A portable Cas13a self-cascading cyclic amplification-integrated system enables multiple respiratory tract viruses...

CRISPR-based molecular detection systems typically require pre-amplification for adequate sensitivity that prolongs reactions and increases risks of nonspecific amplification, primer interference, and aerosol contamination. In this work, we developed a LbuCas13a-based self-cascading cyclic amplification assay (SCC-Cas13a) for simultaneous detection of multiple respiratory tract viruses in a pre-amplification-free manner. It was achieved by designing CRISPR RNAs (crRNAs) that target specific viral RNA sequences and hairpin probes (HP) that contain multiple uracil structures. The sequence-specific hybridization of crRNA and target RNA activates trans-cleavage activity of LbuCas13a, which cleaves the HP at its poly-U-rich stem-loop region. This results in the release of single-stranded RNA activators that are identical to the target RNA from the HP. This triggers another round of sequence-specific hybridization with crRNA, and therefore a self-cascading cyclic amplification. In a proof-of-concept demonstration, respiratory syncytial virus (RSV), influenza A (Flu A), and SARS-CoV-2 were detected within 3 min, exhibiting limit of detections (LODs) of 230 aM (RSV), 310 aM (Flu A), and 420 aM (SARS-CoV-2), respectively. Notably, these LODs represent a 10⁴-fold enhancement over conventional CRISPR-based systems. The SCC-Cas13a eliminates reverse transcription and pre-amplification steps, streamlining workflow into a single-step reaction. Furthermore, a Radial microfluidic chip (R-chip) was engineered to integrate this SCC-Cas13a for simultaneous detection of RSV, Flu A, and SARS-CoV-2 in clinical nasopharyngeal swab samples. Assisted by a smartphone-based device which can stably excite and accurately collect fluorescence signals from R-chips, this research established an innovative solution for multiplexed pathogen identification and precise molecular diagnostics in resource-limited settings.