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

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Digital PCR (dPCR) offers absolute nucleic acid quantitation but typically requires expensive, complex instrumentation.
• Previous work established a dPCR system using a poly(dimethylsiloxane) (PDMS) microwell array (MWA) chip for DNA quantitation.
• There is a need for more accessible dPCR methods, especially for viral load quantification like SARS-CoV-2.

Purpose of the Study:

• To adapt and validate a previously developed PDMS-based dPCR system for RNA quantitation.
• To assess the feasibility of using this simplified dPCR for quantifying SARS-CoV-2 genetic material.
• To demonstrate the application of this system for molecular diagnostics and gene expression analysis.

Main Methods:

• A power-free pumping technique using degassed PDMS was employed to introduce samples into the MWA chip.
• One-step reverse transcription PCR (RT-PCR) was integrated for RNA analysis, using artificial SARS-CoV-2 N gene samples.
• Thermal cycling and fluorescence image acquisition were performed using standard laboratory instruments (thermal cycler, fluorescence microscope).

Main Results:

• The dPCR system successfully differentiated between positive and negative microwells based on fluorescence.
• Quantitation of SARS-CoV-2 RNA concentrations showed excellent agreement with input values across a wide dynamic range (1.0–10,000 copies/μL).
• The reverse transcription step was confirmed as essential for accurate RNA quantitation.

Conclusions:

• The PDMS-based dPCR system is effective for absolute RNA quantitation, including viral RNA.
• This approach provides a cost-effective and accessible alternative to conventional dPCR systems.
• The findings support the potential for facile digital RT-PCR in gene expression studies and molecular diagnostics.