Residual DNA detection method
Sep 01, 2023
Source: Cainiu Health
Dr. Lv Aiming
Introduction
The commonly used clinical method for residual DNA detection is fluorescent staining. Turn on the UV lamp of the biosafety cabinet and irradiate for at least half an hour, then remove the samples to be tested and transfer them into the biosafety cabinet. Use a sterile pipette to withdraw the supernatant and transfer it into a test tube. Add the culture supernatant, antibiotic-free culture medium, and mycoplasma-positive cells separately into the bottle. After disinfection, place the bottle in an incubator and culture for 3–5 days.
The commonly used clinical method for residual DNA detection is fluorescent staining. The specific procedure is as follows:
1. Turn on the ultraviolet (UV) lamp in the biosafety cabinet and irradiate for at least half an hour. After irradiation, turn off the UV lamp and turn on the ventilation fan.
2. Once the biosafety cabinet light is on, use an alcohol spray bottle to disinfect the work surface and sterile disposable pipettes. Place sterilized items such as pipette tip boxes and cell culture plates into the biosafety cabinet.
3. Remove the sample to be tested and transfer it into the biosafety cabinet. Use a sterile dropper to collect the supernatant and transfer it into a test tube.
4. Prepare three T25 flasks of Vero cells with 30–40% confluency, and add the culture supernatant, antibiotic-free culture medium, and mycoplasma-positive cells into each flask, respectively.
5. Disinfect the three flasks with alcohol and place them in the incubator for 3–5 days. Then inoculate the cultured Vero cells into culture plates, add appropriate amounts of culture medium into each well, cover the plates, and shake gently to ensure even cell dispersion.
6. Place the culture plates in the incubator and culture for 3–5 days. Remove the culture medium and use a vacuum pump to completely aspirate it. Add sterile PBS solution evenly into the wells, shake gently to rinse, then aspirate; repeat this washing step three times.
7. Finally, add 5 mL of fresh fixative to each well, incubate for 15 minutes, then aspirate and allow the plates to air-dry naturally.
8. Add staining solution into each well, protect from light, and stain for 30 minutes before aspirating the stain. Add purified water to the culture plate, shake gently to wash, then aspirate; repeat this washing step three times, then allow the plate to air-dry naturally.
9. Place the naturally dried culture plate under a microscope, turn on the fluorescence excitation light source, launch the fluorescence imaging software, and observe the culture plate.
If this procedure is required, it is recommended to visit a qualified hospital and perform the operation under the guidance of a professional physician to ensure the accuracy of the test results.
