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Separation and application of lytic escherichia coli phage RDP-EC-16029

A technology of RDP-EC-16029 and Escherichia coli, which is applied in the field of microbiology, can solve the problems of stagnant research on bacterial diseases and low acceptance of virus preparations, and achieve good acid-base tolerance and high-efficiency infection

Active Publication Date: 2020-05-05
QINGDAO RUNDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to people's low acceptance of virus preparations, with the emergence of antibiotics such as penicillin during World War II, scientists basically gave up research on phage therapy and began to invest in the torrent of antibiotic research. From 1950 to 1980, about phage therapy Bacterial disease research grinds to a halt

Method used

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  • Separation and application of lytic escherichia coli phage RDP-EC-16029
  • Separation and application of lytic escherichia coli phage RDP-EC-16029
  • Separation and application of lytic escherichia coli phage RDP-EC-16029

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Isolation and identification of pathogenic avian Escherichia coli E10

[0035] Sampling was taken from the diseased farm, and the liver of the diseased poultry was taken aseptically, and a line was drawn on the selective medium (MacConkey agar). After culturing at 37°C for 18-24 hours, a round, flat, neat edge, and surface were formed on the medium. Smooth and wet red colonies, pick typical colonies and continue to streak and purify 3 times, then pick a single colony and inoculate in 5mL LB broth, shake and culture at 37°C, 200rpm for 8h to obtain a uniform turbid bacterial suspension. After 16sRNA molecular identification and serotype identification, it was determined to be pathogenic Escherichia coli, named as E10, and stored in a -80°C refrigerator.

Embodiment 2

[0036] Example 2 Isolation and identification of phage RDP-EC-16029:

[0037](1) Manure treatment: Take manure from the farm, weigh 5g of chicken manure and add it to 10mL of sterile water to soak overnight, then centrifuge the overnight leaching solution at 10,000rpm for 5min, take the supernatant and pass it through a 0.22μm filter, and use the filtrate for later use;

[0038] (2) Preparation of mixed bacterial suspension: Take 0.2mL of bacterial suspension and 0.1mL of filtrate into 5mL of LB broth, culture at 37°C, shake at 200rpm overnight, then centrifuge at 10,000rpm for 5min, and pass the supernatant through a 0.22μm filter. The filtrate is ready for use;

[0039] (3) Separation of bacteriophages: Separation of phages using the double-plate method, take 0.1mL of the filtrate of the mixed bacterial suspension and 0.2mL of the host E. After culturing in a 37°C incubator for 6-8 hours, pick the transparent spot and put it in 1mL of normal saline in a 37°C water bath for ...

Embodiment 3

[0041] Example 3 Electron Microscopic Observation of Phage

[0042] Take 20 μL of the liquid containing crude phage particles and drop it on the copper grid, let it settle naturally for 15 min, absorb the excess liquid from the side with filter paper, add a drop of 2% phosphotungstic acid (PTA) on the copper grid to stain the phage for 10 min, and then Use filter paper to absorb the staining solution from the side, and observe the phage morphology with an electron microscope after the sample is dry, as shown in figure 1 shown.

[0043] The bacteriophage RDP-EC-16029 has a polyhedral stereosymmetric head, wrapped in nucleic acid, with a diameter of about 70nm, a tail with a length of about 120nm, a tail sheath, and a neck connecting the head and tail. According to the Ninth Report of the International Virus Taxonomy Organization Virus Classification, the bacteriophage is classified as Myoviridae of the order Cauviridae.

[0044] Whole-genome sequencing and analysis of phage R...

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Abstract

The invention discloses separation and application of a lytic escherichia coli phage RDP-EC-16029. The host of the phage is avian escherichia coli E10, and the phage can form a plaque with a diameterof 0.5-1mm on double plates. Through observation by an electron microscope, the phage has a polyhedral stereosymmetric head which wraps nucleic acid and has a diameter of about 70nm; the phage has a tail with a length of about 120nm and has a tail sheath; a neck is connected with the head and the tail; and the phage belongs to myoviridae of caudovirales. The phage can maintain good activity in a pH range of 6.0-9.0, and has the highest phage titer when the pH value is 8.0, and the phage titer is basically unchanged in a range of 30-40 DEG C. The phage can efficiently infect the host strain avian escherichia coli E10, has a strong lysis effect on the avian escherichia coli E10 in a culture environment, and provides a phage source for industrial production of phages for prevention and treatment of the avian escherichia coli E10.

Description

technical field [0001] The invention relates to the field of microbes, in particular to the isolation and application of a lytic colibacillus phage RDP-EC-16029. Background technique [0002] In recent years, my country's animal husbandry has developed rapidly, and large-scale and intensive livestock and poultry farms have shown a sharp upward trend. As a result, many problems such as excessive stocking density and declining breeding environment have occurred, causing a large number of pathogenic bacteria to proliferate and reducing animal resistance. Pathogen resistance. Therefore, the use of antibiotics has become very common. Although antibiotic treatment has a significant effect, the resistance of animals after the use of antibiotics is also very significant. The emergence of superbugs is one of the serious results of the abuse of antibiotics. All countries are now aware of the problems caused by the abuse of antibiotics. Antibiotic-free farming has become popular in dev...

Claims

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Application Information

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IPC IPC(8): C12N7/00
CPCC12N7/00C12N2795/10121C12N2795/10151
Inventor 杜新永李先胜马如霞
Owner QINGDAO RUNDA BIOTECH
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