peak value, obtains correction data;
After H, detection finish, pump into 70～75% concentration spirituous solutions, clean instrument and sample loop.
2. the fast detection method of β-Nei Xiananmei in a kind of dairy products as claimed in claim 1, contain the scope and the analytic target that detect and analyze and comprise:
β-Nei Xiananmei in A, all milk-product.All milk-product are defined as herein: comprise raw dairy and finished product breast, and liquid milk and solid-state breast, absolutely composition breast or part contain milk-content finished product, Chinese or foreign milk product;
B, the present invention can extend to other non-liquid-food that contains milk-content, beverage, medicine, makeup, chemicals, fermentation preparation, waste water etc.;
The detection of C, β-Nei Xiananmei is defined as: the activity of β-Nei Xiananmei, content, qualitative analysis or quantitative analysis, successive analysis or discontinuous analysis, Braking Analysis or manual analyzing, direct analysis or indirect analysis, pre-treatment or undressed sample analysis etc.
3. the fast detection method of β-Nei Xiananmei in a kind of dairy products as claimed in claim 1, detection and analytical procedure comprise:
A, based on its substrate of biological enzyme (test analyte) the principle that changes of the heat content that produces;
B, measurement are detection signal by the temperature variation that this heat content changes the reactive system that is produced.This temperature signal can difference or non-differential mode measure;
C, the analysis of flow injection sample introduction combine with the hot bioanalysis of enzyme;
D, testing sample (as milk liquid) that contains β-Nei Xiananmei and the damping fluid (seeing for details as follows) that contains its substrate (as penicillin) respectively two independently flow cavity enter reactive system, mix the back at reactor and produce catalyzed reaction;
E, this enzyme-substrate catalyzed reaction can comprise: single enzyme to the catalysis of single substrate, single enzyme simultaneously to the catalysis of many substrates, multienzyme to the catalysis of single substrate, multienzyme simultaneously to catalysis of many substrates etc.
4. the fast detection method of β-Nei Xiananmei in a kind of dairy products as claimed in claim 1, the beta-lactam enzyme analyser comprises:
A, two-pass flow Controlling System and mixing tank, reactor and detector;
B, detecting sensor are temperature sensor, comprise thermistor, thermopair, thermopile, temperature-sensitive transistor etc.;
C, the hot analyzing electrode of enzyme comprise: flow system, reaction column and temperature sensor.Reactor is replaceable and not replaceable two kinds, and can fill and non-filling dual mode;
D, measurement system can be single measurement enzyme thermode and measure enzyme thermode mode more;
E, measurement system can be full-automatic type or type hand, integrated or non-integrated flow control, signal alarm or non-warning;
F, electronic measurement are comparison bridge, difference temperature survey mode.Its detection sensitivity can reach 10
-5℃.
5. the fast detection method of β-Nei Xiananmei in a kind of dairy products as claimed in claim 1, detection method and step:
A, two independently flow cavity by peristaltic pump simultaneously with phosphate buffered saline buffer (as 0.1M, pH7.0-7.4) and the same buffer that contains penicillin (as 1mM Penicillin G) with as, 0.5mL/ minute flow velocity be injected into the input terminus of the hot biological analyser of enzyme (ENZYME THERMISTOR INSTRUMENT) respectively, and in the reactor of this analyser, mix and flow out output terminal through heat sensitive sensor;
B, testing sample (as containing β-Nei Xiananmei milk liquid) by the sample feeding valve will as, 350 μ L sample sizes are sneaked into the only flow cavity of phosphoric acid salt buffer, and mix at reactor with its phosphate buffered saline buffer that contains penicillin, the heat that its catalyzed reaction produces passes through temperature sensor measurement.
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103940865A (en) * | 2014-04-10 | 2014-07-23 | 山东理工大学 | Rapid detector for beta-lactam antibiotic residue |
| CN106526106A (en) * | 2016-12-07 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection method for beta-lactam in milk products |
-
2009
- 2009-06-26 CN CN2009100628522A patent/CN101928748A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103940865A (en) * | 2014-04-10 | 2014-07-23 | 山东理工大学 | Rapid detector for beta-lactam antibiotic residue |
| CN106526106A (en) * | 2016-12-07 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection method for beta-lactam in milk products |
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Application publication date: 20101229 |