A Rapid Digital PCR System with a Pressurized Thermal Cycler

Abstract

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.

Keywords

• digital PCR
• static droplets array (SDA)
• high-pressure thermal cycler