Abstract
Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Highlights
Accurate and precise nucleic-acid quantification is fundamental in many fields, from basic research to molecular diagnostics, and in preclinical and clinical research and industrial processes
Earlier studies have shown that differences in droplet volumes can arise due to different supermix types [18, 19], with potential differences seen for different droplet generator [18]
For the purpose of an inter-laboratory comparison of how different factors affect droplet volume, the mean droplet volume was measured independently both at National Institute of Biology (NIB) and Instituto Nazionale di Ricerca Metrologica (INRiM) using the same droplet measurement protocol
Summary
Accurate and precise nucleic-acid quantification is fundamental in many fields, from basic research to molecular diagnostics, and in preclinical and clinical research and industrial processes. Absolute DNA quantification allows the reduction of measurement bias between laboratories, which is essential for target DNA measurements in medicine, viral load analysis in diagnostics, and microbial quantification in microbiology. The most used method for DNA quantification is real-time polymerase chain reaction, which depends on the standard curve approach for quantification. Absolute quantification of nucleic acids without the need of a standard curve can be achieved with digital PCR (dPCR). Taking into account the high potential of absolute quantification by dPCR, this technique represents a good candidate for reference methods for nucleic-acid copy-number determination [1, 2]
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