Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology
A ring-mediated isothermal, Vibrio vulnificus technology, applied in the field of pathogenic bacteria diagnosis, can solve problems such as detection errors
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0040] Embodiment 1: seawater sample 1
[0041] 1. DNA extraction
[0042] Pick suspected colonies isolated from seawater and add them to 0.4mL TE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then add 10 μL of proteinase K solution and incubate at 55° C. for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL TE solution, and store at -20°C.
[0043] 2) Loop-mediated isothermal amplification (LAMP)
[0044] Add the following reagents to the amplif...
Embodiment 2
[0067] Embodiment 2: clinical isolate sample 1
[0068] 1. DNA extraction
[0069] Pick the colonies and add them to 0.4mL TE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then add 10 μL of proteinase K solution and incubate at 55° C. for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL LTE solution, and store at -20°C.
[0070] 2) Loop-mediated isothermal amplification (LAMP)
[0071] Add the following reagents to the PCR reagent tube to make a...
Embodiment 3
[0085] Embodiment 3: clinical sample 2
[0086] 1. DNA extraction
[0087] Pick suspected colonies and add them to 0.4mL TE buffer (pH8.0) for shaking and mixing. Add 40 μL of sodium dodecylsulfonate (10%), and then add 10 μL of proteinase K solution and incubate at 55° C. for 1-2 hours. Add the same volume of Tris-saturated phenol (pH 8.0), shake vigorously, centrifuge at 8000 rpm for 3 minutes, take the supernatant, and repeat the phenol extraction. Take the supernatant, add 0.1 times the volume of sodium acetate (3mol / L), mix well, add an equal volume of ice ethanol, mix well, let stand at low temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, Wash once with 70% cold ethanol, centrifuge at 12,000 rpm for 5 minutes at room temperature, discard the supernatant, add 50 μL LTE solution, and store at -20°C.
[0088] 2) Loop-mediated isothermal amplification (LAMP)
[0089] Add the following reagents to the PCR reagent tube to make a t...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More