Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements.

Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.

Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.

There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.