Whale Alexandra S, Huggett Jim F, Tzonev Svilen
Molecular and Cell Biology Team, LGC, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
Digital Biology Centre, Bio-Rad Laboratories Inc., 5731 West Las Positas Boulevard, Pleasanton, CA 94588, United States.
Biomol Detect Quantif. 2016 May 27;10:15-23. doi: 10.1016/j.bdq.2016.05.002. eCollection 2016 Dec.
Over the past decade numerous publications have demonstrated how digital PCR (dPCR) enables precise and sensitive quantification of nucleic acids in a wide range of applications in both healthcare and environmental analysis. This has occurred in parallel with the advances in partitioning fluidics that enable a reaction to be subdivided into an increasing number of partitions. As the majority of dPCR systems are based on detection in two discrete optical channels, most research to date has focused on quantification of one or two targets within a single reaction. Here we describe 'higher order multiplexing' that is the unique ability of dPCR to precisely measure more than two targets in the same reaction. Using examples, we describe the different types of duplex and multiplex reactions that can be achieved. We also describe essential experimental considerations to ensure accurate quantification of multiple targets.
在过去十年中，众多出版物都展示了数字PCR（dPCR）如何在医疗保健和环境分析的广泛应用中实现对核酸的精确且灵敏的定量。这与分区流体技术的进步同步发生，该技术能使反应被细分为越来越多的分区。由于大多数dPCR系统基于两个离散光学通道中的检测，迄今为止大多数研究都集中在单个反应中对一两个靶标的定量。在此，我们描述“高阶多重分析”，即dPCR在同一反应中精确测量两个以上靶标的独特能力。通过实例，我们描述了可以实现的不同类型的双链和多重反应。我们还描述了确保对多个靶标进行准确定量所需考虑的基本实验因素。
