Accuracy of Digital Polymerase Chain Reaction

Accuracy of Digital Polymerase Chain Reaction

Abstract
Digital polymerase chain reaction (dPCR) is a method for measuring nucleic acid concentration which unlike qPCR, can provide precise measurements without an external calibrant. It is therefore potentially an ideal method for providing high accuracy measurements needed for the certification of reference materials or to provide clinical reference ranges. One advantage of using dPCR, compared to methods like spectroscopy, is that dPCR only measures accessible, amplifiable targets, similar to qPCR and NGS. Unlike spectroscopy, dPCR results do not include small, degraded fragments. dPCR can be used to compare different reference materials and to examine reference materials for purity issues (such as duplications and deletions). In general, when using dPCR to certify reference materials, it is recommended that multiple assays be used.
In metrology, precision refers to the closeness of repeated measurements to each other and accuracy refers to the closeness of the measured value to the true value. Although dPCR can be accurate and sensitive, it can still be negatively impacted by a variety of factors including PCR inhibitors, flawed primer design, sub-optimal primer concentrations and annealing temperatures. Reverse-transcription dPCR (RT-dPCR) has additional considerations for factors that may affect reverse transcription efficiency such as 1 step vs 2 step reverse transcription. The CCQM Nucleic Acid Working Group (NAWG) has done multiple interlaboratory studies, including using orthogonal methods, to demonstrate that dPCR is both accurate and precise. This talk will explore recent work that has been conducted to investigate dPCR accuracy and discuss how this can be applied to improve nucleic acid analysis in a variety of fields.