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Novel bacteriophage, composition thereof, preparation and application thereof

A technology of phage and composition, applied in the field of microorganisms

Active Publication Date: 2019-04-23
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This makes the control of Salmonella pathogens face severe challenges

Method used

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  • Novel bacteriophage, composition thereof, preparation and application thereof
  • Novel bacteriophage, composition thereof, preparation and application thereof
  • Novel bacteriophage, composition thereof, preparation and application thereof

Examples

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Effect test

Embodiment 1

[0075] The separation and purification of embodiment 1 myotail phage BP-66, myotail phage BP-63 and long tail phage BP-12

[0076] Collect Canada Montreal sewage treatment plant water sample 50ml, get 9ml supernatant after centrifuging 10min at 3500rpm, mix it with 1ml 10 times of LB liquid culture medium and 1ml in logarithmic phase Salmonella (10 8 cfu / ml) were evenly mixed and cultured overnight at 37°C to enrich the phage. The sample enrichment solution was centrifuged at 3500 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm microporous membrane to obtain a filtrate containing phage. Take 50 μl of the filtrate and mix it evenly with 300 μl of the host Salmonella liquid, and let it stand for 15 minutes to fully bind with the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled to 47°C, spread it on the solidified agar plate immediately after mixing, wait for the agar to solidify and incubate at 3...

Embodiment 2

[0080] The determination of embodiment 2 bacteriophage BP-66, BP-63 and BP-12 potency

[0081] Use SM solution as the diluent, and dilute the phage BP-66, BP-63 and BP-12 stock solution by 10 times to 10 times respectively. 7 times. Take l0 respectively 5 , l0 6 and l0 7 100 μl of the diluted phage culture solution was evenly mixed with 300 μl of the host bacterium multidrug-resistant Salmonella typhimurium (ATCC 700408), and allowed to stand for 15 minutes to allow it to fully bind to the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled to 47°C, mix evenly and spread it on the solidified agar plate immediately, and incubate it upside down at 37°C for 6-8h after the agar solidifies. Three parallel samples are required for each dilution, and the average of the three parallel samples of this dilution is used for counting. Among them, phage titer (PFU / ml) = average number of plaques × dilution factor × 10

[0082...

Embodiment 3

[0085] The determination of embodiment 3 phage BP-66, BP-63 and BP-12 to Salmonella optimal multiplicity of infection (MOI)

[0086] A single colony of Salmonella enterica was picked, inoculated into a test tube filled with 3ml of LB culture solution, and shaken at 160rpm in a shaker at 37°C for 12h to obtain a host bacterial suspension. The bacterial suspension was transferred to 10ml LB culture medium at a ratio of 1:100, and cultured with shaking at 160rpm at 37°C to the pre-logarithmic phase. Add phage BP-66, BP-63 and BP-12 pure culture solution and host bacteria (MOI=number of phages / number of bacteria) according to the multiplicity of infection ratio, and add LB liquid medium to make the total volume of each tube the same. Shake culture at 160rpm in a shaker at 37°C for 4h. After culturing, centrifuge at 10000 g for 10 min and collect the supernatant to determine the phage titer. Each point was cultured in duplicate and the average value was taken, and the MOI that pr...

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Abstract

The invention relates to the field of microorganisms, and provides a novel salmonella phage, a composition thereof, and a preparation method and application thereof. The novel salmonella phage is a myoviridae BP-66, the preservation number is CCTCC NO: M2015146, the preservation number of the myoviridae BP-63 is CCTCC NO: M2015145, the preservation number of chilikevirus BP-12 is CCTCC NO: M2015141. The novel bacteriophage is a strict virulent bacteriophage, has high toxicity to host bacteria, has a wide host range, and still has high toxicity to the host bacteria at low concentration; the DNAof the phage cannot encode protein that can pose a potential health risk; the bacteriophage stably survive in a culture solution at room temperature, and can live for more than 6 months below 4 DEG C; the bacteriophage can be well proliferated on a non-pathogenic bacterial host; large-scale industrial production can be achieved. The salmonella phage can provide excellent strain resources for theapplication of phage therapy.

Description

[0001] This application is the application number 201510308565.0, the application date is June 09, 2015, and the title of the invention is "new bacteriophages, their compositions, their preparation methods and applications". technical field [0002] The invention belongs to the technical field of microbes, and specifically relates to new Salmonella phages, their composition, their preparation method and application. Background technique [0003] Salmonella is considered to be one of the most important food-borne pathogens in the world. According to statistics, 70%-80% of food-borne diseases in my country are caused by Salmonella. Because there are many serotypes of Salmonella, they are widely distributed in nature, and can cause food poisoning to humans by contaminating eggs, meat, milk and other foods; poultry infected by Salmonella will develop diseases such as avian typhoid, avian paratyphoid, and pullorum. There are currently two species of Salmonella: Salmonella enteric...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCY02A50/30
Inventor 霍茨蒙德·曼德维尔
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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