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Pseudomonas aeruginosa capable of secreting fluorescent iron carrier and application thereof

A technology of Pseudomonas aeruginosa and fluorescent siderophore, which is applied to Pseudomonas aeruginosa and its application fields, can solve problems such as endangering the healthy breeding of fish, chronic poisoning of fish and shrimp, and gene mutation, and achieves high speed and specificity. High performance and simple bacterial culture

Inactive Publication Date: 2013-02-06
烟台海上传奇生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that furazolidone can cause genetic mutations and induce cancer in experimental animals
Furazolidone residues in the aquaculture environment lead to chronic poisoning of fish and shrimp, seriously endangering the healthy farming of fish

Method used

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  • Pseudomonas aeruginosa capable of secreting fluorescent iron carrier and application thereof
  • Pseudomonas aeruginosa capable of secreting fluorescent iron carrier and application thereof
  • Pseudomonas aeruginosa capable of secreting fluorescent iron carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Acquisition of 16S rDNA of Pseudomonas aeruginosa PA1

[0020] The Pseudomonas aeruginosa PA1 in the stable phase was collected, and the genomic DNA of the bacterium was extracted. The specific steps are:

[0021] (1) The PA1 strain cultured overnight at 30°C was inoculated into a test tube containing 10 mL of LB medium at a volume ratio of 1%, and the OD of the bacteria was 600 When ≈1.0 or so, take 1 mL of the bacterial solution and centrifuge at 10,000 g for 1 min in a 1.5 mL centrifuge tube to collect the bacterial cells.

[0022] (2) Add 1 mL of 0.9% NaCl to the above obtained bacteria to wash the precipitate, and centrifuge at 10,000 g for 3 min.

[0023] (3) Discard the supernatant, resuspend the pellet in 450μL TE buffer (12mM Tris-HCl, 12mM EDTA, pH 8.0), then add 50μL 20% SDS, gently invert the centrifuge tube up and down to mix, and place in a 75℃ water bath Place in the middle for 5min, until the bacterial solution is lysed and becomes clear. ...

Embodiment 2

[0030] Example 2: Purification of fluorescent siderophore

[0031] (1) PA1 culture: select a ring of Pseudomonas aeruginosa PA1 colony from the plate and insert it into the inorganic salt plus succinic acid medium, the liquid volume of the shake bottle is 30%, the culture temperature is 28-30°C, and the culture speed is 180-200rpm / min, the culture time is 48h. The composition of the inorganic salt plus succinic acid medium is 6.0 g of dipotassium hydrogen phosphate, 3.0 g of potassium dihydrogen phosphate, 1.0 g of ammonium sulfate, 0.1 g of magnesium sulfate heptahydrate and 4.0 g of succinic acid per liter of water.

[0032] (2) Purification of fluorescent siderophores: Centrifuge the cultured bacteria at 10,000 rpm for 5 min, and take the supernatant. Add 2 times the volume of binding buffer to the supernatant, add the supernatant with the binding buffer to the copper ion chelation chromatography column, then add 5-10 times the volume of binding buffer to wash, so that th...

Embodiment 3

[0033] Example 3: Observation of the fluorescence quenching effect of furazolidone on fluorescent siderophores under ultraviolet light.

[0034] DDT, trichlorfon, sulfadiazine, sulfadoxine, sulfathiazole, sulfamethazine, sulfamethazine, furazolidone, bahxahix, carbendazim, diethylstilbestrol, diazinon, ethyl bromthiophos, Brothion-methyl was added to the HEPES solution containing fluorescent siderophore respectively so that the final concentration was 50ppm respectively, and the reaction was carried out in a 96-well plate. The 96-well plate containing the above-treated mixed solution was placed under an ultraviolet lamp (wavelength: 256 nm), and the change of fluorescence was observed with naked eyes. Experimental results such as figure 2 , it was found that the addition of furazolidone can quickly cause the fluorescence quenching of fluorescent siderophores, while the addition of other pesticides did not affect the fluorescence intensity of the fluorescent siderophores, so ...

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Abstract

The invention relates to application of a microorganism material in detection of environmental pollutants, in particular to a fluorescent iron carrier secreted by pseudomonas aeruginosa (Pseudomonas aeruginosa) PA1 and application thereof. The pseudomonas aeruginosa PA1 is registered and preserved in China General Microbiological Culture Collection Center (CGMCC); the preservation date is April, 28th, 2011; and the number is CGMCC No.4799. When the fluorescent iron carrier secreted by pseudomonas aeruginosa (Pseudomonas aeruginosa)PA1, related by the invention, is used for detecting furazolidone in the environment, the fluorescent iron carrier has the advantages of high specificity and fastness, and the furazolidone is quickly and sensitively detected on the basis of optimization of experimental conditions.

Description

technical field [0001] The invention relates to the application of microbial materials in the detection of environmental pollutants, in particular to a pseudomonas aeruginosa capable of secreting fluorescent siderophore and the application thereof. Background technique [0002] Furazolidone, also known as furazolidone, has the molecular formula C 8 h 7 N 3 o 5 , is a synthetic broad-spectrum antibacterial drug with the basic structure of 5-nitrofuran. Because furazolidone has the advantages of cheap price and quick drug effect, it is often used to prevent and control infections caused by bacteria and protozoa such as cholera, coccidiosis, swine enteritis and turkey blackhead disease, and it is also used in aquaculture to a certain extent. However, studies have shown that furazolidone can cause gene mutations and induce cancer in experimental animals. The residue of furazolidone in the aquaculture environment leads to chronic poisoning of fish and shrimp, which seriously...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P1/04C07B63/00G01N21/64C12R1/385
Inventor 陈令新张卫卫殷堃牛宗亮潘婷赵君才
Owner 烟台海上传奇生物技术有限公司
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