Trends and Advances in Digital PCR Viral Titer Quantification for CGT
Digital PCR (dPCR) goes beyond quantitative polymerase chain reaction (qPCR) and offers sensitive and accurate absolute quantification of viral infectivity
Knowing the number of viruses in a given sample can go a long way in helping researchers understand infectious diseases and their pathogenesis. Viral titers do exactly this and are integral assays for quantifying viral concentrations. Armed with this knowledge, researchers can further the development of downstream assays concerning antiviral therapeutic discovery, vaccine development, antibody neutralization, and unique cell and gene therapies. Viral titers are now critical to clinical research and testing, featuring prominently in commercial and academic labs. Traditional approaches to viral titers are inherently subjective, time-consuming, and laborious. Due to manual experimentation, the lack of ample data points becomes a further liability. Recent innovations and advances in polymerase chain reaction (PCR) methods have created a platform of new solutions that provide robust, sensitive, and accurate quantitation of DNA templates for reliable results and greater efficiency while reducing the need for expensive equipment.
Viral quantification and clinical diagnostics
PCR methods generally quantify viruses by considering the amount of viral DNA, RNA, or proteins found in a sample. Quantitative PCR (qPCR) has been used extensively in clinical viral diagnostics, with fluorescence-based qPCR techniques deemed the gold standard for viral load quantification and pathogen detection. Despite being a driver for nearly 30 years of major advances in clinical disease diagnostics, qPCR is accompanied by its share of limitations. With the use of a standard curve for quantification and the subjective choice of a signal threshold, qPCR results often vary, especially at lower template copy numbers, depending on a laboratory’s specific approach even with standardized kits and protocols. The advent of digital PCR (dPCR) in 1999 provided solutions to the shortcomings found in qPCR, all the while transforming the technique from its original analog and exponential nature to a digital and linear platform.
dPCR is primarily characterized by its ability for absolute and variation-less quantification of nucleic acids. The latter is possible thanks to the technique’s lower sensitivity to contaminants that can directly impact amplification, and by doing away with a reference standard. These features set it apart from qPCR, which only provides relative quantification with respect to the expression of a housekeeper gene or a standard curve produced via serial dilutions. dPCR can be incorporated into various applications, including absolute quantification, rare mutant allele detection, single-cell sample analysis, identification of low-frequency variants, detection of very low viral loads, chromosomally integrated viral genomes, etc. Yet, despite its status as a powerful tool for nucleic acid analysis, dPCR is largely limited by technical difficulties surrounding low throughput of extraction, high risk of sample contamination, and unsuitable for large amplicons.
Beyond qPCR and dPCR – Droplet Digital PCR
In Droplet Digital PCR (ddPCR), the separation of reaction mixtures combines microfluidic technology with creating microreactors of nucleic acids partitioned into water-in-oil droplets. The individual droplets undergo PCR separately where a two-color optical detection system is used to read the output signals and measure fluorescence. Commercialized ddPCR systems involve standardized procedures that ensure the droplets are even in volume and selected accordingly. The subsequent results are then interpreted using Poisson statistics to determine the target DNA template concentrations.
Despite similar workflows, ddPCR technology far surpasses the dPCR technique with its ability to massively partition samples in the fluid phase in one step. This rectifies earlier issues in dPCR surrounding the lack of scalable and practical technologies, the laborious nature of serial dilution, the risk of pipetting errors, and complex fluidic partitioning schemes. As a result of creating tens of thousands of droplets, ddPCR technology makes way for a single sample to generate tens of thousands of data points versus a single result, successfully elevating and scaling the statistical analysis of dPCR for practical application.
Bio-Rad ddPCR Solutions
Bio-Rad ddPCR systems and software are at the crux of a comprehensive portfolio of ddPCR assays and kits available for numerous applications such as mutation detection, copy number determination, genome edit detection, gene expression analysis, residual DNA quantification, and many more. Meeting the needs of GMP manufacturing and QC environments alongside FDA compliance, Bio-Rad’s ddPCR solutions enable viral quantification solutions essential to quality checks on incoming plasmids, conducting gene therapy biodistribution studies, and safety and efficacy testing around gene-modified cell therapies such as chimeric antigen receptor (CAR) T cells, to name a few. Assisted by QX software, a family of powerful software packages to acquire and analyze ddPCR data, Bio-Rad’s platform of ddPCR solutions automates the ddPCR workflow of droplet generation, thermal cycling, droplet reading, and data analysis, thus making the technology easily accessible to the working research laboratory.
The full power of Bio-Rad’s platform of ddPCR solutions can be observed in successful case studies of viral genome quantification for recombinant adeno-associated virus (rAAV) development. Using qPCR with a plasmid DNA reference standard opens the door to variable results as primers can bind to the standards. ddPCR technology is the ideal alternative as it works in the absence of standard curves and can help determine vector integrity through multiplexing of different probes targeting different areas of the viral genome. The transition from qPCR to ddPCR technology can be quite simple, given that certain steps are taken to bridge process-related and experimental factors contributing to differences in viral titers between the two methods. Bio-Rad demonstrates the same in an AAV inverted terminal repeat (ITR) assay. To begin, maximum separation between positive and negative droplets is achieved with a temperature gradient during the annealing/extension step. The addition of a restriction enzyme also helps increase amplicon accessibility. Together, these two standard optimizations ensure a robust and consistent transition in results from qPCR to ddPCR technology and set the stage for studies that bridge the use of both tools.
A direct implementation of the same, using Bio-Rad’s ddPCR solutions, has also been reported by Martin Lock, director of the Process Development Gene Program at the University of Pennsylvania, “We compared an AAV genome titer assay based on ddPCR technology with a standard and an optimized qPCR assay…We demonstrated absolute quantification of single-stranded AAV vector genomes by ddPCR technology with up to fourfold increased sensitivity over a standard qPCR titration but with equivalent sensitivity to an optimized qPCR assay.” The team would go on to confirm that genome tittering based on ddPCR technology, is far superior to qPCR, observing a tenfold more resistance to PCR reaction inhibitors than qPCR.
Conclusion
ddPCR technology is a highly precise method for viral quantification that is not afflicted by issues surrounding standard curves or inefficient amplifications. The lack of requirement of a reference standard helps save on expenses, and time, as well as avoid preparatory errors. A direct comparison between ddPCR technology and qPCR leans toward the former being a superior alternative to the latter with greater sensitivity, higher reproducibility, lower variability, and consistent inter-assay precision. As such, ddPCR technology is highly suitable for various applications surrounding viral genome quantification in clinical and commercial settings. Bio-Rad’s platform of ddPCR solutions provides ultrasensitive and absolute nucleic acid quantification. With cell and gene therapies rapidly gaining popularity for treating infectious diseases, Bio-Rad’s ddPCR technology is a reliable resource to overcome the challenges and limitations of existing methods for a range of viral quantification applications.
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