Mutant staphylococcus aureus phage and application thereof
A technology for staphylococcus and staphylococcus infection, applied in the field of mutant Staphylococcus aureus bacteriophage, can solve problems such as antibiotic resistance crisis, and achieve the effects of good in vivo treatment effect, wide host spectrum and strong bacteriostatic ability
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Embodiment 1
[0032] Example 1 Isolation and Screening of Staphylococcus aureus Phage 4PHSA25
[0033] The detailed steps are as follows:
[0034] (1) Sample collection
[0035] Pig endometritis swab samples were collected from a pig farm in Guangxi Zhuang Autonomous Region.
[0036] (2) Isolation of Staphylococcus aureus phage
[0037] The pig endometritis swab samples were soaked in physiological saline overnight at 4°C, centrifuged at 8000r for 10min, and the supernatant was filtered through a 0.22μm filter. This filtrate was used as a pretreatment sample for phage isolation, and was stored overnight at 4°C. In 10mL TSB liquid medium (tryptone 17g, sodium chloride 5g, soytone 3g, glucose 2.5g, dipotassium hydrogen phosphate 2.5g, pH7.3±0.2), add 100ul host in a volume ratio of 1:100 Bacteria (Staphylococcus aureus SA25), cultured at 37°C to proliferate to the logarithmic phase. Add 10 mL of the pretreated fecal water sample and 40 mL of TSB liquid medium to the aforementioned logarith...
Embodiment 2
[0040] Electron Microscopic Observation of Example 2 Phage 4PHSA25
[0041] Phage staining was observed by negative staining with phosphotungstic acid (Clokie and Kropinski 2009). Firstly, the phage was concentrated, and the phage 4PHSA25 was proliferated in large quantities, and 1 mL of the phage stock solution was added to 100 mL of the logarithmic growth phase host bacterial liquid (Staphylococcus aureus SA25) for 3-4 hours until the bacterial liquid became clear. Centrifuge at 8000r for 10min at 4°C, take the supernatant and filter it with a 0.22μm filter to sterilize. The filtered phage was further balanced by adding 5 mL of 30% sucrose solution with a long needle, centrifuged at 4°C and 30,000r for 2 h to concentrate the phage, discarded the supernatant, and added 200 μL of TEN solution to the precipitate to resuspend. The concentrated and resuspended phages were sent to the transmission electron microscope platform, and the samples were stained with conventional phosph...
Embodiment 3
[0043] Example 3: Extraction and Whole Genome Sequencing of Phage Genome
[0044] The phage genome was extracted using proteinase K / SDS method. Take 1 mL of the concentrated phage stock solution after ultracentrifugation, add 20ul of RNaseI, 2ul of DNaseI, and 10ul of Buffer, mix well and lyse at 37°C for 1h. Add 10% SDS to 0.5% according to 1 / 5 of the volume of the phage concentrate, mix 50ul proteinase K by inversion and then bathe in 56°C water bath for 1h. Add 1 mL of protein extract with a volume ratio of 25:24:1 phenol:chloroform:isoamyl alcohol (prepared for use, stored away from light) and mix it upside down. Centrifuge at 13000r for 10 min. Pipette the supernatant into a new 2mLEP tube with a 200uL broken pipette tip, and repeat 3-4 times. For the last time, transfer the collected supernatant to a 10mL EP tube, add it to pre-cooled absolute ethanol at a ratio of 1:2 by volume, and let it stand overnight at -20°C. The next day, use a broken pipette tip to put the fl...
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