Real-Time and Digital PCR for Nucleic Acid Quantification

Abstract

Quantitative measurements of the nucleic acid of pathogens are commonly used to determine the level of viral pathogens, such as cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus in immunocompromised patients, and as a measure of the efficacy of antiviral treatment for human immunodeficiency virus and hepatitis C virus. Quantitative real-time PCR is the most common type of PCR for these applications and is used in both laboratory-developed and commercial assays. Quantitative real-time PCR is convenient as well as reasonably accurate and precise; however, the design of the assay and the practical implementation of the assay must be carefully considered. In particular, the selection of the target sequence, the design of the primers and probes, and the use of appropriate quantitative standards will each contribute to the final assay performance characteristics. Digital PCR, while less frequently used, is an emerging technology for quantitative measurement of nucleic acids. While this method provides less variable results than real-time PCR, it is generally less sensitive than real-time PCR, and the current platforms are not suitable for high-throughput testing [1]. In this chapter, the theories underlying these methods of quantitative nucleic acid detection are discussed, the practical considerations of assay implementation are considered, and the advantages and disadvantages of these two methods of quantitative PCR are described.