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Vibrioharveyi bacteriophage, and bacteriophage composition and application thereof

A technology of Vibrio harveii and bacteriophage, applied in the field of microorganisms, can solve problems such as antibiotic residues, and achieve the effects of easy industrial production, reduction of morbidity and mortality of aquatic products, and beneficial to healthy development

Active Publication Date: 2020-09-18
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The phage and its phage composition are safe to use and have no side effects, and are used to solve the infection caused by Vibrio harveyi and the water problem caused by the large proliferation of Vibrio harveyi in water, avoiding the antibiotics caused by the use of antibiotics. Residues, and the problem of induction of drug-resistant Vibrio harveii

Method used

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  • Vibrioharveyi bacteriophage, and bacteriophage composition and application thereof
  • Vibrioharveyi bacteriophage, and bacteriophage composition and application thereof
  • Vibrioharveyi bacteriophage, and bacteriophage composition and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] The isolation and cultivation of embodiment 1 phage

[0033] (1) Preparation of host bacterial suspension:

[0034] The host bacterium K005 was divided and marked on the TCBS plate, and a single colony was picked and inoculated into 5ml 2216E liquid medium, and multiplied to the logarithmic growth phase at 170rpm and 37°C to obtain a fresh bacterial solution.

[0035] (2) Isolation and purification of phage

[0036] (1) Isolation of phage

[0037] Sewage collected from a seafood market in Qingdao City, Shandong Province was centrifuged at 11,000 r / min for 5 minutes, and the supernatant obtained by centrifugation was filtered with a 0.22 μm filter membrane, then added with 2216E medium and 100 μl of host bacteria suspension, and the mixture was heated at 170 rpm 1. After culturing at 37°C for 16 hours, centrifuge at 11,000 r / min for 5 minutes, and finally filter with a 0.22 μm filter membrane to obtain the phage stock solution.

[0038] The phage stock solution was di...

Embodiment 2

[0046] The biological characteristic determination of embodiment 2 bacteriophage

[0047] (1) Electron microscope detection of phage

[0048] Take 20μl for a titer of 10 10 The phage of PFU / ml was dropped on the copper grid, precipitated for 15 minutes, absorbed the excess liquid with filter paper, stained with 2% phosphotungstic acid for 30 minutes, and observed with electron microscope after drying.

[0049] The result is as figure 1 As shown, the length of the phage head is 80nm, the head width is 75nm, and the retractable tail length is 125nm. According to the classification standard identification of the ninth report of the International Committee on Taxonomy of Viruses (ICTV), it can be It was determined that the phage belonged to the family Myocaudidae, and it was named vB_KaS_PK08.

[0050] (2) Determination of lysis profile of phage

[0051] Experimental method: In this example, the double-layer plate method was used to measure the bacteriophage lysis spectrum. Ta...

Embodiment 3

[0073] Embodiment 3 phage lysis experiment in vitro

[0074] The phage suspension and the host bacteria were added to 100ml 2216E liquid medium according to the ratio of MOI0.01, and the final concentration of clinically isolated pathogenic Vibrio harveyi strain KO11 was 5.00×10 6 CFU / ml, the final concentration of phage was 5.00×10 4 PFU / ml, add the same amount of sterile 2216E medium as the phage solution to the control group, mix the bacteriophage and phage, shake and culture in a shaker at 37°C and 170rpm, measure the OD value at regular intervals until the mixture becomes clear .

[0075] The result is as Figure 5 Shown: the phage can effectively inhibit the host bacteria, OD after 2h 600 Significantly decreased, indicating that the bacterial concentration decreased sharply, and the OD at 3h 600 has dropped to 0.3, and at this time the OD of the host bacteria in the control group 600 The value has risen to more than 1.0, and the mixed solution added to the phage sus...

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Abstract

The invention discloses a vibrioharveyi bacteriophage, and a bacteriophage composition and application thereof. The bacteriophage is named as vB_KaS_PK08, and is preserved in China General Microbiological Culture Collection Center on 15th May, 2020, and the preservation No. is CGMCC No.19973. The bacteriophage not only has wide lysis spectrum, is high in specificity, and can effectively prevent and control vibrioharveyi diseases of in-home aquatic farms, and invasion probability of various diseases caused by the vibrioharveyi can be greatly reduced. The vibrioharveyi bacteriophage can also bewidely applied to respects of daily disinfection for links and breeding environment, to which loss is easy to cause due to infection with the vibrioharveyi in the aquaculture process, bacteriostasis of aquatic products and fresh foods, and the like, and is beneficial for healthy development of aquaculture trade.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a Vibrio harveyi phage, a composition containing the phage and applications thereof. Background technique [0002] Bacterial disease is a very common type of disease in aquaculture, and Vibrio harveyi is a pathogenic bacterium that is widely distributed in marine and salt lake environments. Exist in nearshore seawater, seabed sediments and various marine organisms. Vibrio harveyi is one of the main pathogenic bacteria of aquatic economic animals such as fish, shrimp and shellfish. In recent years, aquatic animal diseases caused by the outbreak of Vibrio harveyi have also appeared frequently, and phages have proposed to solve this problem. Program. [0003] In the past few decades, antibiotics have been considered the best solution to bacterial infections. However, with the increasing abuse of antibiotics in recent years, the problem of bacterial resistance has become inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A23K10/18A23K50/80A01N63/40A01P1/00A23B4/22A23L3/3571A01K61/13C12R1/92
CPCC12N7/00A61K35/76A61P31/04A23K10/18A23K50/80A01N63/40A23B4/22A23L3/3571A01K61/13C12N2795/10121C12N2795/10131C12N2795/10132A61K2300/00Y02A40/81
Inventor 潘强任慧英孙虎芝闫艳新王春阳
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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