Abstract
Droplet Digital™ polymerase chain reaction (ddPCR™) is a promising technique that quantitates the absolute concentration of nucleic acids in a given sample. This technique utilizes water-in-oil emulsion technology, a system developed by Bio-Rad Laboratories that partitions a single sample into thousands of nanoliter-sized droplets and counts nucleic acid molecules encapsulated in each individual particle as one PCR reaction. This chapter discusses the applications and methodologies of ddPCR for development of Rift Valley fever (RVF) vaccine, using an example that measures RNA copy numbers of a live-attenuated MP-12 vaccine from virus stocks, infected cells, or animal blood. We also discuss how ddPCR detects a reversion mutant of MP-12 from virus stocks accurately. The use of ddPCR improves the quality control of live-attenuated vaccines in the seed lot systems.
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Acknowledgements
We thank Dr. Raymond Miller (Bio-Rad) for initial setup of ddPCR experiment. This study was supported by NIH grant R01 AI087643, and the funding from the Sealy Center for Vaccine Development at The University of Texas Medical Branch at Galveston.
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Ly, H.J., Lokugamage, N., Ikegami, T. (2016). Application of Droplet Digital PCR to Validate Rift Valley Fever Vaccines. In: Thomas, S. (eds) Vaccine Design. Methods in Molecular Biology, vol 1403. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3387-7_10
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DOI: https://doi.org/10.1007/978-1-4939-3387-7_10
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