CN111198266A

CN111198266A - Rapid detection kit for sulbactam

Info

Publication number: CN111198266A
Application number: CN202010021550.7A
Authority: CN
Inventors: 邓兴朝; 陈欢; 曾祥富
Original assignee: Shenzhen Boao Tongke Biological Products Co ltd
Current assignee: Shenzhen Boao Tongke Biological Products Co ltd
Priority date: 2020-01-09
Filing date: 2020-01-09
Publication date: 2020-05-26

Abstract

The embodiment of the invention discloses a sulbactam rapid detection kit. Wherein, this kit includes: the reagent kit comprises an ELISA plate and a plurality of reagents packaged in different reagent bottles; a plurality of reaction holes are formed in the ELISA plate; the reagent comprises: sulbactam standard, diluent, substrate diluent and enzyme. The rapid detection kit detects the sulbactam remained in the foods such as dairy products and the like by adopting the principle of an enzyme inhibition method, has the characteristics of short sample processing time, suitability for high-throughput sample screening, high specificity, high sensitivity, high accuracy and the like, has the lowest detection content lower limit of 100ug/kg, can be well popularized and applied, and has good development prospect.

Description

Rapid detection kit for sulbactam

Technical Field

The invention relates to the technical field of food detection, and particularly relates to a sulbactam rapid detection kit.

Background

β -lactamases are a class of bacterial enzymes that hydrolyze antibiotics containing a β -lactam ring, such as penicillins, cephalosporins, monobactams, carbapenems, and the like, thereby reducing the potency of these antibiotics.

Although the supervision on antibiotics is more and more strict, the abuse phenomenon still exists in the food industry, and in order to avoid the detection of the antibiotics, some lawbreakers add β -lactamase to the dairy to mask the residues of the antibiotics, so that β -lactamase remains in foods such as dairy.

β -lactamases have been shown to cause increased bacterial resistance and are a major clinical threat, and since β -lactamases have been regulated by government regulators, β -lactamase inhibitors have come into force, some β -lactam derivatives have been shown to act as β -lactamase inhibitors (including clavulanic acid, sulbactam and tazobactam).

The existing detection methods for the sulbactam residue in the food comprise High Performance Liquid Chromatography (HPLC), liquid mass spectrometry (LC-MS), liquid mass tandem mass spectrometry (LC-MS-MS) and enzyme-linked chemical analysis (IA).

In the process of implementing the invention, the inventor finds that the following problems exist in the related art: the existing common instrument detection method has the defects of expensive instrument, high requirement on operators, difficult popularization and unsuitability for high-throughput sample screening. Although the research on the sulbactam detection method has been carried out at home and abroad in recent years, the time consumption is long in the aspect of sample treatment, the overall detection process takes long time, and the use requirement is difficult to be well met.

Disclosure of Invention

Aiming at the technical problems, the embodiment of the invention provides a sulbactam rapid detection kit, which aims to solve the problems that the existing sulbactam residue detection method is long in time consumption and cannot well meet the use requirements of users.

The first aspect of the embodiment of the invention provides a sulbactam rapid detection kit. Wherein, this kit includes: the reagent kit comprises an ELISA plate and a plurality of reagents packaged in different reagent bottles;

a plurality of reaction holes are formed in the ELISA plate; the reagent comprises: sulbactam standard, diluent, substrate diluent and enzyme.

Optionally, the diluent is phosphate buffer, the substrate diluent is dimethyl sulfoxide, the substrate is nitrocefin, and the enzyme is β -lactamase.

Optionally, the concentration of the phosphate buffer is 0.01mol, the concentration of the nitrocefin is 90%, the concentration of the dimethyl sulfoxide is 98%, and the concentration of the β -lactamase is 10 MU.

Optionally, the reagents include a three-vial phosphate buffer, two vials of β -lactamase, one vial of ceftiofur and one vial of dimethyl sulfoxide.

Optionally, the nitrocefin, dimethyl sulfoxide, and β -lactamase are all packaged in glass bottles.

Alternatively, the phosphate buffer solution content per vial was 20ml, the β -lactamase content per vial was 2mg, the nitrothiophene content per vial was 2.1mg, and the dimethylsulfoxide content per vial was 0.3 ml.

Optionally, the microplate comprises: a plastic support and a plurality of microporous strips; each micropore strip is provided with a plurality of reaction holes which are detachably arranged on the plastic bracket.

Optionally, the microplate comprises 12 microwell strips, and each microwell strip is provided with 8 reaction wells side by side.

Optionally, the method further comprises: the box body and the fixed sponge arranged in the box body; the fixing sponge is provided with a plurality of cylindrical slots for inserting the reagent bottles to fix the positions of the reagent bottles in the box body.

Optionally, in use, the sulbactam standard substance is prepared into sulbactam standard solutions with various concentrations through a blank extracting solution; the concentration of the sulbactam standard solution comprises 0, 0.27ug/ml, 0.82ug/ml, 2.47ug/ml, 7.41ug/ml and 22.2 ug/ml.

In the technical scheme provided by the embodiment of the invention, the method for detecting the residual sulbactam in the foods such as dairy products and the like by adopting an enzyme inhibition method has the characteristics of short sample processing time, suitability for high-throughput sample screening, high specificity, high sensitivity, high accuracy and the like, the lower limit of the minimum detection content can reach 100ug/kg, and the method can be well popularized and applied.

Drawings

FIG. 1 is a schematic diagram of one embodiment of a solvent dispensing method according to an embodiment of the invention;

FIG. 2 is a schematic diagram of an embodiment of the sulbactam detection method according to an embodiment of the invention;

FIG. 3 is a schematic diagram of an embodiment of a calibration curve according to an embodiment of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may be present. As used in this specification, the terms "vertical," "horizontal," "left," "right," "up," "down," "inner," "outer," "bottom," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the invention and for simplicity in description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The embodiment of the invention provides a sulbactam rapid detection kit. The kit detects sulbactam residues in foods such as dairy products and the like based on the principle of an enzyme inhibition method. Which consists of an enzyme label plate and reagent consumables required to be used when detecting sulbactam residues.

The enzyme label plate is provided with a plurality of reaction holes for providing places for reaction incubation and the like of a sample and a reagent.

Specifically, the ELISA plate can be composed of a plastic support and a plurality of micropore strips. Each micropore strip is provided with a plurality of reaction holes which are detachably arranged on the plastic bracket.

The micropore strip is disposable, and is packaged in an aluminum foil bag for storage. When in use, the microporous strips with proper number are taken out from the aluminum foil bag and fixed on the plastic bracket according to the requirement of actual situation. The micro-porous strip can be detachably fixed on the plastic bracket by adopting any suitable form such as a buckle. After the use, the microporous strip is detached and replaced by a new microporous strip.

The micro-holes can be arranged on each micro-hole strip according to the requirement of actual conditions. For example, each microporous strip can be provided with a row of 8 reaction holes, and each plastic bracket is provided with 12 microporous strips to meet the requirement of practical use.

The reagent consumable comprises a sulbactam standard substance, a diluent, a substrate diluent and an enzyme, which are respectively packaged in different reagent bottles so as to be used in detection. The effect of each reagent in the assay is described in detail below:

the sulbactam standard is configured into sulbactam standard solutions with various concentrations when in use, and is used for constructing a standard curve between the content and the absorbance of sulbactam. The dilution is used to dilute the sample to the appropriate fold. The substrate diluent is used to dilute the substrate to the appropriate concentration. The substrate may be enzymatically decomposed to produce the corresponding decomposition product.

Sulbactam remaining in the sample inhibits the activity of the enzyme and thus reduces the rate of decomposition of the substrate by the enzyme. That is, the sulbactam content in the sample is inversely proportional to the content between the decomposition products. The higher the sulbactam content, the lower the content of decomposition products.

The dilution solution is Phosphate Buffered Saline (PBS), the substrate dilution solution is dimethyl sulfoxide, the substrate is the cefotaxime, and the enzyme is β -lactamase, so that sulbactam can generate an inhibiting effect on β -lactamase, the decomposition efficiency of the cefotaxime is reduced, and the amount of red products generated by the decomposition of the cefotaxime is reduced.

In some embodiments, the phosphate buffer is at a concentration of 0.01mol, the nitrocefin is at a concentration of 90%, the dimethyl sulfoxide is at a concentration of 98%, and the β -lactamase is at a concentration of 10 MU.

Wherein, each sulbactam rapid detection kit comprises three bottles of phosphate buffer solution, two bottles of β -lactamase, one bottle of nitrocefin and one bottle of dimethyl sulfoxide.

The content of each bottle of phosphate buffer solution is 20ml, the content of each bottle of β -lactamase is 2mg, the content of each bottle of nitrothiophene is 2.1mg, and the content of each bottle of dimethyl sulfoxide is 0.3ml so as to meet the use requirement.

Specifically, the multi-bottle reagent can be placed in a square box body. The case body can be made of materials such as hard paper cards and the like. The inside of the box body is provided with a fixed sponge. The fixing sponge can be provided with cylindrical slots with corresponding size and number for inserting reagent bottles so that the reagent bottles can be fixed.

The sulbactam rapid detection kit provided by the embodiment of the invention has the advantages of high sensitivity, simple structure, short sample processing time, convenience in use and carrying and the like, can be used for analyzing and detecting sulbactam residues in dairy products by dairy product production units, and has a good application prospect.

FIG. 1 is a flow chart of a solvent configuration method of a sulbactam rapid detection kit provided by the embodiment of the invention. Fig. 2 is a flowchart of a detection method according to an embodiment of the present invention. In this embodiment, the sulbactam rapid detection kit is used for quantitative detection of sulbactam residue in a dairy product sample.

As shown in fig. 1, the solvent preparation method may include the steps of:

step 110: preparing a blank extracting solution. The blank extract is a dairy product without sulbactam residue and can be used as a blank reference.

Step 120: preparing a sample extracting solution. The sample extract may be prepared in the same manner as the blank extract except that the processed object is a dairy product to be tested. Step 110 and step 120 may be performed simultaneously.

Step 130: dissolving the sulbactam standard product by using a phosphate buffer solution to prepare a sulbactam standard stock solution.

Step 140: and preparing the sulbactam standard stock solution into sulbactam standard solutions with various concentrations through the blank extracting solution.

Wherein, the concentration of the sulbactam standard solution to be prepared comprises 0, 0.27, 0.82, 2.47, 7.41 and 22.2 ug/ml. At least 0.5ml of each concentration configuration was used to make a standard curve.

Step 150, diluting the β -lactamase to a set multiple by using a phosphate buffer solution.

Step 160: and adding a set amount of dimethyl sulfoxide into the nitrocefin to prepare a substrate solution.

Step 170: the substrate solution was added to the phosphate buffer and mixed well.

As shown in fig. 2, the reagent prepared based on the solvent configuration method shown in fig. 1 can be used for detecting sulbactam residue in a sample by the following steps:

step 210: and taking out a sufficient number of the microporous strips, and installing and fixing the microporous strips on the plastic bracket.

Step 220, setting standard wells and sample wells in the reaction wells of the microwell strip, and adding an appropriate amount of β -lactamase solution to each standard well and each sample well.

Step 230: and respectively adding a proper amount of blank extracting solution and sample extracting solution into the standard sample hole and the sample hole.

Step 240: incubate at room temperature in the dark for a preset time.

The preset time is an empirical value and can be set according to the requirements of actual conditions. During incubation, the reaction holes on the microporous strip can be sealed by using a sealing plate membrane so as to meet the requirement of incubation in a dark place.

Step 250: adding a proper amount of substrate solution diluted by a proper time into each reaction hole, oscillating, uniformly mixing and incubating in a dark place.

The specific time for the incubation in the dark can also be set according to the needs of the actual situation. The incubation time is longer than in step 240 to ensure that the reaction is complete.

Step 260: the absorbance of each reaction well was measured by a microplate reader at a wavelength of 488-492 nm. The absorbance (i.e., OD) may indicate the degradation of the substrate, reflecting the concentration of the degradation product.

In this embodiment, reagent 1 is β -lactamase with a concentration of 10MU, reagent 2 is ceftiofur thiophene with a concentration of 90%, reagent 3 is dimethyl sulfoxide with a concentration of 98%, and reagent 4 is phosphate buffered saline with a concentration of 0.01 mol.

11) Preparing a blank extracting solution:

accurately measuring 2ml of blank milk in a centrifuge tube, adding 3ml of acetonitrile, uniformly mixing by vortex, centrifuging at 4000 rpm for 5 minutes, adding 1ml of n-hexane into 3ml of supernatant, carrying out vortex for 1 minute, carrying out 4000 rpm for 5 minutes, removing the n-hexane, collecting subnatant, drying by blowing with 60-DEG nitrogen, redissolving with 2ml of reagent 4, carrying out vortex for 3 minutes, and passing through a 0.22-micron water-system filter membrane (matched with a syringe), thus obtaining a blank sample extracting solution.

12) Preparing a sample extracting solution:

taking 2ml of milk sample to be detected in a centrifuge tube, and obtaining a sample extracting solution by adopting the same preparation method as the blank extracting solution. The obtained sample extract is stored for later use in an environment with the temperature of 2-8 ℃, and the validity period is one day.

13) Preparing a sulbactam standard stock solution:

adding 0.6ml of reagent 4 into the sulbactam standard product for dissolving to obtain sulbactam standard stock solution with the concentration of 10 mg/ml. The sulbactam standard stock solution is subpackaged (preferably 180ul in one bottle) according to a single dosage and is stored at the temperature of 20 ℃ in a dark place. When in use, 820ul of reagent 4 is added and mixed evenly, and the mixture is diluted to the concentration of 1800 ug/ml.

14) Preparing a sulbactam standard solution:

diluting the sulbactam standard stock solution obtained in the step 13) with blank extracting solution to obtain sulbactam standard solutions with various concentrations of 0, 0.27, 0.82, 2.47, 7.41 and 22.2 ug/ml. Wherein each concentration is not less than 0.5ml, and the preparation can be used as it is.

15) Preparing β -lactamase solution:

according to the amount of the reagent, the reagent 1 is diluted by 100 times by using the reagent 4 to obtain β -lactamase solution which is prepared for use.

16) Preparing a substrate solution:

210ul of reagent 3 was added to reagent 2, and the mixture was dissolved to obtain a substrate solution. The substrate solution is subpackaged according to single dosage and stored at minus 20 ℃ in a dark place.

17) Preparing a substrate dilution solution:

the substrate solution is taken once and added into the reagent 4 with 9 times of volume along the wall slowly and mixed evenly, and the mixture is prepared for use.

Based on all the solutions prepared in the above steps 11) to 17), the quantitative detection of sulbactam can be carried out by the following method:

first, after equilibrating at room temperature for 20 minutes, the desired microwell strip was removed from the aluminum foil pouch (the remaining microwell strip was returned to 4 ℃ for storage with a valve pouch seal) to form an elisa plate with sufficient wells.

Secondly, standard holes and sample holes are set on the enzyme label plate, wherein β -lactamase solution with 20 μ l is added into each standard hole and each sample hole.

And thirdly, adding 160 mu l of blank extracting solution and 160 mu l of sample extracting solution to be detected into the standard sample hole and the sample hole respectively, sealing the reaction hole by using a sealing plate film, and incubating for 15 minutes at room temperature in a dark place.

After incubation for 15 minutes in the absence of light, 20ul of substrate dilution was added to each reaction well, mixed by gentle shaking, and incubated for 30 minutes in the absence of light at room temperature.

Finally, the absorbance (OD value) of each well is measured by using a 488-492nm wavelength microplate reader, and the quantitative analysis of the sulbactam residue of the sample is realized according to a pre-drawn standard curve. Of course, in order to ensure the effect of the measurement, the absorbance should be measured within 15 minutes.

Fig. 3 is a schematic diagram of a calibration curve provided by an embodiment of the present invention. As shown in fig. 3, the abscissa of the calibration curve represents the concentration value and the ordinate represents the absorbance value. When calculating the final result, it is necessary to perform corresponding conversion according to the dilution factor of the sample.

In the sulbactam rapid detection method provided by the embodiment of the invention, the sample processing methods (namely, steps 11) and 12)) are adjusted, so that the subsequent required incubation time is shortened, and the sample processing time is reduced.

The whole detection process is realized based on an enzyme inhibition method, has the characteristics of high specificity, high sensitivity, high accuracy and the like, has the lowest detection limit of 100ug/kg, and is suitable for high-throughput sample screening (an enzyme label plate with a large number of reaction holes can be provided).

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Claims

1. A sulbactam rapid detection kit is characterized by comprising: the reagent kit comprises an ELISA plate and a plurality of reagents packaged in different reagent bottles;

2. The sulbactam rapid detection kit according to claim 1, wherein the diluent is phosphate buffer, the substrate diluent is dimethyl sulfoxide, the substrate is nitrocefin, and the enzyme is β -lactamase.

3. The sulbactam rapid detection kit according to claim 2, wherein the concentration of the phosphate buffer is 0.01mol, the concentration of the nitrocefin is 90%, the concentration of the dimethyl sulfoxide is 98%, and the concentration of the β -lactamase is 10 MU.

4. The sulbactam rapid detection kit according to claim 3, wherein the reagents comprise three vials of phosphate buffer, two vials of β -lactamase, one vial of ceftriaxone and one vial of dimethyl sulfoxide.

5. The sulbactam rapid detection kit according to claim 4, wherein the nitrocefin, the dimethyl sulfoxide and the β -lactamase are all packaged in a glass bottle.

6. The sulbactam rapid detection kit according to claim 5, wherein the content of phosphate buffer solution in each bottle is 20ml, the content of β -lactamase in each bottle is 2mg, the content of nitrothiophene in each bottle is 2.1mg, and the content of dimethyl sulfoxide in each bottle is 0.3 ml.

7. The sulbactam rapid detection kit according to claim 1, wherein the elisa plate comprises: a plastic support and a plurality of microporous strips;

each micropore strip is provided with a plurality of reaction holes which are detachably arranged on the plastic bracket.

8. The sulbactam rapid detection kit according to claim 7, wherein the enzyme label plate comprises 12 microwell strips, and each microwell strip is provided with 8 reaction holes side by side.

9. The sulbactam rapid detection kit according to claim 1, further comprising: the box body and the fixed sponge arranged in the box body;

the fixing sponge is provided with a plurality of cylindrical slots for inserting the reagent bottles to fix the positions of the reagent bottles in the box body.

10. The sulbactam rapid detection kit according to claim 1, wherein in use, the sulbactam standard is prepared into sulbactam standard solutions with various concentrations through a blank extracting solution;

the concentration of the sulbactam standard solution comprises 0, 0.27ug/ml, 0.82ug/ml, 2.47ug/ml, 7.41ug/ml and 22.2 ug/ml.