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A method for detecting β-lactamase in dairy products

A technology of lactamase and lactam, which is applied in the field of immunoassay, can solve the problems of inaccurate quantitative detection, poor sensitivity, and low sensitivity, and achieve the effects of broadening the scope of application, reducing detection costs, and improving sensitivity

Active Publication Date: 2017-10-20
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has strict requirements on reaction time and other conditions, and there are false positives and false negatives. Each test needs to set up positive and negative controls, and the sensitivity is poor and the reproducibility is poor.
Although the iodometric method is classic and fast, its reagent preparation is more complicated, and the starch reagent is prone to failure, so it must be prepared and used immediately
[0006] (3) The acidometric method is simple and easy to perform, has good repeatability, is the easiest to obtain results, is more sensitive, has good result stability, and has fewer false positive and false negative results. It is suitable for most laboratories. Although rapid detection, the detection Low sensitivity and cannot be used for enzyme kinetic analysis
[0007] (4) High-performance liquid chromatography has the advantages of short detection time and quantitative analysis, but has disadvantages such as low sensitivity, high detection cost, and complicated pretreatment operations.
[0008] (5) Cephalosporin chromogenic method: This method is more convenient and fast, and the operation is simple, but the reagents used are expensive and the quantitative detection is not accurate
[0009] (6) The immunoassay method can directly identify the target molecule itself through the preparation of specific antibodies. It has strong specificity, high sensitivity, and low cost. It can be carried out without large and complicated equipment, but the traditional ELISA method requires multiple steps. Washing is time-consuming and labor-intensive, so its use is also subject to certain restrictions
However, these methods require the preparation of antibodies and multiple washings, which are time-consuming and labor-intensive.

Method used

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  • A method for detecting β-lactamase in dairy products
  • A method for detecting β-lactamase in dairy products
  • A method for detecting β-lactamase in dairy products

Examples

Experimental program
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Effect test

Embodiment 1

[0054] In this example, a fluorescent substrate probe is used to detect penicillinase in milk.

[0055] Add 100 μL of 1 μM fluorescent substrate probe to milk spiked with a series of different concentrations of penicillinase (the volume of the milk sample is 1000 μL, the concentrations of penicillinase are 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0ng / mL), shake the reaction at room temperature for 15min, and then measure the fluorescence value of each sample. Through the linear relationship between the fluorescence value and the concentration of penicillinase added, the content of penicillinase in milk can be obtained.

Embodiment 2

[0057] In this example, a fluorescent substrate probe is used to detect cephalosporinase in milk.

[0058] 100 μL of 1 μM fluorescent substrate probe was added to milk spiked with a series of different concentrations of cephalosporinase (the volume of milk sample was 1000 μL, and the concentrations of cephalosporinase were 1000 ng / mL, 100 ng / mL, 10 ng / mL, respectively. mL, 1ng / mL, and 0ng / mL), shake the reaction at room temperature for 15min, and then measure the fluorescence value of each sample. Through the linear relationship between the fluorescence value and the concentration of added cephalosporinase, the content of cephalosporinase in milk can be obtained.

Embodiment 3

[0060] In this example, a fluorescent substrate probe is used to detect cephamycinase in milk.

[0061] 100 μL of 1 μM fluorescent substrate probe was added to milk spiked with a series of different concentrations of cephamycinase (the volume of milk sample was 1000 μL, and the concentrations of cephalosporinase were 1000 ng / mL, 100 ng / mL, 10 ng / mL, respectively. mL, 1ng / mL, and 0ng / mL), shake the reaction at room temperature for 15min, and then measure the fluorescence value of each sample. Through the linear relationship between the fluorescence value and the concentration of cephamycinase added, the content of cephamycinase in milk can be obtained.

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Abstract

The invention relates to a method for detecting beta-lactamase in a dairy produce. The method is characterized in that a fluorescence substrate probe which can be specifically catalyzed by beta-lactamase is designed, and the main structure of the fluorescence substrate probe is chemically synthesized through using the mother ring of fluorescein and beta-lactam antibiotic. The beta-lactamase can catalyze the fluorescence substrate probe to emit strong fluorescence, and the content of beta-lactamase is calculated according to the fluorescence intensity obtained through a reaction. 0.1ng / mL of Beta-lactamase can be detected through the method, the sensitivity of the method is two orders of magnitude higher than that of traditional ELISA methods, the detection time is shortened to 45min, and the method also has the advantages of strong specificity, high sensitivity, simplicity, rapid detection, and great reduction of the detection cost.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a method for detecting β-lactamase in dairy products, in particular to a method for quantitatively detecting β-lactamase in dairy products based on the fluorescence value of a fluorescent substrate probe Background technique [0002] At present, penicillins and cephalosporins are the first choice for the treatment of bovine mastitis as β-lactam drugs. They are the most common residual antibiotics in milk, and the residual antibiotics in dairy products endanger food safety and human health. my country's pollution-free raw milk product standards clearly stipulate that ampicillin in raw fresh milk should not exceed 10 μg / kg, and penicillin should not be detected. Therefore, most domestic dairy companies adopt the principle of reducing the price of milk with excessive antibiotic residues. Driven by economic interests, some illegal milk stations artificially use some biological ag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N21/64
CPCG01N21/6428
Inventor 蒋兴宇陈翊平吴景曹丰晶张晓青牛亚静
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA