Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification, in which the diluted sample and reaction components are partitioned into hundreds, or even millions, of individual, parallel reaction chambers so that each contains one or no copy of the templates [1] [2] [3] [4] . After endpoint amplification, the template concentration in the original sample is determined by a Poisson statistical analysis of the number of "positive" partitions (in which the amplified target is detected) versus "negative" partitions (in which it is not). dPCR offers many advantages over quantitative PCR (qPCR) [5] , such as absolute quantification without dependence on cycle thresholds or external references, and much higher accuracy and sensitivity [6] [7] [8] [9] . Applications of dPCR span many areas of biology, including liquid biopsy [10] [11] [12] , copy number variation analysis [13, 14] , rare sequence detection [15, 16] , gene expression analysis [5, 17, 18] , single-cell genomics analysis [19] [20] [21] , pathogen detection and microbiome analysis [2, 22, 23] , as well as calibration for next-generation sequencing [24] . dPCR has been successfully performed in a variety of formats, such as multiwell plates [1, 3] , an emulsion PCR [25] [26] [27] , microdroplets [19, [28] [29] [30] [31] , microfluidic chambers [32] , a spinning disk platform [33] , and a SlipChip [34, 35] , and these can be summarized as chip-based or droplet-based dPCR [4] . However, most of the reported microfluidic chips still require a complex fabrication process, many tubes for liquid transportation, syringe pumps for pressuredriven flow, and a pneumatic system for microvalve control [36] . Current droplet-based approaches also require pumping equipment and an external rapid readout device. To ensure a homogeneous droplet size distribution, the flow rate of droplet production by T-junctions [37, 38] or flow focusing [39] [40] [41] must be precisely controlled. Moreover, all of these dPCR methods still require thermal cycling and accurate temperature control.