Takara LVpro™ Provirus qPCR Quantitation Kit, 100 Rxns

The LVpro Provirus Quantitation Kit measures the number of vector copies (provirus copy number) incorporated into the genome when genes are transferred into human cells by lentiviral vectors using real-time PCR. This kit uses two probes—one for the internal control and another for the provirus copy—providing an accurate measurement of the proviral copies.

LVpro Provirus qPCR Quantitation Kit workflow.A calibration curve is created using the Provirus Control Template, which contains one copy of each of the lentiviral sequence and a part of the human IFNγ sequence. The average number of copies of the provirus per cell is calculated by quantifying the copy number of lentiviral and human IFNγ in the genomic DNA of the target sample.

Close correlation of provirus copy numbers with WGS using LVpro Provirus qPCR Quantitation Kit.Autosomal diploid cells transduced with a lentiviral vector were cloned, and the proviral copy number was determined by whole-genome sequencing (WGS) from the extracted genomic DNA. In addition, 100 ng of genomic DNA extracted from each clone was used to measure the number of proviral copies by the LVpro Provirus qPCR Quantitation Kit or other companies products. The results showed that the number of proviral copies determined by the LVpro Provirus qPCR Quantitation Kit and the number of proviral copies obtained by WGS analysis were almost equivalent, suggesting that the quantification of this product is highly accurate.

High correlation between MOI and proviral copy number.(Top) Lentiviral vectors containing the ZsGreen1 gene were phased and infected with HT1080 cells with various multiplicity of infection (MOIs). Three days after infection, the cells were treated with DNase I and then genomic DNA was extracted using Nucleospin Tissue. (Bottom) Lentiviral vectors carrying the CD19-CAR gene were phased and infected with SUP-T1 cells with various MOIs. Three days after infection, the cells were treated with DNase I, and then genomic DNA was extracted using Nucleospin Tissue. Real-time PCR was performed using 100 ng of genomic DNA for each sample. The correlation between the average number of proviral copies per cell and MOI was confirmed. The data present the average of the three independent trials. As a result, the combination of different cell lines and different carrier genes showed that the MOI and proviral copy number R2exhibited a high correlation with values above 0.98.