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A stable and efficient Salmonella virulent phage rdp-sa-21004 and its application

A technology of RDP-SA-21004 and Salmonella, which is applied in the direction of phage, virus/phage, medical raw materials derived from virus/phage, etc., can solve the problems of drug resistance of bacteria, untimely treatment, ineffective medication, etc., and achieve a wide cracking spectrum , good acid and alkali tolerance, and strong cracking ability

Active Publication Date: 2022-07-22
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the present invention is to provide a stable and highly effective Salmonella phage RDP-SA-21004, aiming to solve the problem of rapid onset of salmonella infection in farm poultry and untimely treatment, and ineffective medication due to drug resistance of pathogenic bacteria. question

Method used

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  • A stable and efficient Salmonella virulent phage rdp-sa-21004 and its application
  • A stable and efficient Salmonella virulent phage rdp-sa-21004 and its application
  • A stable and efficient Salmonella virulent phage rdp-sa-21004 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Isolation and identification of pathogenic Salmonella BS-20079

[0046] Sampling from the diseased farm, aseptically taking the diseased poultry liver, streaking on the Salmonella color selective medium, and after culturing at 37°C for 18-24 hours, it will form a round, flat, neat edge, smooth and moist surface on the medium. The lavender colonies were picked, and the typical colonies were picked for streak purification for 3-5 times, then single colonies were picked and inoculated into 5 mL of LB liquid, and cultured with shaking at 37°C and 200 rpm for 4-6 hours to obtain a uniform turbid bacterial suspension. Through 16S rRNA molecular identification and serotype identification, it was determined to be a pathogenic Salmonella, named BS-20079, and stored in a -80°C refrigerator.

Embodiment 2

[0047] Example 2 Isolation and purification of phage RDP-SA-21004

[0048] (1) Feces treatment: Take feces from the farm, weigh 3 g of diseased poultry livers, add them to 10 mL of SM buffer, grind and soak them overnight, then centrifuge the overnight leachate at 10,000 rpm for 5 min, take the supernatant and filter it with a 0.22 μm filter. Store the effluent at 4°C for later use;

[0049] (2) Phage enrichment: add 0.2 mL of bacterial suspension and 1 mL of filtrate to 5 mL of LB broth, shake and culture at 37°C at 200 rpm overnight, then centrifuge at 10,000 rpm for 5 min, filter the supernatant with a 0.22 μm filter, and filter out liquid backup;

[0050] (3) Phage separation: The double plate method was used to separate the phage, and 0.1 mL of the host Salmonella suspension was mixed with 0.7% soft agar and spread on the LB plate. After the soft agar solidified, 0.1 mL of the filtrate was placed on the On the plate, let it stand until the mixture is absorbed. After cul...

Embodiment 3

[0053] Example 3 Morphological observation of bacteriophage

[0054] 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. Remove the staining solution from the side, and observe the phage morphology with an electron microscope after the sample is dry, such as figure 1 shown.

[0055] Depend on figure 1 It can be seen that the phage RDP-SA-21004 has a polyhedral three-dimensional symmetrical head, which wraps the nucleic acid, with a diameter of about 57.259 nm, a tail of about 112 nm in length, a tail sheath, and the neck connects the head and the tail. According to the ninth report on the classification of viruses by the International Organization for Taxonomy of Viruses, the phage is classified as Myoviridae in the order Auroviridae.

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Abstract

The invention discloses a stable and efficient Salmonella virulent phage RDP-SA-21004, the preservation number is CGMCC No. 22483. After the whole gene is sequenced, it is found that it does not have virulence genes and lysogenic genes; Stored for 10 months under conditions, its activity remains at 10 8 Above pfu / mL, it has strong stability; it can efficiently infect host bacteria, and its optimal multiplicity of infection is 0.1; it has a broad spectrum of ability to kill Salmonella, and also has a certain ability to lyse Escherichia coli. The phage can be used alone or in combination with other Salmonella phages and probiotics, which can effectively treat, prevent and reduce poultry salmonellosis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a stable and efficient Salmonella virulent phage RDP-SA-21004 and its application in the infection of pathogenic Salmonella in avian animals. Background technique [0002] Salmonella pullorum belongs to the Enterobacteriaceae family, Gram-negative enterobacteria, and is the causative agent of salmonellosis. Widespread in nature, about 1000 strains have been found so far. Salmonella can be isolated from most vertebrates, and it is mainly parasitic in the intestinal tract of humans and animals. The size of the bacteria is (0.6-0.9)×(1-3)μm, generally without spores or capsules, most of them have flagella, and the antigenic structure and biochemical characteristics are relatively complex. [0003] Salmonella species are diverse, and more than 2500 Salmonella enterica serotypes have been described based on antigenic properties defined by antigens on the bacterial surface. Poultry infe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/40A23K10/18A01P1/00A01P3/00C12R1/92
CPCC12N7/00A61K35/76A61P31/04A01N63/40A23K10/18C12N2795/10221C12N2795/10231C12N2795/10232Y02A50/30
Inventor 杜新永崔翠王立坤马如霞李先胜
Owner RECOM QINGDAO BIOTECH CO LTD
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