Method for quickly enriching, separating and detecting bacteria
A bacteria and enrichment technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of complicated operation, complicated detection equipment, and residual magnetic beads affecting the test, etc., to slow down Settling speed, fast settling speed, effect of optimized mass fraction
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Embodiment 1
[0029] This example is the rapid isolation and detection of Escherichia coli, the specific steps are as follows:
[0030] (1) Escherichia coli liquid preparation
[0031] Pick and inoculate Escherichia coli JM109 inoculation loop into 60mL sterilized LB liquid medium, incubate at 37°C for 12h, centrifuge at 8000rpm for 3min, remove the medium, resuspend in PBS, adjust the concentration of bacteria solution to 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 CFU / mL, spare. Another 2 mL of PBS without bacteria solution was taken as a blank control.
[0032] (2) Magnetic bead enrichment
[0033] Take 2mL of Escherichia coli liquid of each concentration described in (1) and blank PBS into corresponding glass bottles, add 50μL of magnetic beads (1mg / mL) respectively, place on a shaker at 150rpm, mix and incubate at 37°C for 1h, and magnetically After enrichment for 3 minutes, the supernatant was removed and resuspended in 150 μL of PBS solution with a pH of 3.8.
[0034] (3) PEG ...
Embodiment 2
[0038] This example is the rapid isolation and detection of Staphylococcus aureus, the specific steps are as follows:
[0039] (1) Preparation of Staphylococcus aureus bacterial liquid
[0040]Pick and inoculate the Staphylococcus aureus inoculation loop into 60mL sterilized LB liquid medium, incubate at 37°C for 12h, centrifuge at 8000rpm for 3min, remove the medium, resuspend in PBS, and adjust the bacterial concentration to 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 CFU / mL, spare. Another 2 mL of PBS without bacteria solution was taken as a blank control.
[0041] (2) Magnetic bead enrichment
[0042] Take 2mL of the Staphylococcus aureus bacterial solution of each concentration mentioned in (1) and blank PBS into corresponding glass bottles, add 100μL of magnetic beads (1mg / mL), place on a shaker at 150rpm, and incubate at 37°C for 1h. After magnetic enrichment for 3 min, the supernatant was removed and resuspended in 150 μL of PBS with a pH of 5.0.
[0043] (3) PE...
Embodiment 3
[0047] (1) Escherichia coli liquid preparation and magnetic bead enrichment
[0048] Carry out with reference to step (1), (2) in embodiment 1.
[0049] (2) Gelatin separation
[0050] Take 400 μL of gelatin (molecular weight: 10,000) with a mass fraction of 20% in a plastic separation tube with a diameter of 5 mm and a length of 3 cm, accurately pipette 150 μL of the sample in (2) above the gelatin, and place the separation tube vertically on the magnetic separation rack Put it aside for 10 minutes, and observe the separation result.
[0051] Measurement results: Observe the bottom of the separation tube. When using gelatin with a mass fraction of 20% to separate different concentrations of E. coli bacterial liquid, put the separation tube on the magnetic separation stand for 10 minutes, and the 2 ~10 7 Within the range of CFU / mL bacterial solution, with the increase of the concentration of the bacterial solution, the black precipitate separated at the bottom of the separa...
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