A highly integrated real-time digital PCR device for accurate DNA quantitative analysis

Misclassification of positive partitions in microfluidic digital polymerase chain reaction (dPCR) can cause the false positives and false negatives, which significantly alter the resulting estimate of target DNA molecules. To address this issue, establishing real-time fluorescence interrogation of each partition in microfluidic arrays is an effective way in which false positive and false negative partitions can be eliminated. However, currently available devices for real-time fluorescence interrogation are either not competent for microfluidic digital array, or they are bulky, expensive and entail peripheral equipment due to low integration. Therefore, in this study, a Raspberry Pi based, low-cost and highly integrated device is presented to achieve real-time fluorescence detection for microfluidic digital array, termed real-time dPCR device. In the device, uniform thermocycler, streamlined real-time fluorescence imaging setup, and compact data processing system are all integrated to undergo on-chip dPCR amplification, real-time fluorescence detection, and data analysis. Using this real-time dPCR device, the accuracy of DNA absolute quantification by dPCR is improved, since the misclassification of positive partitions is efficiently reduced based on the characteristic real-time fluorescence curves of positive partitions in a self-priming microfluidic chip. Compared with end-point dPCR on our device and commercialized QuantStudio™ 3D dPCR system, the real-time dPCR on our device exhibits a higher accuracy for DNA quantification. In addition, this real-time dPCR device is much smaller and cheaper than the commercialized Digital PCR system, but not sacrificing the capability of error correction for absolute quantitation analysis. Conclusively, this highly integrated real-time dPCR device is very beneficial for DNA quantitative analysis where the determination accuracy is pivotal.