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Digital PCR: Quantitative quality control for plasmid and viral vector development
Quality control is essential at every stage of the development process to create reliable cell and gene therapies. With the ability to provide an absolute count of nucleic acids, digital PCR (dPCR) is used for many applications, including:
 | Vector copy number—assess the number of integrated vector genomes during characterization studies and monitor product stability and identity |
 | Virus quantification—precisely quantitate genomic titer, viral particle titer, and full/empty capsid ratios during purification and lot release to accurately asses dose |
 | Purity & safety testing—identify product and process-related impurities such as residual DNA or microbial contamination for in-process and release testing |
Benefits of digital PCR for cell and gene therapy development
Precise absolute quantification across a range of nucleic acid concentrations
For cell and gene therapy workflow steps that require absolute quantification, such as viral titer and vector copy number, dPCR provides accurate measurements without the use of a standard curve. This removes a source of variability, which makes the process more robust and efficient.
Figure 1. Expected vs. observed AAV concentrations across a dilution series, obtained using the Absolute Q AAV Viral Titer dPCR Assay on the QuantStudio Absolute Q AutoRun Suite.
More consistent than droplet digital PCR
Unlike ddPCR platforms that generate micro-reactions by droplet formation, microfluidic array plate (MAP) technology facilitates automated sample distribution consistently into over 20,000 microchambers and utilizes >99% of the bulk reaction to minimize sample loss.
Poster: Comparison of Absolute Q technology against ddPCR for AAV viral titer quantification
Figure 2. The QuantStudio Absolute Q dPCR System demonstrates higher correlation than ddPCR between expected stock concentration and calculated values for quantification of AAV.
Simple and scalable automated dPCR workflows
Reclaim valuable time and resources while helping to maximize throughput with an automated system that enables flexible configurations and walk-away workflows for high throughput digital PCR.
The Applied Biosystems QuantStudio Absolute Q AutoRun Digital PCR Suite is an integrated system solution that scales precise nucleic acid quantification while keeping it simple.
Vector copy number (VCN) refers to the number of vector genomes that have been incorporated into the genome of target cells. Because viral transduction can be variable, precise measurement of vector copy number assumes pivotal significance, serving as a vital metric for monitoring sustained transgene expression and for determining therapeutic dose.
dPCR enables robust absolute quantification for precise copy number calculation
Digital PCR, which utilizes simple counting of target molecules to achieve absolute quantification, enables higher precision and accuracy to detect and quantify copy number variations, even if the changes are small.
Application Note: Lentiviral vector characterization using the QuantStudio Absolute Q Digital PCR System
Learn more in the education center ›
Figure 3: Representative dPCR fluorescent plots measuring proviral copies in cells transduced with lentivirus at the specified MOI and subsequent proviral concentration calculations, obtained with the ViralSEQ Lentivirus Proviral DNA Titer Kit on the QuantStudio Absolute Q System.
Gene and cell therapies utilize a variety of viral vectors for gene transfer, including adeno-associated viruses (AAV), lentiviruses, retroviruses, herpes viruses, adenoviruses, and others. Controlling the concentration of a potential therapeutic is difficult since viral particles are produced in living cells and then purified. The viral titer must be measured accurately, precisely, and consistently to ensure correct formulation..
dPCR provides high precision for genome titer, viral particle titer and full/empty capsid ratio calculation
Applied Biosystems Absolute Q Viral Titer dPCR assays enable easy and accurate quantification of viral vectors. The assays can be run individually or multiplexed using a custom assay with your target gene of interest.
Poster: Measuring rAAV Genomic Titer, Viral Particle Titer and Full/Empty Capsid Ratio on the QuantStudio Absolute Q dPCR System
Learn more in the education center ›
Figure 4: Representative dPCR fluorescent plots measuring proviral copies in cells transduced with lentivirus at the specified MOI and subsequent proviral conentration calculations, obtained with the ViralSEQ Lentivirus Proviral DNA Titer Kit on the QuantStudio Absolute Q System
The process of generating and purifying viral vectors and cell therapies is also susceptible to contamination by host cell DNA, mycoplasma, and other contaminants. Such product and process-related impurities must be detected and screened out with the highest sensitivity to maintain the highest safety standards and meet regulatory requirements.
dPCR enables quantitative detection residual host DNA or plasmid contaminants and microbial agents
Applied Biosystems Absolute Q Viral Titer dPCR assays and SEQ-technology kitted solutions provide a simple and accurate way to test for contamination in a broad range of sample types, from raw materials and in-process samples to purified final products.
Application Note: Assessment of using the QuantStudio Absolute Q Digital PCR System to quantify residual E. coli DNA
Learn more in the education center ›
Figure 5: Concentration of plasmid DNA determined by KanR target quantification, run on the Absolute Q dPCR System using the resDNASEQ Quantitative Plasmid DNA - Kanamycin Resistance Gene (pDNA-KanR) Kit.
Absolute Q dPCR assays for viral titer
Applied Biosystems Absolute Q Viral Titer dPCR assays enable easy and accurate quantification of viral vectors with the following features:
- Simple—streamlined workflow for ease-of-use with your digital PCR instrument
- Fast—minimal hands-on time; results in 90 minutes when used with the QuantStudio Absolute Q Digital PCR System
- Dependable—analyze your data with confidence using verified assays backed by a performance guarantee*
Ordering information
To order an Absolute Q Viral Titer Assay please add it to cart by entering the Catalog Number and Assay ID (if applicable) provided below into ThermoFisher.com’s Quick Order tool.
| Target | Dye | Catalog No. | Assay ID |
Absolute Q Viral Titer Assay | eGFP | VIC-MGB/20X | A53736 | DT2W7KW |
Absolute Q Viral Titer Assay | GFP | VIC-MGB/20X | A53736 | DTFVKJZ |
Absolute Q Viral Titer Assay | SV40 polyA signal | VIC-MGB/20X | A53736 | DTZTDZY |
Absolute Q Viral Titer Assay | AAV ITR-2 | VIC-MGB/20X | A53736 | DTH49PU |
Absolute Q Viral Titer Assay | CMV promoter region | VIC-MGB/20X | A53736 | DTYMJF2 |
Other assays and kits for digital PCR analysis
Custom dPCR assays
Can’t find the assay you’re looking for? Design a digital PCR assay with a self service tool or get help with our design services
Lentivirus characterization solutions
- ViralSEQ Lentivirus Physical Titer Kit—quantitate the total number of genome-containing lentiviral particles by targeting the conserved long terminal repeat (LTR) region of the genome
- ViralSEQ Lentivirus Proviral DNA Titer Kit—measure vector integration in transduced cells by targeting LTR in the provirus
Residual DNA testing solutions
- resDNASEQ dPCR E1A DNA Fragment Length Kit—quantify residual DNA fragment sizes in HEK293 production systems and other cell lines transformed with the E1A gene
- resDNASEQ dPCR E. coli. DNA Kit—quantify residual DNA in E. coli production systems, commonly used for production of recombinant proteins and plasmids
- resDNASEQ Quantitative Plasmid DNA - Kanamycin Resistance Gene Kits—detect residual plasmid containting kanamycin-resistant genes
TaqMan Assays
Adapt an existing TaqMan Assay for use on the QuantStudio Absolute Q Digital PCR System.
Download quick reference guide ›
Resources
dPCR and qPCR workflow tool
Explore workflows, view product recommendations, and find regulatory documentation for cell and gene therapy.
Customer testimonial
See why Senti Biosciences is using Absolute Q dPCR System to characterize their gene circuit technology.
Talk to a digital PCR specialist
Have questions about implementing dPCR in your lab? Let us help you get started.
To order an Absolute Q Viral Titer Assay for adeno-associated virus (AAV), please add it to cart by entering the Catalog Number and Assay ID (if applicable) provided below into ThermoFisher.com’s Quick Order Tool.
| Target | Dye | Catalog No. | Assay ID |
Absolute Q Viral Titer Assay | eGFP | VIC-MGB/20X | A53736 | DT2W7KW |
Absolute Q Viral Titer Assay | SV40 polyA signal | VIC-MGB/20X | A53736 | DTZTDZY |
Absolute Q Viral Titer Assay | AAV ITR-2 | VIC-MGB/20X | A53736 | DTH49PU |
Absolute Q Viral Titer Assay | CMV promoter region | VIC-MGB/20X | A53736 | DTYMJF2 |
More resources
*Terms and conditions apply. For complete details of the guarantee, go to thermofisher.com/absoluteqassayguarantee
For Research Use Only. Not for use in diagnostic procedures.
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