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Tetracycline drug detection method based on TetR protein steric hindrance and gene cleavage technology

A tetracycline and derivative technology, applied in the field of tetracycline drug detection, can solve the problems of complex operation, unfavorable promotion and application, etc., and achieve the effects of high sensitivity, reduced operation steps, and simple operation

Pending Publication Date: 2022-01-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method based on TetR and tetO has been reported, but the traditional ELISA method needs multi-step incubation, and needs to catalyze the substrate to generate the detection signal, the operation is complicated, and it is not conducive to popularization and application. Therefore, it is urgent to develop a new detection technology.

Method used

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  • Tetracycline drug detection method based on TetR protein steric hindrance and gene cleavage technology
  • Tetracycline drug detection method based on TetR protein steric hindrance and gene cleavage technology
  • Tetracycline drug detection method based on TetR protein steric hindrance and gene cleavage technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1, TetR and restriction endonuclease competitive binding tetO-X

[0035] 1. Preparation of double-stranded DNA molecule tetO-X for detection of tetracycline and its derivatives

[0036] Design and synthesize a DNA fragment (tetO-X) containing the tetracycline operator gene tetO and a restriction endonuclease recognition site. The 3' end of one chain is coupled with biotin, and the 3' end of the other chain is coupled with fluorescein. The two strands are complementary hybridized in vitro to form a double-stranded DNA for future use.

[0037]The sequences of the two chains of tetO-X are as follows:

[0038] tetOF: 5'-GTTATTTTACCACGGAATTCCTCCCTATCAGTGATAGAGAAA-3'-TAMRA (SEQ ID No. 1)

[0039] tetOR: 5'-TTTCTCTATCACTGATAGGGAGGAATTCCGTGGTAAAATAACTTTTTTTTTTTTTTT-3'-biotin (SEQ ID No. 2).

[0040] Wherein, the sequence of the tetracycline control gene tetO is shown in 22-40 positions in SEQ ID No.1, and the sequence of the restriction endonuclease EcoRI recognition...

Embodiment 2

[0065] Example 2, using tetO-X to detect tetracycline and its derivatives

[0066] 1. Determine the optimal working concentration of TetR and EcoRI

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Abstract

The invention discloses a tetracycline drug detection method based on TetR protein steric hindrance and a gene cleavage technology, a double-stranded DNA molecule for detecting tetracycline drugs, a composition, a kit and application of the double-stranded DNA molecule. The double-stranded DNA molecule comprises a tetracycline manipulating gene tetO and a restriction endonuclease recognition site, one end of the double-stranded DNA molecule is connected with a fluorophore, and the other end of the double-stranded DNA molecule is connected with biotin. The double-stranded DNA molecule can realize detection of tetracycline drugs.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a tetracycline drug detection method based on TetR protein steric hindrance and gene cutting technology. Background technique [0002] Tetracyclines are broad-spectrum antibiotics widely used in animal husbandry, which can effectively improve feed utilization, promote growth, and prevent and control diseases. However, the large-scale use, abuse or even illegal use of drugs will lead to their residues in animal-derived foods and the environment, endangering human health. Efficient, rapid, sensitive, and multi-target detection of them is an important means to ensure food safety. [0003] The currently reported detection methods for tetracycline drug residues mainly include microbiological methods, spectroscopic methods, chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, and immunological methods. Among them, high-performance liquid chromatography and l...

Claims

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Application Information

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IPC IPC(8): C12N15/11G01N21/64
CPCC07K14/00G01N21/6486Y02A50/30
Inventor 王战辉沈建忠于雪芝温凯江海洋余文博
Owner CHINA AGRI UNIV
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