Phage and application thereof

Abstract

The invention discloses a bacteriophage and application thereof, the bacteriophage is sent to the China General Microbiological Culture Collection Center, is classified and named as salmonella bacteriophage S142, the preservation number is CGMCC No.23083, and the preservation time is August 16, 2021. According to the invention, the S142 bacteriophage belonging to the long-tail bacteriophage family is separated from excrement or sewage of a chicken (duck) field, the outbreak amount of the bacteriophage is up to 1.8 * 10 < 4 > PFU / mL, the bacteriophage has strong multiplication capacity, and can be multiplied in a short time to obtain a large number of bacteriophage offspring, so that the preparation time is greatly shortened, and meanwhile, the S142 bacteriophage has relatively good tolerance to temperature, PH and ultraviolet light, and can be used for preparing the long-tail bacteriophage. According to the present invention, the salmonella of chicken origin, the salmonella of duck origin and the salmonella of swine origin can be cracked, the cracking rates are respectively 72.8%, 69.6% and 100%, and the salmonella of chicken origin, the salmonella of duck origin and the salmonella of swine origin have strong treatment effects on infectious diseases caused by at least one of the salmonella of chicken origin, the salmonella of duck origin and the salmonella of swine origin, and provide good development values.

Description

technical field

[0001] The invention relates to the technical field of cultivation, in particular to a bacteriophage and its application. Background technique

[0002] Salmonella is a genus of Enterobacteriaceae characterized by Gram-negative, facultatively anaerobic, non-spore-forming, rod-shaped bacteria, most of which move by flagella. Salmonella is a pathogenic microorganism that causes infection in domestic animals and humans. Salmonella enterica—a species of Salmonella—has several serotypes, including Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella typhi, Salmonella choleraesuis, and Salmonella derby ).

[0003] Salmonellosis, also known as paratyphoid, is a general term for various animal diseases caused by Salmonella bacteria. Clinically, it is often manifested as sepsis and enteritis, and it can also cause abortion in pregnant dams. Salmonellosis in chickens and salmonellosis in ducks are two of the more com...

Examples

Embodiment 1

[0033] The separation (dot plate method) of embodiment 1 S142 bacteriophage

[0034] Screen 96 strains of Salmonella strains from the strain bank as the host bacteria for the isolation of phages, and carry out resuscitation culture: use an inoculation loop to pick up a ring of Salmonella bacterial liquid frozen in glycerol at -80°C, and separate them by streaking on ordinary nutrient agar medium Single colonies were cultured in an incubator at 37°C for 16 hours. On the next day, 96 strains of Salmonella were picked and inoculated into a 2ml 96-well deep-well plate for overnight culture.

[0035] Add 200ml of LB to 200ml of LB and soak overnight in an incubator at 37°C. Filter out excess impurities with gauze for later use, and sterilize with 0.22um filter. The 96 strains of Salmonella cultured above were added to the enriched feces, with 100 ul of each strain of bacteria, and cultured in a 37°C incubator for 18-24 hours. The next day, the cultured feces were centrifuged at 1...

Embodiment 2

[0038] Example 2 Identification of S142 phage

[0039] (1) Take 20 μL of S142 phage proliferation solution for ten-fold dilution, take 50 μL of the proliferation solution with a suitable dilution gradient and 50 μL of the host bacteria 21-1944 solution, and add them to the melted upper agar (temperature is about 50-60°C) , and rub the test tube by hand to make it mix evenly, quickly pour it into the prepared bottom agar plate, rotate the plate to mix the upper layer of agar evenly, wait until the agar is solidified, put it in a constant temperature incubator at 37°C for cultivation, and observe the phage after 3 hours spot shape. The result is as figure 2 As shown, it can be seen from the figure that the S142 phage can form a round transparent phage plaque on the double-layer plate, with a diameter of about 4mm.

[0040] (2) Take 200 μL of the phage proliferation solution stored for later use, put the copper grid on the sealing film, add 20 μL of sample to the copper grid a...

Embodiment 3

[0044] The determination of the optimal multiplicity of infection (MOI) of embodiment 3 S142 phage

[0045] The S142 phage and the host bacterium 21-1944 were respectively diluted ten-fold, and mixed according to the appropriate ratio (as shown in Table 1 below). Add 5mL of sterilized nutrient broth, then put it into a 37°C full-temperature shaking incubator for shaking culture for 3h, take it out and centrifuge at 12000rpm for 20min, then take the supernatant, and filter it with a 0.22μm filter to obtain bacteriophage proliferation fluid. The phage proliferation solution was diluted to an appropriate concentration, and then the optimal multiplicity of infection and titer of the phage were determined by the double-layer plate method, and the results were recorded in Table 1.

[0046] Table 1

[0047]

[0048] The results showed that when the multiplicity of infection was 0.001, the highest titer of phage S142 reached 6.2×10 10 PFU / mL. Therefore, the optimal MOI for this...

technical field

[0001] The invention relates to the technical field of cultivation, in particular to a bacteriophage and its application. Background technique

[0002] Salmonella is a genus of Enterobacteriaceae characterized by Gram-negative, facultatively anaerobic, non-spore-forming, rod-shaped bacteria, most of which move by flagella. Salmonella is a pathogenic microorganism that causes infection in domestic animals and humans. Salmonella enterica—a species of Salmonella—has several serotypes, including Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella typhi, Salmonella choleraesuis, and Salmonella derby ).

[0003] Salmonellosis, also known as paratyphoid, is a general term for various animal diseases caused by Salmonella bacteria. Clinically, it is often manifested as sepsis and enteritis, and it can also cause abortion in pregnant dams. Salmonellosis in chickens and salmonellosis in ducks are two of the more com...

Examples

Embodiment 1

[0033] The separation (dot plate method) of embodiment 1 S142 bacteriophage

[0034] Screen 96 strains of Salmonella strains from the strain bank as the host bacteria for the isolation of phages, and carry out resuscitation culture: use an inoculation loop to pick up a ring of Salmonella bacterial liquid frozen in glycerol at -80°C, and separate them by streaking on ordinary nutrient agar medium Single colonies were cultured in an incubator at 37°C for 16 hours. On the next day, 96 strains of Salmonella were picked and inoculated into a 2ml 96-well deep-well plate for overnight culture.

[0035] Add 200ml of LB to 200ml of LB and soak overnight in an incubator at 37°C. Filter out excess impurities with gauze for later use, and sterilize with 0.22um filter. The 96 strains of Salmonella cultured above were added to the enriched feces, with 100 ul of each strain of bacteria, and cultured in a 37°C incubator for 18-24 hours. The next day, the cultured feces were centrifuged at 1...

Embodiment 2

[0038] Example 2 Identification of S142 phage

[0039] (1) Take 20 μL of S142 phage proliferation solution for ten-fold dilution, take 50 μL of the proliferation solution with a suitable dilution gradient and 50 μL of the host bacteria 21-1944 solution, and add them to the melted upper agar (temperature is about 50-60°C) , and rub the test tube by hand to make it mix evenly, quickly pour it into the prepared bottom agar plate, rotate the plate to mix the upper layer of agar evenly, wait until the agar is solidified, put it in a constant temperature incubator at 37°C for cultivation, and observe the phage after 3 hours spot shape. The result is as figure 2 As shown, it can be seen from the figure that the S142 phage can form a round transparent phage plaque on the double-layer plate, with a diameter of about 4mm.

[0040] (2) Take 200 μL of the phage proliferation solution stored for later use, put the copper grid on the sealing film, add 20 μL of sample to the copper grid a...

Embodiment 3

[0044] The determination of the optimal multiplicity of infection (MOI) of embodiment 3 S142 phage

[0045] The S142 phage and the host bacterium 21-1944 were respectively diluted ten-fold, and mixed according to the appropriate ratio (as shown in Table 1 below). Add 5mL of sterilized nutrient broth, then put it into a 37°C full-temperature shaking incubator for shaking culture for 3h, take it out and centrifuge at 12000rpm for 20min, then take the supernatant, and filter it with a 0.22μm filter to obtain bacteriophage proliferation fluid. The phage proliferation solution was diluted to an appropriate concentration, and then the optimal multiplicity of infection and titer of the phage were determined by the double-layer plate method, and the results were recorded in Table 1.

[0046] Table 1

[0047]

[0048] The results showed that when the multiplicity of infection was 0.001, the highest titer of phage S142 reached 6.2×10 10 PFU / mL. Therefore, the optimal MOI for this...

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