Fully automatic integrated continuous-flow digital PCR device for absolute DNA quantification

Abstract

Existing digital polymerase chain reaction (PCR) devices consist of multiple independent devices, such as a droplet generator, a PCR thermocycler, and a droplet reader, which have the disadvantages of low integration, complex equipment structure, and high operation difficulty. This paper proposes a fully automatic integrated digital PCR device based on continuous-flow digital PCR theory. By simply adding the sample to the entrance of the integrated instrument, a series of procedures required for digital PCR detection can be fully automated, including sample injection, droplet generation, PCR thermal cycling, fluorescence acquisition, and signal analysis. In contrast to traditional techniques, sample testing requires only one integrated device rather than three separate instruments. For full automation, we design complete control and data processing software, which can complete a test by one-step operation. Therefore, the disadvantages of traditional instruments, such as multi-step operation, and hence, potential environmental pollution, are avoided. Moreover, the system can be powered by solar cells and does not require an external power supply.As a proof of concept, the proposed device is used for absolute quantitative detection of the hepatitis B virus in serum samples. The capacity of the system is validated by absolute quantification of three orders of magnitude from 103 to 105 IU/mL. The results have a good linear correlation (0.9986) with those of the traditional quantitative (qPCR), thus confirming the reliability of the instrument. In summary, we believe that our work can promote the development of integrated digital PCR systems.