Rapid, tunable, and multiplexed detection of RNA using convective array PCR.

Detection of RNA targets is typically achieved through RT-qPCR or RNAseq. RT-qPCR is rapid but limited in number and complexity of targets detected, while RNAseq is high-throughput but takes multiple days. We demonstrate simultaneous amplification and detection of 28 distinct RNA targets from a single unsplit purified RNA sample in under 40 minutes using our convective array PCR (caPCR) technology. We integrate tunable strand displacement probes into caPCR to allow detection of RNA species with programmable sequence selectivity for either a single, perfectly matched target sequence or for targets with up to 2 single-nucleotide variants within the probe-binding regions. Tunable probes allow for robust detection of desired RNA species against high homology background sequences and robust detection of RNA species with significant sequence diversity due to community-acquired mutations. As a proof-of-concept, we experimentally demonstrated detection of 7 human coronaviruses and 7 key variants of concern of SARS-CoV-2 in a single assay.