Microfluidic toolbox using padlock probes and rolling circle amplification for direct detection and genotyping of viral RNA.

Rolling circle amplification (RCA) combined with padlock probes presents a promising tool for direct detection and genotyping of viral RNA, offering advantages over conventional methods like RT-PCR. This isothermal process enables highly sensitive and specific amplification of nucleic acids without the need for thermal cycling, making it suitable for point-of-care applications. In this study, we demonstrate a microfluidic and RCA-based method for the direct detection of SARS-CoV-2 RNA and variant profiling, bypassing the reverse transcription step. Our approach allows for the identification of single nucleotide polymorphisms (SNPs) specific to viral variants, enhancing the detection sensitivity through the circle-to-circle amplification (C2CA) technique. This methodology shows potential as a robust, cost-effective platform for viral diagnostics, capable of being fully automated and integrated with miniaturized detection systems for efficient use in both resource-rich and resource-limited settings.