Experimental study on inactivation of disposable virus sampling tube
Virus sampling tube:
Lentivirus (gkai gene, gcnl0319432), HEK293T cells
Nucleic acid extraction kit and extractor:
Refer to the detection method described in the virus inactivation test in the technical specification for disinfection (2002 Edition) to determine the inactivation capacity of the virus preservation tube.
Step 1: cell culture
HEK293T cells in good growth state were digested and counted, and diluted with 1 × 104 / ml, according to 100 μ L / well volume, make three multiple wells for each gradient of virus, calculate the required number of inoculation wells, and slowly and evenly inoculate the cells into 96 well plates. Put it into a 5% CO2 incubator at 37 ° C and incubate overnight
Step 2: lentivirus dilution
The lentivirus with a titer of 107-108tu / ml was diluted by 10 fold gradient, and three gradients were continuously diluted.
Step 3: virus inactivation
Place the diluted virus at room temperature, mix it with the inactivated virus preservation solution (batch number: 220201 / 220207 / 220211) in a volume ratio of 1:1, and place it at room temperature for 1min, 5min and 10min respectively for inactivation.
Step 4: stop inactivation
The virus isolation medium (which can be replaced by DMEM complete medium) was diluted 1000 times to neutralize the inactivation preservation solution and terminate the inactivation.
Negative control: the mixture of virus isolation medium and inactivated virus preservation solution diluted 1000 times;
Positive control: the mixed solution of virus isolation medium diluted 1000 times of virus original solution.
Step 5: virus incubation
The cell culture medium was aspirated from the previously cultured HEK293T cells, and 100 μ L of the prepared virus mixture, and the corresponding positive control and negative control. The virus was incubated for 3 hours, and was removed from the incubator and shaken every 30 minutes.
Step 6: cell culture
After the incubation, the virus solution was aspirated, and 100 μ L DMEM complete medium, place 96 well plates in a 5% CO2 incubator at 37 ° C, and observe the cell state.
Step 7: observation and counting of fluorescent cells
Continue to culture for 48-72h, and observe the cell state during the period. If the cell culture solution turns yellow, change the solution, count the wells with the appropriate number of fluorescent cells, and calculate the virus titer.
It can be seen from the data in Fig. 1 and table 1 that after lentivirus was inactivated for 1min, 5min and 10min, HEK293T cells grew normally under the microscope bright field; Three batches of inactivated virus storage tubes can effectively inactivate the virus after interacting with the virus for 5min.