Basic Information
| Virus Skeleton | Insert Sequence Information | Titer | Other Features |
|---|---|---|---|
| Lentivirus | 2019-nCov-N，1260bp，the reference sequence is NC_045512. | ≥1×108 copies/mL | Inactivated |
QC
| Specification | Result | Judgement | |
|---|---|---|---|
| Pseudovirus Genome Titer | >=1E8 copy/ml | 9.04 E8 | Pass |
| Ncov-N plasmid residues in Pseudoviruses | ＜3.2% | 0.85% | Pass |
QC data——detection of pseudovirus genomic titer and nCoV-N plasmid residue in pseudovirus
Methods:
RT-qPCR was used to detect N gene of 0.001ul pseudovirus, including RT sample and non RT sample (i.e. RT enzyme was not added)
Results:
the quantitative data were as follows
| nCov-N-RT | nCov-N-nonRT |
| 24.28 | 31.16 |
Calculation:
（1）The residual amount of ncov-n plasmid in pseudovirus 2019-ncov-n is 2^(24.28-31.16)=0.85%
（2）The genomic titer of 2019-ncov-n =（1+95.8%)^(24.13-24.28)*1*10^3*10^3*1000=9.04 E8（VP/ml）
Appendix: establishment of the determination formula of pseudovirus genomic titer:
（1）Take the pseudovirus genome standard and add 1e6 / 1E5 / 1E4 / 1E3 / 1E2 copy number standard to qPCR system;the test data are as follows
| Copy number of virus genome（copies） | ThresholdCycle |
|---|---|
| （Average） | |
| 1.00E+06 | 13.83 |
| 1.00E+05 | 17.32 |
| 1.00E+04 | 20.8 |
| 1.00E+03 | 24.13 |
| 1.00E+02 | 27.43 |
（2）Drawing of standard curve:
The standard curve of the relationship between the CT value obtained in the previous step and the value of the initial copy number is as follows:
（3）Calculation formula of lentivirus genome titer:
Physical titer of lentivirus（VP/ml）=（1+E）Ct1-Ct2 *copies-std*103*dilution ratio
E——Standard amplification efficiency
Ct1——CT value of standard
Ct1——CT value of sample
Copies-std——Copies of standards，E=95.8%