Salmonella bacteriophage CKT1 without transduction of drug-resistant gene and application of salmonella bacteriophage CKT1
A Salmonella phage and drug-resistant gene technology, applied to viruses/phages, medical raw materials derived from viruses/phages, applications, etc., can solve the problems of different strain characteristics and different effects, and achieve high outbreaks and low mutation rates , the effect of protecting health
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Embodiment 1
[0035] Example 1: Isolation and culture of Salmonella virulent phage CKT1
[0036] Take 10 mL of centrifugally filtered sewage collected from the farm, add it to 2-fold concentrated LB liquid, add Salmonella pullorum at a ratio of 1%, shake at 37 °C for 8 to 10 hours, centrifuge at 10,000 rpm / min for 3 minutes, take the supernatant with 0.22 A sterile phage-enriched solution was obtained by filtration through a μm filter. Dilute with SM buffer according to a 10-fold gradient ratio, spot plate 10 μL on a double-layer agar plate containing the host Salmonella pullorum, incubate at 37°C for 6-8 hours, and observe the phage plaques. Pick a single phage plaque in SM buffer, shake and mix well and then double-dilute, take 10 μL of each dilution gradient and spot it on a double-layer agar plate. The bacteriophage CKT1 ( figure 1 A). The enriched phages were stained with negative staining method, and the morphology of the phages was observed under a Hitachi H7650 transmission elect...
Embodiment 2
[0039] Example 2: Determination of biological characteristics of bacteriophage CKT1
[0040] Host profile determination: take 10 μL of phage enrichment solution (10 9 PFU / mL) were spotted on double-layer agar plates of different Salmonella, and incubated at 37°C for 6-8 hours. If transparent plaques appeared, it indicated that the host bacteria could be lysed by phage CKT1.
[0041] Table 1: Lysis ability of bacteriophage CKT1 against different Salmonella and Enterobacteriaceae
[0042]
[0043] Note: ++ means it has cleavage ability, - means no cleavage ability
[0044] The results are shown in Table 1. The results show that the phage CKT1 has the ability to lyse the Salmonella enteritidis, Salmonella pullorum and Salmonella typhi and other group D Salmonella of the Salmonella genus.
[0045] Temperature stability assay: will contain 1 mL of 10 8 The centrifuge tubes of PFU / mL phage dilution solution were incubated at 37°C, 50°C, 60°C, 70°C, and 80°C for 2 h, and then d...
Embodiment 3
[0049] Example 3: Analysis of phage CKT1 transduction ability and determination of anti-phage mutation rate
[0050] 1. Verification of the transduction ability of bacteriophage CKT1:
[0051] 10 to culture to stationary phase 8 CFU / mL Salmonella pullorum SPLH19 (containing the drug resistance gene bla TEM , streptomycin resistance gene aadA), add bacteriophage CKT1 to the final concentration of 10 5 PFU / mL, lysed at 37°C for 6 h, centrifuged and filtered, and the phage titer was determined by the double-layer plate method. Take the above phage preparation solution (10 9 PFU / mL) 1 μL, 5 μL and 20 μL were added to 200 μL overnight Salmonella pullorum SPLH12 (sensitive to ampicillin and streptomycin, without resistance gene bla TEM , streptomycin resistance gene aadA), vortex and mix, and incubate at room temperature for 15 minutes to fully adsorb the phage to the bacteria, then add 1 mL of liquid LB, shake at 37 °C for 1 h, centrifuge at 10,000 rpm to discard the supernatan...
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