Evaluating Droplet Digital Polymerase Chain Reaction for the Quantification of Human Genomic DNA: Lifting the Traceability Fog

摘要

Digital polymerase chain reaction (dPCR) end point platforms directly estimate the number of DNA target copies per reaction partition, lambda, where the partitions are fixed location chambers (cdPCR) or aqueous droplets floating in oil (ddPCR). For use in the certification of target concentration in primary calibrant certified reference materials (CRMs), both lambda and the partition volume, V, must be metrologically traceable to some accessible reference system, ideally, the International System of Units (SI). The fixed spatial distribution of cdPCR chambers enables real-time monitoring of PCR amplification. Analysis of the resulting reaction curves enables validation of the critical dPCR assumptions that are essential for establishing the SI traceability of lambda. We know of no direct method for validating these assumptions for ddPCR platforms. The manufacturers of the cdPCR and ddPCR systems available to us do not provide traceable partition volume specifications. Our colleagues at the National Institute of Standards and Technology (NIST) have developed a reliable method for determining ddPCR droplet volume and have demonstrated that different ddPCR reagents yield droplets of somewhat different size. Thus, neither dPCR platform by itself provides metrologically traceable estimates of target concentration. We show here that evaluating split samples with both cdPCR and ddPCR platforms can transfer the lambda traceability characteristics of a cdPCR assay to its ddPCR analogue, establishing fully traceable ddPCR estimates of CRM target concentration.