Validation of Preamplification to Improve Quantification of Cytomegalovirus DNA Using Droplet Digital Polymerase Chain Reaction.

Abstract

Subclinical cytomegalovirus (CMV) replication is associated with strong cellular immune response and chronic inflammation, which could contribute to aging-related conditions such as cardiovascular disease and frailty. However, because of very low levels of CMV DNA present in people with chronic CMV infection, it has been difficult to explore the virologic and immunologic mechanisms of chronic low-level CMV infection and a sensitive method to monitor CMV replication is needed. Droplet digital PCR (ddPCR) has been shown to have higher precision and reproducibility than real-time quantitative PCR (qPCR) in quantifying low levels of CMV DNA, but it is not always sensitive enough for this purpose. Through rigorous validation experiments, we demonstrated that sensitivity and precision of quantification of very low levels of CMV DNA by ddPCR can be significantly increased by preamplification of samples with 10-20 cycles of conventional PCR, especially when testing CMV DNA in the presence of cellular DNA. With preamplification, we could reliably quantify down to two copies of CMV DNA, as opposed to five copies without preamplification. Further studies are needed to determine if ddPCR with preamplification can facilitate mechanistic studies of the characteristics and consequences of chronic CMV infection in aging adults.