Need to robustly validate your VCN assay?

Raymond Wu, PhD

Raymond Wu, PhD

Sr. Scientist, Early Phase Dev

An Efficient Approach to Check Accuracy of Viral Copy Number Assay in Droplet Digital PCR


Viral Copy Number (VCN) assay is a critical assay in cell and gene therapy because it is used to monitor toxicity, pharmacokinetics, and durability of virus modified cell therapy products. However, the assay needs to be validated for its accuracy before it can be used in clinical settings. Here, we describe an approach to check the accuracy of this assay by using a commercially available synthetic DNA called gBlocks. gBlocks are traditionally used as a positive control for quantitative PCR and Next Generation Sequencing experiments. We synthesized a gBlock containing the primer and probe binding sites of Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and Ribonuclease P Protein subunit p30 (RPP30). The two DNA fragments are connected by Hind III endonuclease cutting site. WPRE is commonly included in many lentiviral vectors. RPP30 is commonly used as a housekeeping gene in droplet digital PCR (ddPCR) because it is validated to have two copies in mammalian cells. WPRE and RPP30 assays are also commonly used in ddPCR to measure viral copy number integrated in cells after lentivirus infection. Using a multiplex WPRE/RPP30 ddPCR assay, we amplified WPRE and RPP30 from the WPRE-RPP30 gBlock with the same efficiency. Therefore, the VCN generated from WPRE/RPP30 is approximately two (2) regardless of input number of DNA. The VCN is accurately detected until the input gBlock number is as low as 4.38 copies. In conclusion, gBlocks can be used as a quality control material to monitor the VCN assay and the approach can be robustly applied to other Chimeric Antigen Receptor (CAR) specific VCN assays.

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