No abstract

Digital PCR (polymerase chain reaction) is a powerful and attractive tool for the quantification of nucleic acids. However, the multiplex detection capabilities of this system are limited or require expensive instrumentation and reagents, all of which can hinder multiplex detection goals. Here, we propose strategies toward solving these issues regarding digital PCR. We designed and tested a self-priming digital PCR chip containing 6plex detection capabilities using monochrome fluorescence, which has six detection areas and four-layer structures. This strategy achieved multiplex digital detection by the use of self-priming to preintroduce the specific reaction mix to a certain detection area. This avoids competition when multiple primer pairs coexist, allowing for multiplexing in a shorter time while using less reagents and low-cost instruments. This also prevents the digital PCR chip from experiencing long sample introduction time and evaporation. For further validation, this multiplex digital PCR chip was used to detect five types of EGFR (epidermal growth factor receptor) gene mutations in 15 blood samples from lung cancer patients. We conclude that this technique can precisely quantify EGFR mutations in high-performance diagnostics. This multiplex digital detection chip is a simple and inexpensive test intended for liquid biopsies. It can be applied and used in prenatal diagnostics, the monitoring of residual disease, rapid pathogen detection, and many other procedures.

The flow of Mask R-CNN model for processing digital polymerase chain reaction (dPCR) fluorescence images.