Kit for quantitatively detecting beta-lactamase residue in milk

A technology for quantitative detection and lactamase, which is applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problem of measuring undecomposed enzymatic reaction substrates for a long time, cannot be quantified, and is difficult to be specific Sexual separation and other issues, to achieve the effect of simple detection steps, good precision, good specificity and sensitivity

Inactive Publication Date: 2013-10-09
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are certain defects in the above two types of detection methods.
No matter which of the above methods can not be quantified, and the method of measuring the undecomposed enzymatic reaction substrate has the defects of long time and high cost
At the same time, β-lactamase, as a kind of small molecule protein (29kDa~39kDa), is mixed in a large amount of protein and amino acid emulsion (milk), and it is difficult to specifically separate it without effective detection means

Method used

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  • Kit for quantitatively detecting beta-lactamase residue in milk
  • Kit for quantitatively detecting beta-lactamase residue in milk
  • Kit for quantitatively detecting beta-lactamase residue in milk

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Preparation of key components of the kit

[0023] 1) Preparation of ELISA plate

[0024] figure 1 It is a structural schematic diagram of the microplate plate of the present invention. Wherein a is a schematic plan view of a microtiter plate, which is a 96-well panel. Dilute penicillin-binding protein PBP2x or PBP3 to 0.05-0.5 μg / mL with carbonate buffer solution (CBS) at a concentration of 0.05mol / L and pH9.6, and add to the wells of the microplate (see figure 1 b), 150 μL per well, incubated at 37°C for 2 hours and then left at 4°C overnight. Pour off the coating buffer, wash twice with washing solution, 30s each time, pat dry, then add 200 μL of 10% calf serum blocking solution to each well, incubate at 37°C for 1.5 h, pour off the blocking solution, pat dry and use Dry in a vacuum desiccator at 4°C for 4 hours, and finally pack in a vacuum-sealed package with aluminum foil, and store in a dry place at 2-8°C.

[0025] 2) Preparation of enzyme markers

[0026] D...

Embodiment 2

[0028] Composition of the kit

[0029] (1) A microtiter plate coated with penicillin-binding protein PBP2x or PBP2x recombinant protein;

[0030] (2) Enzyme markers, i.e. horseradish peroxidase-labeled ampicillin conjugates, at a concentration of 0.1 μg / mL to 1 μg / mL;

[0031] (3) Substrate control 1 and substrate control 2, 6 bottles of each, 12% skim milk freeze-dried product containing 0ppb penicillin and 12% skim milk freeze-dried product containing 20ppb penicillin;

[0032] (4) 20 times concentrated washing buffer solution, that is, 0.1moL / L PBS solution containing 1% (v / v) Tween-20;

[0033] (5) Enzyme standard diluent, that is, a PBS solution with a concentration of 0.05mol / L and a pH of 7.4;

[0034] (6) Chromogenic solution, including substrate solution A and substrate solution B, wherein substrate solution A is 0.1‰~1‰H 2 o 2 Solution, substrate solution B is 1mg / mL~5mg / mL dimethylbenzidine (TMB) solution;

[0035] (7) Reaction termination solution, that is, a ...

Embodiment 3

[0048] Determination of activity of β-lactamase reference substance

[0049] The method for calibrating the activity concentration of β-lactamase in the present invention refers to the determination method of β-lactamase activity defined in "Chinese Pharmacopoeia" (2005 edition two): 37 ℃, within 60 minutes, β-lactamase converts the substrate ( Penicillin) becomes penicillin, and penicillin competes with starch for binding I 2 . Starch is used as an exclusive indicator; sodium thiosulfate solution is used as a standard solution; the activity concentration of β-lactamase is accurately determined.

[0050] The present invention adopts the above determination method to calibrate the calibrator, standard product and quality control product of β-lactamase.

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Abstract

The invention belongs to the field of food detection and in particular relates to a kit for quantitatively detecting beta-lactamase residue in milk. The kit for quantitatively detecting beta-lactamase residue in milk is characterized in that the kit comprises an elisa plate enveloped with penicillin-binding proteins, marker coupled beta-lactam antibiotics, a skim milk powder freeze dried product of which the substrate reference 1 is beta-lactam antibiotics and which has the concentration of 0ppb, a skim milk powder freeze dried product of which the substrate reference 2 is beta-lactam antibiotics containing calibration concentration, and a beta-lactam antibiotic standard substance. The kit has high specificity and sensitivity, beta-lactam with the concentration of about 0.5U/ml can be detected, and the linear detection range is 1-16 U/ml; intra-batch CV percent of the kit is less than 10 percent, the inter-batch CV percent is less than 20 percent, and high precision is reflected; the detection steps are simple, the time consumption is low, and the cost is low; according to the kit, the beta-lactamase residue can be accurately, quantitatively and rapidly detected at high throughput, and the requirements of enterprises and detection mechanisms are met.

Description

technical field [0001] The invention belongs to the field of food detection, and in particular relates to a kit for quantitative detection of beta-lactamase residues in milk. Background technique [0002] β-lactamase is a food additive that has only attracted attention in recent years. It is mainly added to milk to produce antibiotic-free milk (referring to milk produced from raw materials that do not contain antibiotics). β-lactamase, as an antibiotic decomposition agent (antagonist) in milk, was originally introduced by researchers as a scientific research result. This practice can effectively break down the residual β-lactam antibiotics in milk. However, the risks of using β-lactamase to decompose antibiotics in milk are: (1) A large amount of β-lactamase ingested by the human body may lead to bacterial resistance in the human body, so the administrative department has explicitly banned the addition of antibiotics in raw milk ; (2) After decomposing β-lactam drugs, othe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/573G01N33/543
Inventor 黄士新王蓓商军顾欣
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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