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Method for quantitatively detecting chloramphenicol by utilizing photobacterium leiognathus

A quantitative detection and chloramphenicol technology, which is applied in the determination/inspection of microorganisms, chemiluminescence/bioluminescence, and analysis by making materials undergo chemical reactions, can solve the problems of environmental pollutant detection without luminescent bacteria

Inactive Publication Date: 2010-03-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Except for Luminescent bacteria, there is no report on the use of other species of luminescent bacteria to detect environmental pollutants.

Method used

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  • Method for quantitatively detecting chloramphenicol by utilizing photobacterium leiognathus
  • Method for quantitatively detecting chloramphenicol by utilizing photobacterium leiognathus
  • Method for quantitatively detecting chloramphenicol by utilizing photobacterium leiognathus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Inoculate P.leiognathi YL single colony in 150mL liquid medium (medium composition: peptone 5g, yeast extract 1g, FePO 4 0.1g, NaCl 20g, glycerol 3ml, distilled water 1000ml), 26°C, 150r / m shaker culture for about 6h, until the luminescence value reaches 2.0×10 5 -4.0×10 5 When the concentration of chloramphenicol is 0, 0.1ng / mL, 0.2ng / mL, 0.4ng / mL, 0.6ng / mL, 0.8ng / mL, 1.0ng / mL test tubes, the reaction system Chloramphenicol 5mL, bacteria solution 5mL. After mixing evenly, incubate at 26°C and 150r / m shaker for 30min, and measure the luminescence value. Draw the standard curve for chloramphenicol detection ( figure 1 )

Embodiment 2

[0012] Fish pretreatment method

[0013] (1) Take out the fish viscera, peel off the fish skin, and homogenize. Pack 20.0g of each bag of fish paste into small packaging bags for later use.

[0014] (2) Thaw the frozen fish, weigh 5.0g into a 50mL centrifuge tube, and add 50uL of standard CAP solution (concentration: 10ng / mL) to the centrifuge tube, which is equivalent to the concentration of chloramphenicol in the fish after adding the standard is 0.1ng / g, do three groups in parallel.

[0015] (3) Mix the samples in each centrifuge tube for 1 min.

[0016] (4) Add 20 mL of ethyl acetate to each centrifuge tube, mix for 1 min, sonicate for 10 min, and centrifuge for 5 min (4000 r / m).

[0017] (5) Pour the supernatant into a chicken heart bottle, and evaporate to dryness in a water bath at 40°C.

[0018] (6) Add 1 mL of methanol and vortex to dissolve the residue.

[0019] (7) Add 15 mL of n-hexane and mix evenly, then add 25 mL of 4% NaCl solution, shake and mix, and ext...

Embodiment 3

[0025] Thaw the frozen fish, weigh 5.0g into a 50mL centrifuge tube, add standard CAP solution (concentration: 10ng / mL) 50μL, 100μL, 200μL, 30μL, 400μL, 500μL to the centrifuge tube in turn, the The concentration of chloramphenicol was 0.1ng / g, 0.2ng / g, 0.4ng / g, 0.6ng / g, 0.8ng / g, 1.0ng / g, and six parallel groups were made for each concentration.

[0026] After adding chloramphenicol solution to the sample, pretreatment was carried out according to Example 2.

[0027] Inoculate a single colony of P.leiognathi YL into 150mL of liquid medium, put it into a shaking table for culture, and shaker parameter settings: 26°C, 150r / m. The luminous intensity of the P.leiognathi YL bacterial solution was at 3.0×10 5 Add 5mL of bacterial solution to each test tube containing 5mL of chloramphenicol solution recovered by standard addition, and make a set of standard solution blank control for each treatment method, that is, add 5mL of bacterial solution to a test tube containing 5mL of doubl...

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Abstract

The invention aims to quantitatively detect chloramphenicol by utilizing photobacterium leiognathus (YL). In the invention, the photobacterium leiognathus (YL) from marine environment is separated andpurified, and the photobacterium leiognathus is a gram-negative bacillus. China Center for Type Culture Collection (CCTCC) is authorized to preserve the photobacterium leiognathus strain on December27, 2006 with a preservation number of M 206139. The 16S rDNA gene sequence of the photobacterium leiognathus strain is submitted to the CenBank database with an access number of EF017227. The invention establishes a method system for quantitatively detecting the chloramphenicol by utilizing the characteristics of quick growth, stable luminous performance, low requirement on nutrition and sensitivity to the chloramphenicol of the photobacterium leiognathus strain and the characteristic that the concentration of the chloramphenicol is in a direct proportion to the luminous intensity suppressionratio of the photobacterium leiognathus strain through selecting indexes of an optimal detection wavelength, an optimal growth state, initiative luminous intensity, reaction time, reaction temperature, and the like so as to realize the quantitative detection of the chloramphenicol. The requirement on the quick quantitative detection of the chloramphenicol can be met by utilizing the characteristics of low cost, simple operation, high sensitivity, and the like for detecting the chloramphenicol of the invention.

Description

technical field [0001] The invention relates to a method for detecting chloramphenicol, in particular to a method for quantitatively detecting chloramphenicol by using the marine luminescent bacterium Photobacterium leiognathi YL strain. Background technique [0002] According to the inventor's information and literature search, there are only three known genera of marine luminescent bacteria, which are Vibrio, Photobacterium, and Shewanella. On land, there are different short bacillus (Xenorhabdus). In an aerobic natural environment, luminescent bacteria can produce luciferase (LE), catalyze flavin mononucleotide (FMNH 2 ) and long-chain aliphatic aldehydes undergo oxidation reactions to emit blue-green fluorescence. [0003] At present, Photobacterium phosphoreum is widely used in our country. There are some reports on the use of Photobacterium phosphoreum to detect environmental pollutants. Except for Luminescent bacteria, there is no report on using other species of l...

Claims

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Application Information

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IPC IPC(8): G01N21/76C12Q1/18
Inventor 王静雪林洪朱兰兰王亚群
Owner OCEAN UNIV OF CHINA
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