The Future of Digital Polymerase Chain Reaction in Virology

Abstract

Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

Applications of Digital PCR for Clinical Microbiology

“…The absolute quantification of dPCR, which is achieved without reliance on a calibration curve, is a major advantage of this method (4,6). For the relative quantification of nucleic acids performed by qPCR, the C T value of a sample is compared to a standard curve generated by amplification of dilutions of the same template with assigned values, which must be determined by using another method.…”

“…If amplification is inhibited by impurities in the sample or amplification efficiency is poor due to mismatches between target, primer, and probe sequences, quantification will be underestimated. Because dPCR quantifies using endpoint instead of real-time amplification, quantification is less affected by inhibitors of amplification that may be present in the sample (10,11), and it is also less affected by poor amplification efficiency (4,12,13). In fact, it may be possible to perform dPCR on samples without prior extraction of the nucleic acids (14).…”

“…In fact, it may be possible to perform dPCR on samples without prior extraction of the nucleic acids (14). While qPCR and dPCR are generally equally sensitive given an equivalent input of template (15), dPCR assays have been shown to quantify some targets more precisely and are especially useful for precise quantification of low viral loads for monitoring antiviral therapy (3,4,6,13,(15)(16)(17). Compared to qPCR, the better precision of dPCR is also useful for detection and quantitation of rare variants (6) and for assays measuring ratios of high-and low-copy-number targets in a reaction (18).…”

“…1C and D). Based on the number of positive and negative partitions, the target copy number in the sample can be calculated (3)(4)(5). As the concentration of target increases and it becomes more likely that a given partition will contain two or more copies, Poisson's Law is used to accurately calculate the number of DNA targets per partition and the copy number in the original sample (4,6).…”

“…Partitions should also be of uniform size so that each will contain the same number of target molecules. Finally, amplification must be sufficiently efficient so that all partitions containing target molecules are amplified, and there must be a clear discrimination between positive and negative partitions (4,6).…”