Digital PCR in Virology: Current Applications and Future Perspectives.

Digital PCR (dPCR) has been used in the field of virology since its inception. Technological innovations in microfluidics more than a decade ago caused a sharp increase in its use. There is an emerging consensus that dPCR now outperforms quantitative PCR (qPCR) in the basic parameters such as precision, sensitivity, accuracy, repeatability and resistance to inhibitors. These strengths have led to several current applications in quantification, mutation detection and environmental DNA and RNA samples. In high throughput scenarios, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the cost and throughput still significantly hampered the adaption of dPCR. There is much unexplored potential within the multiplexing capabilities of dPCR. This will allow simultaneous multi-target quantification and can also partially alleviate the throughput and cost drawback. In this review, we discuss the strengths and weaknesses of dPCR with a focus on virology applications and we discuss future applications. Finally, we discuss recent evolutions of the technology in the form of real-time dPCR and digital high-resolution melting.