Method for in-situ detection of coronavirus nucleic acid based on molecular beacon of artificial vesicles
A molecular beacon, coronavirus technology, applied in the biomedical field, can solve the problems of long detection time and high false negatives
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0065] Embodiment one, the construction of ACE2 eukaryotic cell expression plasmid
[0066] The ACE2 eukaryotic expression plasmid pCMV3-Flag-ACE2 was constructed, and the plasmid pCMV3-Flag-ACE2 was transfected into competent Escherichia coli, and PCR was performed to verify whether the transduction was successful.
[0067] 1. Using the kit, construct the ACE2 expression plasmid pCMV3-Flag-ACE2 (its map is as follows figure 1 shown).
[0068] 2. Thaw 50 μL of competent E. coli on ice, mix an appropriate amount of plasmid pCMV3-Flag-ACE2 with competent E. coli, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then place on ice to cool for 2 minutes . Add 500 μL of culture medium and incubate for 1.5 h at 37° C. and 220 rpm. Spread on a nutrient agar solid medium plate containing kanamycin (50 μg / mL), and place it upside down in a 37°C incubator for overnight culture.
[0069] 3. Bacterial culture and verification
[0070] Single clones were picked, inoc...
Embodiment 2
[0075] Example 2, cells transfected to express ACE2 protein
[0076] 1. Inoculate 0.5-2×10 in 500 μL antibiotic-free medium (DMEM / F12 medium containing 10% FBS) 5 CV-1 cells (an African green monkey kidney cell line), ensure that the turbidity of the cells reaches 90%-95% at the time of transfection.
[0077] 2. Dilute the plasmid DNA in Example 1 in 50 μL of serum-free medium and mix gently.
[0078] 3. Gently mix LipofectamineTM 2000 before use, and then dilute the corresponding amount in the culture medium. Let stand at room temperature for 5 minutes.
[0079] 4. After standing at room temperature for 5 minutes, mix LipofectamineTM 2000 and DNA dilution (the total volume is 100 μL). Mix gently and let stand at room temperature for 20 minutes.
[0080] 5. Add 100 μL of the mixed solution to each well of the cell culture plate, and gently shake the cell culture plate back and forth to mix the mixed solution with the culture solution in the well. Cells were transferred to...
Embodiment 3
[0083] Example 3, Preparation of plasma membrane of cells highly expressing ACE2 protein
[0084] 1. Cell treatment: The cells highly expressing ACE2 protein obtained in Example 2 were subcultured. After the cells were fused to 80%, the culture medium was aspirated, washed with PBS buffer, and then aspirated, and treated with EDTA for 30 minutes. Transfer to a centrifuge tube and centrifuge at 1500rpm for 5min, remove the supernatant, resuspend the cells in PBS buffer, centrifuge at 1500rpm for 5min, remove the supernatant, add 1mL hypotonic buffer (Tris, 20mM; MgCl 2 , 2mM; KCl, 10mM; protease inhibitors).
[0085] 2. Place the cell solution in ice for 5 minutes, then place it at 42°C for 90 seconds, repeat 3 times.
[0086] 3. Grinding the cell solution. Put the cell liquid on ice, extract 20 times with a Dounce homogenizer, centrifuge at 3200g for 5min, transfer the centrifuged supernatant to a new EP tube, add 1mL of hypotonic buffer (Tris , 20mM; MgCl 2 , 2mM; KCl, 10...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More