ddPCR Vs. RT-qPCR - The Results Are In!
Blog Apr 05, 2016
Sean: The major benefits of Droplet Digital PCR (ddPCR) include:
2) Absolute quantification. Standard curves are not required for ddPCR technology to quantify the amount of DNA/RNA in samples whereas for qPCR they are not only needed but also a major source of error for labs attempting absolute quantification with this technique.
Furthermore, for dynamic studies requiring sample collection at various time points, ddPCR technology permits the immediate processing of individual samples as they become available without compromising the data or conclusions. This can be particularly important for plant, animal, patient or environmental studies that require study collection over months or years where initial data may help in redirecting the experimental design. Also, since clinical diagnostics are often dynamic and require data from successive samples to make measured decisions to alter treatment options, the sample independence of ddPCR technology offers an excellent solution as it did for the viral samples tested in our paper.
Why are these benefits potentially significant for the management of drug resistant Influenza strains?
In case of drug-resistant Influenza strains, all these benefits become significant because the abundance of the different viral strains must be monitored from very high to very low levels with a high level of precision, reproducibility and confidence. By eliminating Cq values and reducing the efficiency dependency, more sample with more background contaminants can be tested with Droplet Digital PCR to give higher signal to noise when assessing residual infection post treatment to more accurately assess patient recovery.
Absolute concentration data without the requirement for a standard curve assures reliable results from each successive sample taken over the course of infection to make reliable treatment decisions. For qPCR, this would require running a standard curve on separate plates with each collected sample which not only adds significant operating cost but can lead to large inter-assay variability, inaccurate data and poor treatment decisions.
Since this study not only required quantification of viral load but also the specific abundance of the mutated (H275Y) versus the wild type virus, the unique capacity of ddPCR technology to partition the wild type virus from the mutant strain gave more precise and sensitive detection of residual virus. This enabled the appropriate selection of antiviral therapy throughout infection to eliminate disease.
What other applications could ddPCR systems be useful for within the field of virology?
In addition to being well suited for monitoring residual infections, Droplet Digital PCR is gaining traction in the virology market as a method for quality control for gene therapeutic products. Last year in Human Gene Therapy Methods, Boehme et al. demonstrated Droplet Digital PCR's advantages as such a tool and concluded that ddPCR technology could be advantageous for titration of viral vectors commonly used in gene therapy.
Droplet Digital PCR is also useful in the evaluation of commercially available qPCR standards. Last year in the Journal of Clinical Microbiology, for example, Hayden et al. used ddPCR technology to test several commercially produced secondary standards based on the first WHO International Standard for human cytomegalovirus (CMV), which are aimed to improve agreement among labs performing quantitative CMV PCR. The use of digital PCR as a reference method demonstrated the lack of quantitative agreement among different secondary standards on the market. Due to the challenging nature of quantitative standard production, several manufacturers of such standards have adopted Droplet Digital PCR to aid in better standards development.
Have you seen increased interest in ddPCR technology for virology diagnostics since the publication of your research?