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Bacteriophage of fish bacteria and application thereof

A phage and fish technology, applied in the phage field of fish bacteria, can solve the problems that the phage of Aeromonas schtzeri has not been reported.

Inactive Publication Date: 2018-06-12
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report on the research on Aeromonas schutzeri phage at home and abroad.

Method used

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  • Bacteriophage of fish bacteria and application thereof
  • Bacteriophage of fish bacteria and application thereof
  • Bacteriophage of fish bacteria and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Isolation and purification of embodiment 1 phage SD04

[0027] Methods: Collect 450mL fresh water samples from the ponds of Channa chinensis infected with Aeromonas struzeri, centrifuge at 5000rpm / min for 20min, add 50mL of 10-fold concentrated nutrient broth medium to the supernatant and cultivate them to the early logarithmic growth stage. Aeromonas schutzeri GC 1 2 mL, shake overnight at 28°C. Take 10 mL of the sample solution cultured overnight, centrifuge at 10 000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane and place it in a water bath at 56 °C for 1 h, and dilute the filtrate 10 times to 10 -9 . Refer to the double-layer agar plate method reported in the literature and slightly modify it to check the formation of phage plaques. Take 0.2mL of each gradient dilution and GC in the early logarithmic period. 1 Mix 0.2 mL of the suspension and let it stand at room temperature for 15 minutes, then mix it with 6 mL of agar that has been m...

Embodiment 2

[0029] Titer determination of embodiment 2 phage SD04

[0030] Method: The titer of phage SD04 strain was determined by the double-layer agar plate method described in Example 1. Using SM buffer as the diluent, the purified phage SD04 strain was serially diluted 10 times, and diluted to 10 -9 , each dilution was done in 3 parallel replicates. When counting, select a plate with 30-300 phage plaques, and then take the average of 3 parallel repetitions of the dilution to calculate the phage titer. Phage titer (PFU mL -1 )=dilution factor×average number of plaques×5.

[0031] Results: phage SD04 made 10 -9 After dilution, the double-layer agar plate test can form phage plaques of uniform size, and the number of phage plaques on the three parallel-treated plates is 90, 88, and 93, respectively, between 30-300, which can be used for counting. Calculated according to the above-mentioned formula, the titer of this Aeromonas schushii phage is about 4.5 * 10 10 PFUmL -1 .

Embodiment 3

[0032] Example 3 Determination of phage SD04 multiplicity of infection (multiplicity of infection, MOI)

[0033] Method: Culture the host bacteria Aeromonas schtzeri GC 1 Strain to the early logarithmic period, adjust the bacterial concentration to 1.5×10 with a turbidimeter 8 PFUmL -1 . According to the ratio of MOI of 0.001, 0.01, 0.1, 1, 10, 100 and 1000, respectively, add phage pure culture solution and host bacteria, and finally add broth culture solution so that the total volume of each tube is 15 mL. Centrifuge at 10,000×g for 10 minutes at 160 r / min in a shaker at 28°C for 12 to 16 hours, collect the supernatant, filter through a 0.22 μm filter, and measure phage titer by double-layer agar culture method. Each group was cultured in duplicate in multiple tubes to obtain the average value. At the same time, the host bacteria without phage and the phage without host bacteria were used as controls, and the MOI that produced the highest phage titer was considered as the ...

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Abstract

The invention discloses bacteriophage of fish bacteria and application thereof. The bacteriophage is named as Aeromonassa schubertii bacteriophage SD04. The bacteriophage has the advantages that the bacteriophage SD04 is used for treating white spot diseases of inner organs of channa maculatae due to infection of aeromonas schubertii for the first time, and the natural infection mode of the bacteria is simulated; the bacteriophage is sprayed to prevent the white spot diseases, the disease treatment mode of abuse of antibiotics and chemical drugs is broken through, the possible multiple drug-resistant bacteria infection, even threat of 'super-bacteria', and pollution and destruction destroy to breedingculture environment and ecological environment are reduced for the breedingculture industry, and a foundation is laid for treatment of the bacteria diseases by using the bacteriophage as a biological agentpreparation in the later study.

Description

technical field [0001] The invention relates to a phage of fish bacteria and its application. Background technique [0002] Channamaculata, also known as local raw fish, belongs to the genus Chann of the family Ophiocephalidae of the order Perciformes. It is mainly distributed in the south of the Yangtze River Basin. This species grows fast and has high economic benefits. At present, the breeding area in Guangdong Province is about 25,000 mu, and the yield per mu is as high as 2500-4000kg. However, with the high-density intensive farming mode, diseases occur frequently, and the degree of damage is also increasing. [1-3] . According to preliminary investigations, since 2006 in some fish ponds in Guangdong Province every year from May to August, a large number of spotted snakeheads have suffered explosive deaths, with white spots on the liver, spleen and kidneys. The incidence rate in ponds is 6% to 9%, and the mortality rate is as high as 50%. 60%, farmers suffered heavy l...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCA61K35/76C12N7/00C12N2795/10121C12N2795/10132
Inventor 罗霞廖国礼刘春花黄志斌陈总会江小燕赵长臣
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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