A highly lytic Pseudomonas aeruginosa phage rdp-pa-20001 and its application
A technology of RDP-PA-20001 and Pseudomonas aeruginosa, applied in the direction of phage, virus/phage, application, etc., can solve the problems of ineffective medication and untimely treatment, and achieve strong lytic activity and wide acid-base tolerance range , the effect of good application prospects
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Embodiment 1
[0029] Example 1 Isolation and identification of Pseudomonas aeruginosa in BP-20001
[0030] Stool samples from farms with Pseudomonas aeruginosa bacterial disease, the method aseptic, were diluted 1:10 with LB broth sterilized taken 1:10 sample dilution was added to 10ml 90ml nutrient broth medium, cultured for 18-24 hours at 37 ℃. Surface of the growth medium with a thin film of bacteria, culture medium yellow-green.
[0031] Bacteria of the thin film enrichment broth cultures were picked, streaked on hexadecane bromide agar plates, placed in culture at 37 ℃ 18 ~ 24 hours, and appeared gray, flat, surface wetting, and the yellow-green of the surrounding agar colony, diameter of about 2 ~ 4mm. Typical continuously picked colonies streaked on hexadecane bromide agar purified three times, Individual colonies were picked and inoculated in 5ml of LB broth in a liquid medium, 37 ℃, 200rpm shaking 8h, to give uniformly turbid bacterial suspension. 16sRNA through the molecular identific...
Embodiment 2
[0032] Example 2 Isolation and identification of Pseudomonas aeruginosa phages RDP-PA-20001 of the embodiment
[0033] Experiments with the present invention is a stool sample collected in 2020 from a Jimo broiler farms stool pool, a phage separated sample.
[0034] (1) bacteriophage RDP-PA-20001 isolated:
[0035] Take 10ml LB broth stool 10㎎ disposed soaked overnight at 4 ℃, 10000rpm centrifuge 5min, the supernatant was sterilized with 0.22μm filter membrane. 3ml 3ml filtrate is taken and the host logarithmic phase bacterial culture was added to 20ml LB broth autoclaved together, placed in an incubator at 37 ℃ static overnight culture, there may be phage proliferation. A sample broth 3ml, 10000rpm centrifuge 5min, the supernatant was sterilized with 0.22μm filter membrane. Assuming that the above filtrate containing phage. Serially diluted with LB broth, prepared in a bilayer tablet logarithmic growth phase of the host growth medium, and cultured at 37 ° C and 19-23h. Appears on...
Embodiment 3
[0041] Example 3 morphological observation of phage
[0042] Using phosphate tungsungstic acid, 20 μl of phage proliferation solution (10) 9 PFU / ml) On the stone chilly tablets, the copper net was placed at 10 min to adsorb the phage. It was dried at 2-3 min at natural room, stained with 2% phosphotungstic acid hydrogen, and then suction moisture after 2 min, naturally dried 5 min, and observed the phage morphology in electrishry. Electron microscope photo ( figure 2 It can be seen that phage RDP-PA-20001 head is a noodle, and the stereo symmetry is 60.865 nm, the length of the phage is 183.254 nm, according to the latest classification and naming of the phage, belonging to the turtlee, the endococci .
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