Digital reverse transcription PCR using a simple poly(dimethylsiloxane) microwell array chip for detection of...

Digital PCR (dPCR) enables absolute quantitation of nucleic acid without calibration using a standard curve, and is promising for quantitation of SARS-CoV-2 viral load. However, dPCR suffers from the need for complicated and expensive instruments. We previously reported a dPCR system using a poly(dimethylsiloxane) (PDMS) microwell array (MWA) chip and common laboratory tools. This dPCR system had been applied to DNA quantitation. In this paper, application of this dPCR system to RNA quantitation through one-step reverse transcription PCR (RT-PCR) is described. As a model template, SARS-CoV-2 genetic RNA was selected. Artificial standard samples of SARS-CoV-2 N gene were mixed with RT-PCR reagents. The resulting mixture was introduced into the microwells by the power-free pumping technique utilizing degassed PDMS. Thermal cycling and image acquisition were carried out using basic instruments such as a thermal cycler and an inverted fluorescence microscope. The fluorescence images showed distinctive difference between bright (positive) microwells and dark (negative) microwells. The number of the positive microwells was used for estimation of the template concentration in the sample based on the Poisson distribution theory. The estimated template concentrations exhibited excellent agreement with the input template concentrations in the range from 1.0 copy/μL to 10,000 copies/μL. The RT step in the thermal program was confirmed to be indispensable for the accurate quantitation. These results may open up the possibility of facile digital RT-PCR experiments for gene expression analysis and molecular diagnosis without the need for expensive specialized instruments.