High-temperature-resistant Escherichia coli phage RDP-EC-20031 and application of high-temperature-resistant Escherichia coli phage RDP-EC-20031

A technology of RDP-EC-20031 and Escherichia coli, which is applied in the direction of viruses/phages, medical raw materials derived from viruses/phages, applications, etc., can solve the problems of drug resistance of pathogenic bacteria, ineffective medication, and untimely treatment

Active Publication Date: 2021-04-16
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a high-temperature-resistant coliphage RDP-EC-20031, which aims to solve the problem of untimely treatment of poultry in farms due to acute colibacillosis infection and ineffective medication due to drug resistance of pathogenic bacteria and other difficulties

Method used

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  • High-temperature-resistant Escherichia coli phage RDP-EC-20031 and application of high-temperature-resistant Escherichia coli phage RDP-EC-20031
  • High-temperature-resistant Escherichia coli phage RDP-EC-20031 and application of high-temperature-resistant Escherichia coli phage RDP-EC-20031
  • High-temperature-resistant Escherichia coli phage RDP-EC-20031 and application of high-temperature-resistant Escherichia coli phage RDP-EC-20031

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Isolation and Identification of Pathogenic Escherichia coli BE-20036

[0035] Sampling from the diseased chicken farm, aseptically take the liver of the diseased chicken, streak it on the selective medium (MacConkey agar medium), cultivate it at 37°C for 18-24 hours, and form a round, flat, edge on the medium For red colonies with neat, smooth and moist surface, pick typical colonies and continue to streak and purify for 3 times, then pick a single colony and inoculate in 5mL LB broth, shake and culture at 37°C and 200rpm for 8h to obtain a uniform turbid bacterial suspension. Then it was identified as Escherichia coli through 16sRNA molecular identification, and its serotype was further determined to be O78 through serum identification. O78 has strong pathogenicity, and can be transmitted to humans through food chains and other methods, and has strong pathogenic infectivity; Name it BE-20036 and store it in a -80°C refrigerator.

Embodiment 2

[0036] Example 2 Isolation and identification of coliphage RDP-EC-20031

[0037] (1) Sewage treatment: take sewage from the farm, centrifuge at 10,000 rpm for 5 minutes, take the supernatant and pass it through a 0.22 μm filter, and collect the filtered supernatant for later use.

[0038] (2) Preparation of mixed bacterial suspension: Take 0.2mL of bacterial suspension and 0.3mL of filtrate and add it to 5mL of LB broth, culture at 37°C, shake at 200rpm overnight, then centrifuge at 10,000rpm for 5min, take the supernatant and pass it through a 0.22μm filter. Set aside the filtrate.

[0039] (3) Separation of bacteriophages: Separation of phages was carried out using the double-plate method. Take 0.2 mL of the filtrate of the mixed bacterial suspension and 0.2 mL of the Escherichia coli suspension and mix evenly, bathe in water at 37°C for 10 minutes, then spread double plates, and incubate at 37°C for 8 After -12h, observe the result. If there are phages, there will be clea...

Embodiment 3

[0042] Morphological observation of embodiment 3 phage

[0043] Take 20 μL of the liquid containing crude phage particles and drop them on the copper grid, let it settle naturally for 15 minutes, and 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 minutes, and then use the filter paper to remove the The staining solution was sucked off from the side, and the morphology of the phage was observed with an electron microscope after the sample was dried. The result is as figure 1 shown.

[0044] Depend on figure 1 It can be seen that the bacteriophage RDP-EC-20031 has a polyhedral stereosymmetric head, wrapped in nucleic acid, with a diameter of about 70nm, a tail with a length of about 90nm, a tail sheath, and a neck connecting the head and the tail. According to the Ninth Report of the International Virus Taxonomy Organization Virus Classification, the phage can be classified as ...

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Abstract

The invention discloses a high-temperature-resistant Escherichia coli phage RDP-EC-20031 with the preservation number of CGMCC No.21406 and the gene length of 135812bp, which is discovered to have no virulence gene and lysogenic gene after sequencing the whole gene, and the gene of the phage contains a plurality of genes of lytic enzyme. The Escherichia coli phage RDP-EC-20031 provided by the invention has higher temperature tolerance and wider acid-base tolerance range, and is beneficial to industrial production and preparation; and the phage is found to have a lysis rate of 98% by lysis rate test and RTD test, and whenthe phage diluted by 107 times,the phage still has a good cracking effect on pathogenic bacteria. The phage has the characteristics of excellent performance and safety. The phage can be applied to preparation of medicines for preventing and / or treating diseases caused by escherichia coli.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a strain of high-temperature-resistant coliphage RDP-EC-20031 and its application in pathogenic coliform infection of poultry animals. Background technique [0002] Avian colibacillosis refers to the general term for acute or chronic infectious diseases caused by pathogenic Escherichia coli, often leading to extraintestinal diseases in poultry. The morbidity rate in the flock is about 11%-69%, and the mortality rate is about 3.8%-72.9%. With the development of intensive breeding industry, poultry colibacillosis has become an important infectious disease threatening the poultry industry, which poses a serious threat to the poultry industry and causes huge economic losses. At present, antibiotics are widely used to prevent and treat avian E. coli disease, which has led to a rapid increase in the resistance of E. coli. Antimicrobial resistance of Escherichia coli from animal sources n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61K45/06A61P31/04A23K10/18A23K20/195A23K50/75C11D3/38C11D3/48A01N63/40A01P1/00C12R1/92
Inventor 杜新永李先胜王立坤赵丹丹张庆刘玉庆罗成盛盖春云马如霞刘爽
Owner RECOM QINGDAO BIOTECH CO LTD
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