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Pseudomonas aeruginosa bacteriophage yapa and its use

A Pseudomonas aeruginosa and phage technology, applied in the field of microbiology, can solve problems such as destroying non-pathogenic bacteria, reducing bacterial drug resistance, and secondary infection

Inactive Publication Date: 2016-01-13
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibiotics have a broad antibacterial spectrum, but they may destroy non-pathogenic bacteria, lead to an imbalance of human flora, and even cause serious secondary infections, while phages are specific and have fewer side effects than antibiotics
Bacteriophages rapidly reproduce in the host and form mutations, which greatly reduce the drug resistance of bacteria, while antibiotics are static in the body and cannot respond to bacterial drug resistance

Method used

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  • Pseudomonas aeruginosa bacteriophage yapa and its use
  • Pseudomonas aeruginosa bacteriophage yapa and its use
  • Pseudomonas aeruginosa bacteriophage yapa and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Double layer agar plate method to the separation and screening of Pseudomonas aeruginosa phage

[0043] 1) Pretreat the untreated sewage collected from the sewage treatment centers of various hospitals in Beijing. The treatment procedure includes centrifugation once to remove solid impurities and collect the supernatant; filter twice with filter paper, and then use a 0.45 μm filter to treat After the sewage is filtered again to obtain sewage samples;

[0044] 2) Melt 8mL of 0.8% LB culture in a microwave oven water bath, wait for it to cool to about 40°C, add 300μL of host bacteria ATCC27853 grown to the logarithmic phase (this model strain can be purchased at ATCC or CMCC), shake Mix evenly and lay the layer on 1.5% LB plate (mark the grid on the LB plate in advance according to the number of water samples screened);

[0045] 3) After the upper layer of agar is solidified, take 5 μL of the sewage sample and add it to the double-layer agar plate containing th...

Embodiment 2

[0049] The transmission electron microscope morphological observation of embodiment 2 bacteriophage YAPa

[0050] After PEG precipitation, the concentration is about 10 10 10 μL of Pfu / mL phage suspension was dropped on the copper mesh (300 mesh), and after about 3 minutes of precipitation, it was blotted off with filter paper, negatively stained with 10 μL of uranyl acetate for 10 seconds, then blotted and dried on filter paper, and examined by a transmission electron microscope. Hitachi H-7500 for observation, such as figure 1, the strain of phage belongs to the long-tailed phage family, the size of its head is about 100nm, and the length of its tail is about 405nm.

Embodiment 3

[0051] Nucleic acid property analysis of embodiment 3 bacteriophage YAPa

[0052] Extraction of phage genome (using proteinase K / SDS method)

[0053] 1) Add EDTA to the phage suspension to a final concentration of 20 mmol / L, proteinase K to a final concentration of 50 μg / mL, and SDS to a final concentration of 5 g / L;

[0054] 2) Incubate the above mixture at 56° C. for 1 h;

[0055] 3) Add an equal volume of saturated phenol and mix gently;

[0056] 4) Centrifuge at 10000r / min for 5min, transfer the upper aqueous phase to a new centrifuge tube;

[0057] 5) Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and mix gently

[0058] 6) Centrifuge at 10000r / min for 5min, transfer the upper aqueous phase to a new centrifuge tube;

[0059] 7) Continue to extract with equal volume of chloroform until there is no phenol smell;

[0060] 8) Add an equal volume of isoamyl alcohol to the upper aqueous phase, and place it at -20°C for more than 30 minutes;

[0061] 9)...

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Abstract

The invention belongs to the field of microbiology, and particularly relates to a pseudomonas aeruginosa phage YAPa with wide preferendum and application thereof. The preservation number is CGMCC (China General Microbiological Culture Collection Center) No.8951. It is verified that the phage has the splitting action to a type strain and is a double chain DNA (Deoxyribonucleic Acid). The length of the nucleotide sequence is 94588bp, and the phage belongs to siphoviridae. The head of the phage is about 100nm long, and the tail is about 405nm long. The phage is high in absorption efficiency to the host bacterium and great in splitting amount. Through further detection, the phage has the phagocytosis and splitting action to 44 pseudomonas aeruginosa of 42 genotypes. The phage provided by the invention is suitable for various practical purposes for phagocytosis and splitting decomposition of the pseudomonas aeruginosa according to good splitting property, absorption and wide preferendum. Products such as pharmaceutical compositions and environmental detergents which take the phage as one of the active components as well as use are required to be protected.

Description

technical field [0001] The invention belongs to the technical field of microbiology, and in particular relates to the isolation and identification of broad-tropic Pseudomonas aeruginosa phage and its basic biological characteristics. Background technique [0002] Pseudomonas aeruginosa (P.Aeruginosa) is a common opportunistic pathogenic bacterium, which is a Gram-negative non-fermenting bacillus. the presence of bacteria. As an opportunistic pathogen, it can cause fatal infection to fibrocystic patients, burn patients, AIDS patients or other immunocompromised patients. It is also the most important Gram-negative bacteria in nosocomial infections. High rate. [0003] In recent years, with the irregular use of antibiotics, the number of multidrug-resistant or pan-drug-resistant Pseudomonas aeruginosa isolates has been increasing, which has brought great difficulties to clinical treatment. In view of the fact that the speed of research and development of new antibiotics is m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/00A01P1/00C12R1/92
Inventor 李宁代芳芳娄金丽于艳华孙桂珍
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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