阅读说明：本技术 一种定量检测舒巴坦的方法及其试剂盒 (Method for quantitatively detecting sulbactam and kit thereof ) 是由 杨大进 刘龙飞 于 2019-12-03 设计创作，主要内容包括：本发明属于酶法分析、生物检测领域,提供了一种检测舒巴坦的方法以及试剂盒,其简便经济,并能保证测定结果的准确性与精密度。(The invention belongs to the field of enzymatic analysis and biological detection, and provides a method and a kit for detecting sulbactam, which are simple, convenient and economical and can ensure the accuracy and precision of a determination result.)

1. A method for quantitatively detecting β -lactamase inhibitor sulbactam, which is characterized by comprising the following steps:

1) sample treatment, 2) β -lactamase inhibitor standard, 3) enzyme reaction, and 4) comparing the absorbance value corresponding to the sample with the absorbance standard curve of the standard.

2. The method of claim 1, wherein step 1) comprises adding a sample treatment fluid, wherein the sample treatment fluid comprises sample treatment fluid I and/or sample treatment fluid II, wherein the sample treatment fluid I is an acid solution and the sample treatment fluid II is an alkali solution; preferably, the sample treatment solution I is a mixed solution of hydrochloric acid and nitric acid, and the sample treatment solution II is sodium hydroxide; preferably, the sample treatment solution I is 147-; more preferably, the volume of the sample treatment solution I is 149. mu.L, and the volume of the sample treatment solution II is 156. mu.L.

3. The method of claim 1 or 2, wherein the method is used for microplate detection; preferably, the kit is used for 96-well microplate detection.

4. The method according to any one of claims 1 to 3, wherein the standard β -lactamase inhibitor of step 2) has the same matrix as the sample to be tested, preferably wherein the matrix is a milk matrix.

5. The method according to any one of claims 1 to 4, wherein the reaction of step 3) comprises two incubations, a first incubation of the solution containing the β -lactamase inhibitor with β -lactamase and a second incubation of the β -lactamase with a chromogenic substrate.

6. The method of claim 5, wherein the incubation time of the first step is 40 minutes and/or the reaction temperature of the second step is 25 ℃ and the reaction time is 110 min.

7. The method according to any one of claims 1 to 6, wherein in step 3), the β -lactamase in the final reaction solution is 3200IU/mL, and/or the concentration of the chromogenic substrate in the final reaction solution is 75 mg/L.

8. A kit comprises a sample treatment liquid I, a sample treatment liquid II, a chromogenic substrate, β -lactamase, a reaction buffer solution and a β -lactamase inhibitor standard substance, wherein the sample treatment liquid I is an acid solution, the sample treatment liquid II is an alkali solution, preferably, the sample treatment liquid I is a mixed solution of hydrochloric acid and nitric acid, and the sample treatment liquid II is sodium hydroxide.

9. The kit of claim 8, wherein the β -lactamase inhibitor standard has the same matrix as the sample to be tested, preferably wherein the matrix is a milk matrix.

10. A kit according to claim 8 or 9, wherein the β -lactamase inhibitor is sulbactam.

Technical Field

The invention relates to a method and a kit for indirectly and quantitatively detecting β -lactamase inhibitor sulbactam by an enzyme method, belonging to the field of enzyme analysis and biological detection.

Background

In the process of monitoring the veterinary drug residue of milk and dairy products for many years by departments such as the Ministry of agriculture and the market supervision and administration, β -lactam drug residue exceeds the standard, however, some production operators escape monitoring by adding β -lactamase antagonist to degrade β -lactam drug, along with the establishment of a β -lactamase detection method, some production operators begin to illegally add β -lactamase inhibitors such as sulbactam to inhibit the activity of β -lactamase, so that β -lactamase is difficult to detect by related departments, therefore, a rapid and effective method is urgently needed to detect the illegally added β -lactamase inhibitor in food.

At present, methods such as high performance liquid chromatography, liquid chromatography-tandem mass spectrometry and the like are mainly adopted at home and abroad to directly detect β -lactamase inhibitor, for example, the patent CN101852780A relates to the direct detection of sulbactam by High Performance Liquid Chromatography (HPLC). the methods are complex to operate, the price of detection equipment is high, so that the detection cost is high, and the requirement of rapid development of the detection industry cannot be met.

In the food detection method, the cost of the enzyme detection technology is relatively low, but the accuracy and precision of enzyme detection cannot meet the requirements of the industry in many cases due to the complex components and great interference of food samples, for example, when the glycerin content in honey is measured by adopting the enzyme method, such as Liliang and the like (research on the glycerin content in honey measured by the enzyme method under different conditions, Liliang and the like, Chinese agricultural science and technology reports, 2018, 20 (2): 86-92), the detection precision of the enzyme preparation method can only reach the basic requirement, namely, the precision is low, and can only basically meet the requirement of repeatability.

Disclosure of Invention

In order to overcome the disadvantages of the prior methods, the invention aims to provide a method and a related kit for detecting β -lactamase inhibitor sulbactam accurately, with high throughput and standardization, and economy.

In order to achieve the above object, the present invention seeks to avoid direct detection of β -lactamase inhibitors and to employ indirect enzymatic detection of β -lactamase inhibitors.

The method mainly aims to remove various impurities which are relatively high in content and generally exist in a sample, such as some saccharides, lipids or proteins, and the like, and aims to reduce the background of reaction and reduce irrelevant nonspecific binding, however, the content of endogenous β -lactamase in the sample is usually very small, namely the endogenous β -lactamase is not impurity in the general sense, but the endogenous β -lactamase can directly and specifically carry out specific enzyme reaction with a chromogenic substrate like β -lactamase added in the method of the invention, so that the accuracy of a measurement result is greatly influenced.

Different from spectrophotometry, an enzyme-labeled microplate can greatly reduce a reaction system, so that the waste of reagents and the like is avoided, and the enzyme-labeled microplate is more suitable for popularization and use in the food detection industry, but because the volume is too small, details such as reagent preparation errors, enzyme activity and concentration, reaction temperature, reaction time, reaction system composition and the like can greatly influence a determination result, and a detection method meeting the industrial requirements is difficult to form.

Wherein β -lactamase inhibitor is sulbactam, the sample to be detected can be dissolved liquid after dissolving liquid or solid, such as various dairy products, including liquid dairy products, dissolved solid dairy products (such as milk powder, etc.), yoghurt, skim milk, fresh milk, reconstituted milk, etc.

In one example, the kit of the invention comprises a 96-well microplate, β -lactamase inhibitor standard solution, sample treatment solution I, sample treatment solution II, β -lactamase solution, reaction buffer solution and chromogenic substrate solution, preferably, the β -lactamase inhibitor standard has the same substrate as the sample to be detected, so as to eliminate the influence of a milk substrate and improve the accuracy of the detection result.

In one example, the invention relates to an assay method for enzymatic indirect quantitative detection of sulbactam, an illegal additive in milk, which comprises the following steps:

1) preparing a sulbactam standard solution, namely treating defatted powder consistent with a milk matrix by adopting a sample treatment solution I and a sample treatment solution II to form a solvent consistent with the milk matrix, wherein the solvent is used as a 0 standard, other concentrations of standard are formed by diluting mother liquor, and all the standard are stored at the temperature of 2-8 ℃;

2) preparation of chromogenic substrate solution: dissolving ceftiofur by using dimethyl sulfoxide (DMSO) as a solvent to form a homogeneous solution with the concentration of 5mg/mL, and storing the solution at the temperature of minus 20 ℃ in a dark place;

3) preparation of reaction buffer: NaCl: 40g, KCl: 1g, KH₂PO₄：1.2g、Na₂HPO₄: 7.2g of the buffer solution is dissolved in 1L of sterilized water to form a concentrated enzymolysis reaction buffer solution;

4) β -lactamase working solution is prepared by mixing β -lactamase solution and reaction buffer solution at a volume ratio of 4: 6;

5) preparation of a chromogenic substrate working solution: the reaction buffer solution and the chromogenic substrate solution are mixed according to the volume ratio of 57: 3.

Preferably, the kit used for the method comprises a 96-well microplate, a sample treatment solution I and a sample treatment solution II, wherein the concentration of the sulbactam standard solution is respectively 0mg/L, 5mg/L, 20mg/L, 40mg/L, 80mg/L and 160mg/L, 1 mL/bottle is preferred, the concentration of the β -lactamase solution is 16000IU/mL, 4.2 mL/bottle is preferred, the kit contains 15 mL/bottle of reaction buffer solution, the sample treatment solution I and the sample treatment solution II, and the chromogenic substrate solution is 320 muL/bottle.

In one example, the sample preparation method is as follows:

1) 1.5mL of homogeneous milk (8000 g of 4 minutes of degreasing treatment is needed if the sample is fresh milk) is placed in a 2mL centrifuge tube, 147-;

2) centrifuging at 8000g for 10 min;

3) transferring 800 mu L of the supernatant into a 1.5mL centrifuge tube, adding 154 mu L and 158 mu L of the sample treatment liquid II, and uniformly mixing;

4)8000g for 10min, and the supernatant is used for detection.

In one example, the detection method or process of the present invention includes the steps of:

1) all reagents were returned to room temperature (20-25 ℃) prior to use, and the remaining reagents were stored at 2-8 ℃ immediately after use;

2) taking out the micropores with required quantity, placing the micropores on a micropore frame, and recording the positions of the standard substance and the sample;

3) adding 40 mu L of standard solution or test sample into the corresponding micropore;

4) adding 100 μ L of β -lactamase working solution to each well;

5) incubation at 25 ℃ for 40min in a 250rpm shaker;

6) adding 60 mu L of chromogenic substrate working solution into each micropore;

7) incubation at 25 ℃ for 110 minutes in a 250rpm shaker;

8) the absorbance was measured at 490nm with a microplate reader within 10 minutes after the end of incubation.

And (4) calculating a result: and (3) taking the concentration mg/L of the standard substance as an X axis, taking the absorbance value of the standard substance as a Y axis, constructing a standard curve by adopting a 4P algorithm, and considering the factor of 1.32 in the sample calculation result.

The whole detection process is constant in temperature, light-resistant, thorough in reaction, stable in absorbance value and accurate in test result.

In one embodiment, the sulbactam standard solution is prepared by the following steps: the sample treatment liquid is adopted to treat the milk powder with the milk matrix, the treated supernatant is used as a solvent to dissolve the standard substance, and the standard substance solution obtained after dissolution has the same matrix effect as the test sample, so that the accuracy and precision of the detection result are ensured.

In one embodiment, the sample processing method is to use the sample processing liquid to process the milk source so as to remove the endogenous β -lactamase in the milk sample, thereby ensuring the accuracy and precision of the detection result.

In one embodiment, the chromogenic substrate solution is prepared by: the chromogenic substrate solution is concentrated and frozen and stored in a dark place to ensure the titer of the chromogenic substrate, thereby ensuring the effective sensitivity and accuracy.

In one embodiment, the β -lactamase solution is prepared by concentrating β -lactamase to stabilize enzyme activity, thereby ensuring accuracy and precision of detection results.

In one embodiment, the sulbactam standard solution is prepared by the following steps:

1) 1g of defatted powder is restored in 10mL of sterilized water, the restored milk is subjected to sample treatment according to a sample preparation method, and the treated supernatant is used as a solvent of a standard product;

2) dissolving the sulbactam standard substance by the solvent of 1) to prepare 5000mg/L mother solution, and storing at 4 ℃.

In one embodiment, the present invention provides a combination of 6 sets of reagents for detection, consisting of:

1) providing one 96-well microplate;

2) the reagent comprises a sulbactam standard substance (6 bottles, β -lactamase solution (1 bottle, reaction buffer solution (1 bottle, chromogenic substrate solution (1 bottle), sample processing solution I (1 bottle) and sample processing solution II (1 bottle).

In one embodiment, the present invention also provides a microplate reader detection method for enzymatic quantitative detection of sulbactam, comprising the steps of:

1) sample preparation

a) Putting 1.5mL of homogenized milk (8000 g if the sample is fresh milk, and degreasing for 4 minutes) into a 2mL centrifuge tube, adding 149 μ L of sample treatment solution I, and mixing uniformly;

b) centrifuging at 8000g for 10 min;

c) transferring 800 mu L of supernatant into a 1.5mL centrifuge tube, adding 156 mu L of sample treatment solution II, and mixing uniformly;

d) centrifuging at 8000g for 10min, collecting supernatant for detection,

2) detection step

a) Taking out the micropores with required quantity, placing the micropores on a micropore frame, and recording the positions of the standard substance and the sample; adding 40 mu L of standard solution or sample to be detected into the corresponding micropore;

b) adding 100 μ L of β -lactamase working solution to each well;

c) incubation at 25 ℃ for 40min in a 250rpm shaker;

d) adding 60 mu L of chromogenic substrate working solution into each micropore;

e) incubation at 25 ℃ for 110 minutes in a 250rpm shaker;

f) measuring the absorbance value at 490nm by using an enzyme-linked immunosorbent assay within 10 minutes after the incubation is finished,

3) calculation of results

And (3) taking the concentration mg/L of the standard substance as an X axis, taking the absorbance value of the standard substance as a Y axis, constructing a standard curve by adopting a 4P algorithm, and considering the factor of 1.32 in the sample calculation result. Detection limit: the detection limit was 5 mg/L. Detection range: according to the enzymatic detection reaction, the linear detection range of the kit is 0.0-160 mg/L sulbactam.

Drawings

FIG. 1 is a graph of sample treatment fluid volume versus recovery obtained in example 1 of the present invention;

FIG. 2 is a graph showing β -lactamase activity obtained in example 2 of the present invention as a function of a chromogenic substrate;

FIG. 3 is a graph showing the relationship between the chromogenic substrate obtained in example 3 of the present invention and the activity of β -lactamase;

FIG. 4 is a standard curve of sulbactam at a concentration of 0-160 μ g/mL obtained in example 4 of the present invention;

FIG. 5 is a standard curve of sulbactam at a concentration of 0-200 μ g/mL obtained in example 4 of the present invention;

FIG. 6 is a graph showing the relationship between the absorbance obtained in example 5 of the present invention and the temperature and time of the second incubation reaction;

FIG. 7 is a graph showing the relationship between the reaction time and the absorbance in the first step of incubation obtained in example 6 of the present invention;

FIG. 8 is a graph showing the results of the sulbactam spiking recovery experiment obtained in example 7 of the present invention;

FIG. 9 is a standard curve for the quantitative determination of sulbactam by the enzymatic method according to the present invention; and

FIG. 10 is a diagram showing the correlation test results between the detection method and HPLC-MS.

Detailed Description

The present invention is described in detail in the context of sulbactam, and it is contemplated by those skilled in the art that the methods of the present invention are equally applicable to the detection of other β -lactamase inhibitors.

The invention is further described below with reference to the accompanying drawings.

Examples of the experiments

Preparing a sulbactam standard solution:

1) 1g of defatted powder is restored in 10mL of sterilized water, the restored milk is subjected to sample treatment according to a sample preparation method, and the treated supernatant is used as a solvent of a standard product;

2) dissolving the sulbactam standard substance by the solvent of 1) to prepare 5000mg/L mother solution, and storing at 4 ℃.

Diluting the mother liquor obtained in the step 2) with the solvent obtained in the step 1) to obtain standard products with the concentrations of 5mg/L, 20mg/L, 40mg/L, 80mg/L and 160mg/L, and putting the prepared standard product solution into a brown bottle with 1mL of the solution in each bottle, wherein the standard product 0 is the solvent obtained in the step 1).

Preparation of sample treatment solution I: preparing the solution containing 0.2M HCl and 0.002M HNO₃The solution of (1).

Preparation of sample treatment liquid II: NaOH solution with concentration of 0.5M was prepared.

Preparation of reaction buffer:

weighing 40g NaCl, 1g KCl and KH₂PO₄1.2g、Na₂HPO₄: adding 7.2g of the mixture into a 1L volumetric flask, and fixing the volume to 1L by using sterilized water; 15mL of the reaction buffer was taken and filled into a vial, and each kit contained 1 vial of the reaction buffer.

β preparation of lactamase working solution:

β -lactamase solution at a concentration of 16000IU/mL was filled into vials, 4.2mL per vial.

When in use, β -lactamase solution and reaction buffer solution are mixed according to the volume ratio of 4: 6 to obtain β -lactamase working solution of 6400 IU/mL;

preparation of chromogenic substrate working solution:

weighing 100mg of ceftiofur, adding the ceftiofur into 20mL of dimethyl sulfoxide (DMSO), fully and uniformly mixing to obtain a homogeneous solution with the concentration of 5000mg/L, and storing the solution at the temperature of minus 20 ℃ in a dark place; each kit contained 1 vial of chromogenic substrate solution, 320. mu.L per vial.

In use, the reaction buffer and chromogenic substrate solution are mixed according to a 57: 3 to obtain a chromogenic substrate working solution with a concentration of 250 mg/L.

To better illustrate the advances of the inventive sample treatment, the present invention exemplifies the effect of treating 100mg/L of a spiked milk sample with 20% trichloroacetic acid, potassium ferrocyanide/zinc sulfate, potassium ferrocyanide/zinc acetate, and the inventive "sample treatment liquids I and II". Ensuring the milk source to be reliable, respectively adding 20% trichloroacetic acid, potassium ferrocyanide/zinc sulfate, potassium ferrocyanide/zinc acetate and sample treatment liquid (including sample treatment liquid I and sample treatment liquid II) with the same volume into a natural milk sample (40 mu L per hole), detecting the treated sample according to the detection steps provided by the specification, calculating the standard addition recovery rate, performing 6 parallels in each method, and taking an average value of the results. The results are shown in Table A below.

TABLE A results of measurements on different sample treatment solutions

Serial number	Sample treatment liquid	Detection result, mg/L	The recovery rate is high
				1	Inventive sample treatment solutions I and II	100.6	100.6
2	20% trichloroacetic acid	138.7	138.7
				3	Potassium ferrocyanide/zinc sulfate	136.4	136.4
4	Potassium ferrocyanide/zinc acetate	141.3	141.3

As can be seen from Table A, the recovery obtained with the sample treatment solutions I and II according to the present invention was 100.6%, which is closest to 100%, and thus the sample treatment solutions according to the present invention were the most preferable.

Experimental example 1

This experimental example consists in studying the optimal volumes of sample treatment solutions I and II.

Adding a sulbactam standard substance into a milk sample to obtain a sample with the concentration of 100 mg/L. The standard sample (40. mu.L per well) was subjected to sample treatment in which the volume of the sample treatment solution I was A. mu.L and the volume of the sample treatment solution II was B. mu.L, and the other steps were carried out in accordance with the aforementioned sample treatment method. To determine the optimal values for a and B, the treated samples were tested according to the test procedures provided in the specification and the spiked recovery was calculated, 3 replicates of each method were performed and the results averaged. The results are shown in Table 1.

TABLE 1

As can be seen from Table 1 and FIG. 1, when the volume of the sample treatment solution I was 149. mu.L and the volume of the sample treatment solution II was 156. mu.L, the recovery rate was 100.6, which was closest to 100, and therefore, the optimum volume of the sample treatment solution I was 149. mu.L and the optimum volume of the sample treatment solution II was 156. mu.L.

Experimental example 2

This experimental example consisted in studying β -lactamase for optimal activity (see table 2 and figure 2).

Adding 40 mu L of reaction buffer solution into each micropore, then respectively adding 100 mu L of IU/mL, 6400IU/mL, 3200IU/mL, 1600IU/mL, 800IU/mL, 400IU/mL and 0IU/mL of β -lactamase into the micropore, making each concentration in parallel 6, finally adding 60 mu L of 250mg/L of chromogenic substrate into each micropore, oscillating and incubating for 110 minutes at 25 ℃ and 250rpm, and measuring absorbance value by using a microplate reader at 490nm, wherein when the concentration of the chromogenic substrate solution is certain and excessive, the relation between β -lactamase and chromogenic substrate with different activities is shown in Table 2. since the addition amount of β -lactamase is 100 mu L, the total volume of the reaction is 200 mu L, the actual concentration of β -lactamase in the reaction solution is 1/2 of the added standard concentration, and the concentration of 4000IU/mL, 3200IU/mL, 800/mL, 400IU/mL, 200IU/mL, 0IU/mL and the concentration of chromogenic substrate is 64 mg/mL.

TABLE 2 relationship of 2 β -lactamase Activity with chromogenic substrates

According to the above experiment, when the activity of β -lactamase was 4000IU/mL, the OD value was 1.159, and when the activity of β -lactamase was 3200IU/mL, the OD value was 1.126, and there was no significant difference, and it was confirmed that β -lactamase in the final measurement solution (or reaction solution) was 3200IU/mL, and therefore the concentration of β -lactamase working solution was 6400 IU/mL.

Experimental example 3

This experimental example was conducted to examine the optimum concentration of the chromogenic substrate solution (see Table 3 and FIG. 3).

40. mu.L of reaction buffer was added to each well, then 100. mu.L of β -lactamase of 6400IU/mL was added to each well, and finally 60. mu.L of 300mg/L, 250mg/L, 125mg/L, 67.5mg/L, 33.75mg/L, 16.875mg/L, 0mg/L of chromogenic substrate were added to each well in 6 replicates at each concentration, incubated at 25 ℃ for 110 minutes with shaking at 250rpm, and absorbance values were measured at 490nm with a microplate reader since the amount of reaction buffer added was 40. mu.L, the amount of β -lactamase added was 100. mu.L, the amount of chromogenic substrate added was 60. mu.L, and the total volume of the reaction was 200. mu.L, the concentration of the chromogenic substrate solution was 60/200 at the added concentration.

TABLE 3 relationship between chromogenic substrate and the Activity of β -lactamase

According to the above experiment, the OD value was 1.115 when the concentration of the chromogenic substrate solution was 90mg/L, and 1.088 when the concentration of the chromogenic substrate solution was 75mg/L, with no significant difference in the results. Therefore, the concentration of the chromogenic substrate solution in the reaction solution was determined to be 75mg/L, and the concentration of the chromogenic substrate solution added was determined to be 250 mg/L.

Experimental example 4

This experimental example consists in studying the linear range of sulbactam.

When the activity of β -lactamase in the determination solution is set to 3200IU/mL, sulbactam solutions with different concentrations and a chromogenic substrate solution with a certain concentration are added to determine the linear range of sulbactam, and the results are shown in Table 4.

TABLE 4 measurement results of concentration absorbance values of sulbactam standard series

As can be seen from Table 4, according to the standard curve fitting of the sulbactam solution in sections between 0 and 200mg/L, the correlation coefficient can reach 0.998 (see FIG. 4) when the concentration of the sulbactam solution is between 0 and 160mg/L, and the requirement of negative correlation exists, and the correlation coefficient can reach 0.989 (see FIG. 5) when the concentration of the sulbactam solution is between 0 and 200mg/L, the correlation is poor. Thereby determining the range of the standard curve to be 0-160 mg/L.

Experimental example 5

This experimental example is to study the optimum reaction temperature and time.

The determined activity (6400IU/mL) of β -lactamase solution, chromogenic substrate solution (250mg/L) and sulbactam solution (5mg/L) are selected for determining the optimal reaction temperature and time, the reaction needs two-step incubation, after the first step of incubation is to add sulbactam and β -lactamase, the incubation time is temporarily set to be 60 minutes (enough time) for completely inhibiting the enzyme, after the second step of incubation is to add chromogenic substrate, the determination of the incubation temperature and time is more critical, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 40 ℃ and 30min, 60min, 110min and 150min4 time periods are designed together, the method is to measure the absorbance at each experiment temperature according to the time, and the experiment results are shown in Table 5 and FIG. 6.

TABLE 5 relationship between absorbance and reaction temperature and time in the second incubation step

The comprehensive experiment result shows that the reaction is the optimal experiment condition under the conditions of 25 ℃ and 110min of reaction on the basis of considering the laboratory temperature in application.

Experimental example 6

This experimental example consists in studying the incubation time after addition of β -lactamase.

Experimental selection the determined β -lactamase activity (6400IU/mL), chromogenic substrate (250mg/L), sulbactam (5. mu.g/mL) were used to determine the optimal reaction time for the first incubation step, and the second incubation temperature and time were determined as in Experimental example 5 at 25 ℃ for 110 minutes, with 20min, 30min, 40min, 50min and 60min 5 time periods, by measuring the absorbance at 25 ℃ for the above time periods, respectively, and the results are shown in Table 6 and FIG. 7.

TABLE 6 first step incubation reaction time vs. absorbance relationship

As can be seen from fig. 7, the absorbance values gradually decreased with the lapse of time, and the change in absorbance values was smooth when the incubation time was 40 minutes, and there was no significant difference in absorbance values at 40 minutes, 50 minutes and 60 minutes, and thus the incubation time was determined to be 40 minutes.

Experimental example 7

The experimental example is to study the detection limit of the present invention.

When the content of sulbactam is too low to inhibit β -lactamase, so that β -lactamase can not be influenced to degrade chromogenic substrates, 6 parallel tests are respectively carried out on each concentration at 10 concentration points (0, 1, 3, 5, 10, 30, 50, 100, 160 and 200mg/L) at six time points in order to determine the optimal range and detection limit of a sulbactam standard curve, and the data of each test is shown in Table 4.

The absorbance values at concentrations of 0mg/L, 1mg/L, 3mg/L, 5mg/L, 20mg/L, 40mg/L, 80mg/L and 160mg/L were determined in parallel in4 concentrations according to the standard curve range determined in Experimental example 4, and the results are shown in Table 7.

TABLE 7 t pairing experiments

When the concentration of sulbactam is less than 5mg/L, the corresponding absorbance value is 1.052-1.146, and the change is not obvious. T pair test shows that when the concentration of sulbactam is 1mg/L and 3mg/L, t is_d＝0.000723＜t_0.05，32.353; when the concentration of sulbactam is 3mg/L and 5mg/L respectively, t_d＝0.00911＜t_0.05，3When the absorbance value is 2.353, the absorbance value is not significantly different, so that the detection limit of the method is determined to be 5 mg/L.

Experimental example 8

The experimental example is to study the accuracy of the kit of the invention.

And (3) labeling 4 concentrations of 5mg/L of detection limit, 10mg/L of double detection limit, 20mg/L of quadruple detection limit and 50mg/L of ten detection limit, respectively performing labeling test at 3 time points, performing 3 technical repetitions at each concentration at each time point, and obtaining the average detection results shown in the following table 8 and fig. 8.

TABLE 8 Sulbutan addition recovery test results

According to the technical requirements confirmed by the inspection method in GB27404-2008 laboratory quality control Standard food physicochemical inspection, when the content of the detected component is 1-100mg/kg, the recovery rate range is 90-110%; when the content of the measured component is 1-10mg/kg, the coefficient of variation (CV%) in a laboratory should be less than 11%, and when the content of the measured component is 10-100mg/kg, the CV should be less than 7.5%, so that the standardized recovery rate and the coefficient of variation of the method can be judged to meet the technical requirements of GB 27404-2008.

Experimental example 9

The experimental example is to study the precision of the kit of the invention.

Milk spiked samples (total 6 concentrations) were tested using 3 batches of the kit (kit prepared in the examples), 3 replicates per concentration, and the test results averaged and are shown in table 9.

TABLE 9

The coefficient of variation of the 6 concentrations of the milk added with the target is very small (0.07-2.28%), which indicates high precision.

Calibration curve

The range of the detection standard curve is 0-160 mg/L. In order to obtain the sulbactam content of the sample, the sample results read from the standard curve must be multiplied by the dilution factor 1.32. The data processing software contains the dilution factor at the time of final calculation, and the output result is mg sulbactam/L sample. A typical curve is shown in fig. 9.

Experimental example 10

The experimental example is to study the correlation between the detection method provided by the present invention and the HPLC method.

3 blind samples are detected by respectively adopting the detection method and the HPLC-MS method provided by the invention, the three concentrations of 5mg/L, 10mg/L and 20mg/L are labeled, 3 samples and the labeled samples are paralleled, the detection results are averaged, and the results are shown in a table 10 and a figure 10.

TABLE 10 correlation test results of the detection method and HPLC-MS provided by the present invention

The invention has the advantages that:

1. the method is simple and easy to operate, economical and practical, greatly saves cost, and is convenient for industrial popularization and application.

2. A complete set of optimal procedures which have small interference of endogenous β -lactamase and are suitable for standardized detection is developed.

3. The method disclosed by the invention has the advantages that the repeated determination variation coefficient is within 8%, and the accuracy and precision of the determination result are ensured.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, devices, means, methods, or steps.