A new Vibrio parahaemolyticus phage with broad lysis spectrum, its specific primers and its application
A technology of Vibrio hemolyticus and bacteriophage, applied in the direction of bacteriophage, virus/phage, medical raw materials derived from virus/phage, etc. Effective in treating infection with high potency
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Embodiment 1
[0032] Isolation and cultivation of embodiment one phage
[0033] (1) Recovery and proliferation of bacteria
[0034] Pick the frozen bacterial solution of Vibrio parahaemolyticus, line it in three areas on the TCBS plate, and culture it in a 37°C incubator for 16-24h. Pick a single colony, inoculate in 5ml 2216E broth, proliferate in an air shaker at 37°C, shake at 170rpm for 16h, and obtain a single bacterial suspension.
[0035] (2) Isolation and purification of phage
[0036]Take the sewage and culture pond water samples from the seafood market in Chengyang District, Qingdao City, and centrifuge them at 10,000rpm for 5 minutes, prepare 2216E broth with the supernatant, and divide them into triangular flasks. Vibrio hemolyticus was stirred evenly, cultured overnight in an air shaker at 37°C at 170rpm, centrifuged at 10000rpm for 5min, and sterilized by filtration with a 0.22μm bacterial filter. Mix the filtrate and the host bacteria, incubate at 37°C for 5 minutes, pour ...
Embodiment 2
[0037] The strain identification of embodiment two phages
[0038] 1. Morphological observation of phage under electron microscope
[0039] Take 20 μl of 1 x 10 9 The phage sample of PFU / ml was dropped on the microporous copper grid, precipitated for 15 min, and the excess liquid was absorbed with filter paper. Add 15 μl of 2% phosphotungstic acid (PTA) dropwise on the copper grid, stain for 5 minutes, absorb excess dye solution with filter paper, observe and take pictures with transmission electron microscope after drying. Phage morphology results see figure 1 .
[0040] The observation results are: the head of PG07 is a polyhedron with a diameter of about 80nm, and its non-stretch tail is about 150nm in length. According to the ninth report of the International Committee on Taxonomy of Viruses (ICTV), phage PG07 can be determined to be Caudophages.
[0041] Concentrate the phage by PEG-NaCl method, and extract the phage nucleic acid using the viral genome RNA extraction...
Embodiment 3
[0057] The detection of embodiment three bacteriophage biological characteristics
[0058] (1) Proliferation and titer determination of phage
[0059] Take 100 μl of the host bacteria and phage plaque extraction solution and add it to 5ml 2216E broth, incubate in an air shaker at 37°C at 170rpm for 3-4 hours, and wait until the mixed solution becomes clear to obtain a phage proliferation solution. The phage proliferation solution was diluted 10 times, and the titer was measured by the double-layer plate method, and three parallel samples were made for each dilution. When counting, observe the phage plaques in the plate and take 30-300 plates to count and calculate the titer.
[0060] Take the dilution as 10 -7 Counting of 3 parallel samples, the results were: 107, 112, 109 plaques, and the calculated titer was 1.09×10 10 PFU / ml.
[0061] (2) Cleavage profile detection of phage PG07
[0062] 123 strains of Vibrio parahaemolyticus preserved in the laboratory were selected, ...
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