Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof

A detection kit, lactam technology, applied in biochemical equipment and methods, testing food, testing dairy products, etc. Not enough and other problems, to achieve the effect of short detection time, large detection throughput, and easy operation

Active Publication Date: 2013-07-24
上海溯源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its principle is the specific recognition reaction between ligands and receptors. It has the characteristics of strong specificity, high sensitivity, short detection time, and does not require complicated equipment. However, the disadvantage is that there are few such products on the market at present, and the The types of β-lactam antibiotics that can be detected are not enough, and the detection limit is higher than the relevant residue limit standards at home and abroad, which cannot fully meet the needs of daily residue monitoring

Method used

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  • Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof
  • Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof
  • Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Recombinant Penicillin Binding Protein PBP 2x a and PBP 2x b preparation of

[0063] 1) Gene synthesis

[0064] According to the gene sequence of Streptococcus pneumonia R6 strain penicillin-binding protein (PBP 2x) (gene sequence number: GENBANK 18266817), two artificial gene fragments were designed and synthesized: (1) PBP 2x a The artificially synthesized gene sequence is 19-750aa, and the gene sequence of the transmembrane region of 1-19aa is deleted (see SEQ ID No 1 in the sequence listing for details); (2) PBP 2x b The artificially synthesized gene sequence is 266-615aa, which is the binding functional region of PBP 2x and β-lactam antibiotics, and the gene sequences of other non-binding functional regions are deleted (see SEQ ID No 2 in the sequence list for details). In order to facilitate the connection with the expression vector and obtain the recombinant protein with the same function as the penicillin-binding protein PBP 2x, a Bgl II restriction site (und...

Embodiment 2

[0077] Preparation of key components of the kit

[0078] 1) Preparation of enzyme plate

[0079] 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. Penicillin-binding protein PBP 2x was added with a concentration of 0.05mol / L, pH 9.6 carbonate buffer a or PBP 2x b Dilute to 0.05-0.5μg / mL, add to the microplate well (see figure 1 b) 150 μL per well, incubate at 37°C for 2 hours and then stand overnight at 4°C. After pouring off the coating buffer, wash with washing solution twice, each time for 30s, pat dry, then add 200μL of 10% calf serum blocking solution to each well, incubate at 37°C for 1.5h, pour off the blocking solution, and pat dry Use a vacuum desiccator to dry at 4°C for 4 hours, and finally wrap it in a vacuum-sealed package with aluminum foil, and store it in a dry place at 2-8°C.

[0080] 2) Preparation of enzyme markers

[0081...

Embodiment 3

[0083] Composition of the kit

[0084] (1) coated with penicillin binding protein PBP 2x a or PBP 2x b ELISA plate of recombinant protein;

[0085] (2) Enzyme markers, i.e. horseradish peroxidase-labeled ampicillin conjugates, at a concentration of 0.1 μg / mL to 1 μg / mL;

[0086] (3) Ampicillin sodium standard solution, a total of 6 bottles, the concentrations are 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, 8.1 μg / L;

[0087] (4) 20 times concentrated washing buffer solution, that is, a 0.1moL / L PBS solution containing 1% (v / v) Tween-20;

[0088] (5) Enzyme label diluent, that is, a PBS solution with a concentration of 0.05mol / L and a pH of 7.4;

[0089] (6) 20 times concentrated sample extract, that is, a PBS buffer solution with a pH of 7.4 and 0.1moL / L;

[0090] (7) 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 dimethyl benzidine (TMB) solu...

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Abstract

The invention discloses a kit for detecting beta-lactam antibiotic ligand in milk by a receptor method and a detection method thereof. The kit comprises the following components: (1) an enzyme-labeled plate which is coated by penicillin-binding protein PBP2xa or PBP2xb recombinant protein; (2) an enzyme-labeled marker; (3) standard solution of ampicillin sodium; (4) 20 times concentrated cleaning buffer solution; (5) enzyme-labeled diluent; (6) 20 times concentrated sample extract; (7) developing solution; and (8) reaction stop solution. The kit of the invention can simultaneously screen eight beta-lactam antibiotic residues in the milk; the detection limit is lower than the minimum residue limit of China and main developed countries in the world; the detection time is within 50 minutes; and the kit has the characteristics of simple, rapid and accurate operation, low cost and the like and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of micro-analysis of biochemistry, and in particular relates to a detection kit and a detection method of β-lactam antibiotic ligand receptor method in milk. Background technique [0002] With the increasing awareness of food safety, the problem of antibiotic residues in milk has attracted more and more attention from consumers. Residues in milk are mainly caused by antibiotic residues in cow feed, cow drug injection and milk insurance additives. There are many kinds of antibiotics used in veterinary clinics, among which β-lactam antibiotics are the most common, and the residues of β-lactam antibiotics are the most harmful to human body. [0003] At present, controlling the amount of antibiotics used in milk and formulating a standard method for rapid detection of antibiotic content in milk is a necessary guarantee to ensure the quality and safety of milk, the main requirement of food quality and safety control, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/566G01N33/04C12N15/31C12N15/63C07K14/315
Inventor 肖理文胡佳骏李康郭晓梅
Owner 上海溯源生物技术有限公司
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