Basic Information


Virus SkeletonInsert Sequence InformationTiterOther Features
Lentivirus2019-nCov-N,1260bp,the reference sequence is NC_045512.≥1×108 copies/mLInactivated

QC


SpecificationResultJudgement
Pseudovirus Genome Titer>=1E8 copy/ml9.04 E8Pass
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-RTnCov-N-nonRT
24.2831.16

nCoV-N-COA-1

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+0613.83
1.00E+0517.32
1.00E+0420.8
1.00E+0324.13
1.00E+0227.43
nCoV-N-COA-2

(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:


nCoV-N-COA-3

(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%





Related reading:

CERTIFICATE OF ANALYSIS - nCov-1abN pseudovirus

CERTIFICATE OF ANALYSIS - nCov-S pseudovirus

CERTIFICATE OF ANALYSIS - nCov-E pseudovirus