RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

RNA病毒具有很高的遗传变异性和在选择压力下通过突变和重组产生的快速进化的巨大潜力。马铃薯Y病毒也是如此（PVY），其包含高度多样性的不同重组和非重组菌株。因此，很难开发出对所有PVY菌株具有相同扩增效率的逆转录实时定量PCR（RT-qPCR），这将使其平衡定量成为可能。这在混合感染和其他发病机理研究中特别需要。为了实现这一目标，我们最初将PVY通用RT-qPCR测定法转换为逆转录液滴数字PCR（RT-ddPCR）格式。RT-ddPCR是绝对定量方法，不需要校准曲线，并且不易产生抑制剂。在这项研究中开发和验证的RT-ddPCR在五个数量级上实现了动态范围的定量，并且就其灵敏度而言，它与RT-qPCR相当甚至更好。RT-ddPCR显示较低的测量变异性。我们已经显示RT-ddPCR可以用作评估不同RT-qPCR分析的参考工具。此外，它可用于基于内部参考材料的RNA定量，然后可将其用作诊断实验室的校准剂。