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Isolation and Identification of a Novel Enterohaemorrhagic Escherichia coli o157 Phage PE-3 in Sewage Treatment System

A sewage treatment system, Escherichia coli technology, applied in the field of bioengineering, to achieve a wide temperature tolerance range, good physical and chemical resistance, and the effect of preventing and controlling pollution

Active Publication Date: 2021-12-17
UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a new type of biological control with high efficiency and specificity for lysing enterohemorrhagic E. phage control therapy;

Method used

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  • Isolation and Identification of a Novel Enterohaemorrhagic Escherichia coli o157 Phage PE-3 in Sewage Treatment System
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  • Isolation and Identification of a Novel Enterohaemorrhagic Escherichia coli o157 Phage PE-3 in Sewage Treatment System

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, the separation and purification of phage

[0027] The sewage sample used in the test of the present invention was collected in January 2013 as a water sample for separating bacteriophage in the aeration tank of a certain urban sewage treatment plant in Beijing.

[0028] Take 500mL of sewage sample, add solid CaCl 2 To a final concentration of 1mmol / L, centrifuge at 3000g for 10min to remove large impurities, and then filter to sterilize with a 0.22μm microporous membrane. Take 300ml of filtrate, 300mL of 2×LB culture medium and 15mL of host bacterial suspension and put them into a high-pressure sterilized Erlenmeyer flask. After 10 hours of shaking culture at 37°C and 120rpm, centrifuge at 3000g for 10min at 4°C, and take 100mL of supernatant. Add 100mL of 2×LB culture medium and 5mL of the corresponding host bacterial suspension, place on a shaker at 37°C at 120rpm for 12h, then centrifuge at 5000g for 10min at 4°C, and then filter the supernatant throug...

Embodiment 2

[0035] Embodiment 2, the morphological observation of bacteriophage

[0036] Take 20 μL of the phage suspension and drop it on the copper grid, wait for its natural precipitation for 10 minutes, blot it dry from the side with dry filter paper, let it air for about 1 minute, add 1 drop of 1% uranyl acetate on the copper grid, stain for 2 minutes, and then use it carefully to dry Blot the excess dye from the side of the filter paper, let it dry naturally in the dark for 30 minutes, and observe it with a transmission electron microscope (JEM-1400).

[0037] The result is as figure 2 As shown in electron microscope photos, the head of bacteriophage PE-3 presents a regular hexahedral symmetry, the diameter of the head is about 50nm, the tail is short, and the length and width are about 12nm. According to the eighth virus classification of the International Organization for Taxonomy of Viruses (ICTV) According to the report, the EHE coli O157 phage PE-3 can be preliminarily classi...

Embodiment 3

[0038] Example 3, phage genome analysis and identification

[0039] Enzyme digestion identification of phage genome: add DNaseI and RNaseA to the purified phage suspension, incubate at 37°C for 30 minutes, add proteinase K and SDS, mix well, put in a water bath at 56°C overnight, add NaCl solution to the digestion solution, mix well, Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), repeat the extraction twice, collect the hydrophilic phase, and use absolute ethanol to precipitate the nucleic acid. The resulting nucleic acid precipitate is washed with 70% ethanol and then suspended in TE , stored at -20°C for later use. Respectively use restriction endonucleases EcoR I, EcoR V, Hind III, Nde I, Pst I to identify the type of phage nucleic acid, and the digested products are analyzed by electrophoresis in 1% agarose gel at a voltage of 90V for 90min .

[0040] Digestion results such as image 3 shown. After the phage PE-3 genome was treated with EcoR V re...

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Abstract

The invention relates to the prevention and control of highly pathogenic enterohaemorrhagic Escherichia coli O157 in a sewage treatment system, and belongs to the field of bioengineering. A new type of phage for lysing enterohemorrhagic Escherichia coli O157 in the sewage treatment system is provided, which is named PE‑3 (gene sequence SEQ ID NO.1, GenBank Access no. KJ748011). The phage has a strong lytic effect on enterohaemorrhagic Escherichia coli O157, which can effectively prevent and control the production and spread of enterohaemorrhagic Escherichia coli O157, and has high temperature tolerance and wide acid-base tolerance range, which is beneficial to Industrialized production and preparation, the bacteriophage can be used as a safe, reliable and efficient biological disinfectant to quickly and efficiently prevent enterohaemorrhagic Escherichia coli O157 pollution caused by sewage discharge or reuse.

Description

technical field [0001] The invention relates to the prevention and control of highly pathogenic enterohaemorrhagic Escherichia coli O157 in a sewage treatment system, and belongs to the field of bioengineering. In particular, the present invention relates to the isolation and identification of basic biological characteristics of a novel phage PE-3 (gene sequence SEQ ID NO.1, GenBank Access no.KJ748011) from the sewage treatment system, especially its ability to quickly and efficiently Lytic enterohaemorrhagic Escherichia coli O157 and identification of its essential characteristics. Background technique [0002] Recently, the contamination of water bodies by pathogenic bacteria often causes outbreaks of water-borne infectious diseases. The number of deaths caused by water-borne infectious diseases alone has reached 4% of the total number of deaths in the world. Among them, enterohemorrhagic Escherichia coli (EHEC) has become a worldwide problem endangering public health an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/92
Inventor 刘新春易鑫李娟黄京刘红辉
Owner UNIVERSITY OF CHINESE ACADEMY OF SCIENCES
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