Separation and application of high-lysis-rate salmonella phage RDP-SA-17118

A technology of RDP-SA-17118 and RDP-EC-16029, applied in the field of separation and application of Salmonella phage RDP-SA-17118 with high lysis rate, can solve the problems of drug failure and lack of antibiotics, and achieve good acid-base tolerance sexual effect

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, in actual farming, due to the lack of relevant knowledge of antibiotic use by farmers, broad-spectrum antibiotics are often used or a large number of drugs are used, resulting in the gradual increase of bacterial resistance to antibiotics and the gradual ineffectiveness of drugs.

Method used

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  • Separation and application of high-lysis-rate salmonella phage RDP-SA-17118
  • Separation and application of high-lysis-rate salmonella phage RDP-SA-17118
  • Separation and application of high-lysis-rate salmonella phage RDP-SA-17118

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation and Identification of Pathogenic Salmonella Avian S6

[0038] Sampling from diseased farms, aseptically take livers of diseased poultry, streaked on selective medium (SS agar), cultured at 37°C for 18-24 hours, and formed round, flat, neat edges and smooth surface on the medium 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 200rpm at 37°C for 8h to obtain a uniform turbid bacterial suspension. Through 16sRNA molecular identification and serotype identification, it was determined to be pathogenic Salmonella, named S6, and stored in a -80°C refrigerator.

Embodiment 2

[0039] Example 2 Isolation and identification of phage RDP-SA-17118:

[0040](1) Manure treatment: 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;

[0041] (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. Reserve the filtrate;

[0042] (3) Separation of phages: Separation of phages was carried out using the double-plate method. After mixing 0.1mL of the filtrate of the mixed bacterial suspension and 0.2mL of the host Salmonella suspension, they were bathed in water at 37°C for 10 minutes, and then double-plates were spread and placed in a 37°C oven. After culturing in an inc...

Embodiment 3

[0045] Example 3 Electron Microscopic Observation of Phage

[0046] 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.

[0047] The bacteriophage RDP-SA-17118 has a polyhedral three-dimensional symmetrical head wrapped around nucleic acid, with a diameter of about 70nm, a tail about 120nm in length, 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.

[0048] Whole-genome sequencing and analysis ...

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Abstract

The invention discloses separation and application of a high-lysis-rate salmonella phage RDP-SA-17118. The host of the salmonella phage RDP-SA-17118 is salmonella S6, and the phage can form a plaque with a diameter of 2mm-4mm 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 70nm; the phage has a tail with a length of 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 has a high titer of 10<12> pfu/mL and has good tolerance to temperature and pH value, and the titer still keeps 10<12>pfu/mL or above after continuous passage for 29 times. The phage has a good lysis effect on a host after being diluted by 10<6> times, and after the phage is fed, existence of the phage can be detected in heart, liver, lung, kidney, thymus and serum. Through animal experiments, the phage has a good effect in treating chicken salmonella diseases.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to the isolation and application of a Salmonella phage RDP-SA-17118 with a high lysis rate. Background technique [0002] The problem of increased bacterial resistance has long received international attention. The European Union has banned the use of large amounts of antibiotics in feed since 1999, and the European Union, the United States and other regions and countries have begun to monitor the problem of drug resistance in their regions earlier. The results not only show that Salmonella has high resistance to a variety of antibiotics, but also the problem of multi-drug resistance is becoming more and more serious. In my country, the problem of bacterial drug resistance has been paid more attention in recent years, and related drug resistance and drug sensitivity tests are also being carried out continuously. Current research on drug resistance mainly focuses on the molecular level...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCA61K35/76A61P31/04C12N7/00C12N2795/10121C12N2795/10132C12N2795/10151
Inventor 杜新永李先胜马如霞
Owner QINGDAO RUNDA BIOTECH
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