An Escherichia coli phage that can reduce antibiotic resistance and its application
An antibiotic resistance, Escherichia coli technology, applied in the preservation of bacteriophages, viruses/phages, food ingredients as antimicrobials, etc. Cracking activity, good application prospect, high titer effect
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Embodiment 1
[0044] Example 1 Isolation and identification of pathogenic bacteria and bacteriophages
[0045] (1) Isolation and identification of pathogenic Escherichia coli BE-20058
[0046] Samples were taken from the diseased chicken farm in Jimo, Qingdao, Shandong Province, and the livers of the diseased chickens were taken aseptically, streaked on the selective medium (MacConkey agar medium), and after culturing at 37°C for 18-24 hours, a round, circular shape formed on the medium. The red colonies with flat, neat edges and smooth and moist surface were picked out for typical colonies and continued to be streaked and purified for 3 times. Then a single colony was picked and inoculated into 5 mL of LB broth, and incubated with shaking at 37°C and 200 rpm for 8 hours to obtain a uniform and turbid bacterial suspension. liquid. The serotype of Escherichia coli was confirmed by 16sRNA molecular identification, and the serotype was determined to be O78 by serum identification. It was nam...
Embodiment 2
[0054] Example 2 Electron microscope observation of bacteriophage
[0055] Take 20 μL of the liquid containing the crude phage particles and drop them on the copper mesh, let it settle naturally for 15 minutes, and remove the excess liquid from the side with filter paper. The staining solution was aspirated from the side, and the morphology of the phage was observed with an electron microscope after the sample was dried.
[0056] Photo by electron microscope figure 1 It can be seen that the bacteriophage RDP-EC-20059 has a polyhedral stereosymmetric head that wraps the nucleic acid, with a diameter of about 61 nm and no tail.
Embodiment 3
[0057] Example 3 Phage Genome Sequencing
[0058] After enriching and culturing a single phage, centrifuge at 8000g for 15 minutes at 4°C, add 10% PEG8000 and 0.5M NaCl and let stand overnight, then add an equal amount of chloroform to mix well, and centrifuge at 5000g for 10 minutes after standing for stratification. After removing the chloroform layer and the PEG layer, restriction endonuclease digestion was added, and the phage was suspended under the condition of gradient density cesium chloride, and then dialyzed with TM buffer for 3 times, each 30min, and sent to BGI for sequencing The company performs whole-genome sequencing. The results are shown in the sequence listing.
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