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Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium

A technology of fluoroquinolones and luciferase, which is applied in the field of veterinary drug residue analysis and genetic engineering, can solve the problems of insufficient detection range of tissue samples, and achieve the effects of wide application range, simple processing method, good accuracy and precision

Inactive Publication Date: 2014-02-19
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the engineering strains constructed by the Institute have shortcomings in terms of stability, and the detection range of tissue samples is not wide enough, which needs to be further improved in practical applications

Method used

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  • Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium
  • Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium
  • Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of luciferase-labeled genetically engineered bacteria

[0030] Pick a small amount of Escherichia coli K12 (purchased from China. Wuhan. Wuhan University China Type Culture Collection Center, its preservation number is CCTCC NO: AB200068) freeze-dried powder, inoculate into 10mL LB broth medium, 37°C, 250rpm Shake overnight in a constant temperature shaker. Pick a small amount of bacterial solution to streak and inoculate on LB agar plate, overnight at 37°C. Prepare Escherichia coli K12 into competent cells [The method of bacterial competent cells is a common method, see: J. Sambrook, EF Fritsch, T Maniartis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guidelines (Third Edition), Beijing, Science Press, 2002 Edition], cryopreserved at -70°C.

[0031]The plasmid pRecAlux3 (gifted by Professor Amy Cheng Vollmer, Department of Biology, Swarthmore College, USA, and preserved in the National Laboratory of Veterinary Dr...

Embodiment 2

[0032] Example 2 Establishment of Fluoroquinolones Detection Method

[0033] 2.1 Determination of the optimal working concentration of bacterial solution

[0034] Escherichia coli pK12 was cultured in LB liquid medium (containing 25 μg / mL kanamycin) at 26°C and 250 rpm until OD 600 At 0.7 and 1, the sensitivity of Escherichia coli pK12 to enrofloxacin was detected, and the incubation time was 45min; the results showed that the higher the concentration of the bacterial solution in the logarithmic growth period, the higher the sensitivity, so the OD 600 When =1, the bacterial liquid is the working bacterial liquid.

[0035] 2.2 Determination of the optimal incubation time of bacteria solution and drug

[0036] Escherichia coli pK12 was cultured in 10 mL LB liquid medium (containing 25 μg / mL kanamycin) to OD 600 =1, add a series of enrofloxacin (0.025 μg / mL, 0.050 μg / mL, 0.100 μg / mL, 0.200 μg / mL, 0.400 μg / mL, 0.800 μg / mL), respectively Each 50 μL was co-incubated for a certa...

Embodiment 3

[0047] Embodiment 3 The implementation procedure of the method that the present invention establishes

[0048] 3.1 Preparation of reagents

[0049] The preparation method of phosphate buffer solution is: weigh 0.523g KH 2 PO 4 and 16.73g K 2 HPO 4 Dissolve in 800mL distilled water, adjust the pH value to 8.0, dilute to 1L, sterilize under high-pressure steam at 121°C for 20min, and set aside.

[0050] 3.2 Pretreatment of tissue samples

[0051] The pretreatment method of muscle and viscera samples: take muscle, liver, kidney and fish samples of beef, chicken and pig respectively, remove fascia, cut into pieces, and homogenize for 5 minutes. Weigh 1.0 g of homogeneous tissue into a centrifuge tube, add 2 mL of sterile pH 8.0 phosphate buffer, vortex and mix for 5 min, let stand at room temperature for 30 min, centrifuge at 12000 g for 10 min, and take the supernatant for testing.

[0052] Pretreatment method of milk samples: Fresh milk samples can be loaded directly for d...

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Abstract

The invention belongs to the field of veterinary drug residue analysis, and particularly relates to a detection method of fluoroquinolone drug residues based on a luciferase labelled genetically engineered bacterium, and an application. The invention provides construction and the application of the luciferase labelled genetically engineered bacterium capable of simultaneously detecting 11 fluoroquinolone drug residues. The engineered bacterium is escherichia coli pK12; the preservation number is CCTCC NO (China Center for Type Culture Collection Number):M2013385; and the engineered bacterium is obtained by converting escherichia coli K12 with a plasmid pRecAlux3 comprising an recA promoter and a luciferase reporter gene luxCDABE. Compared with the prior art, the constructed genetically engineered bacterium can simultaneously recognize multiple fluoroquinolone drugs; a detection object of the prior art is widened; and the detection method has the prominent advantages of simplicity, rapidness, sensitiveness, accuracy, semiquantitative function and the like.

Description

technical field [0001] The method belongs to the technical fields of veterinary drug residue analysis and genetic engineering. It specifically involves the construction of luciferase-labeled genetically engineered bacteria capable of simultaneously detecting 11 fluoroquinolones, and the establishment of a detection method for simultaneously detecting 11 fluoroquinolones. Background technique [0002] Fluoroquinolones are an important class of chemically synthesized antibacterial drugs. These drugs have a broad antibacterial spectrum and strong antibacterial activity, and are commonly used in veterinary clinics. Due to the use of veterinary fluoroquinolones or the use of human fluoroquinolones on animals in accordance with the drug withdrawal period, serious residues in livestock, poultry and aquatic products have resulted. The residues of fluoroquinolones in food of animal origin not only have a direct harmful effect on the human body, but also affect my country's import a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N21/64G01N21/76G01N33/50
Inventor 袁宗辉董小冰程古月王玉莲彭大鹏郝海红黄玲利陶燕飞陈冬梅戴梦红王旭谢书宇潘源虎刘振利谢长清
Owner HUAZHONG AGRI UNIV
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