Method for detecting beta-lactamase in dairy produce

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 immunoassays, 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, penicillin and cephalosporins, as β-lactam drugs, are the first choice for the treatment of bovine mastitis. They are the most common residual antibiotics in milk, and the residual antibiotics in dairy endanger food safety and human health. my country’s pollution-free fresh milk product standards clearly stipulate that ampicillin ≤ 10μg / kg in fresh milk, and penicillin shall not be detected. Therefore, most domestic dairy companies adopt the principle of reducing prices for milk with excessive antibiotic residues. Driven by economic interests, some illegal milk stations artificially use some biological agents to degrade the residual antib...

Examples

Embodiment 1

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

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

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

Technical field

[0001] The invention relates to the technical field of immunoassays, 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, penicillin and cephalosporins, as β-lactam drugs, are the first choice for the treatment of bovine mastitis. They are the most common residual antibiotics in milk, and the residual antibiotics in dairy endanger food safety and human health. my country’s pollution-free fresh milk product standards clearly stipulate that ampicillin ≤ 10μg / kg in fresh milk, and penicillin shall not be detected. Therefore, most domestic dairy companies adopt the principle of reducing prices for milk with excessive antibiotic residues. Driven by economic interests, some illegal milk stations artificially use some biological agents to degrade the residual antib...

Examples

Embodiment 1

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

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

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

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