One-pot Point-of-Care Nucleic-acid Detection via Padlock DNA Ligation and Rolling Circle Transcription

The demand for swift, reliable, and precise nucleic acid detection methods is pressing across numerous sectors, including clinical diagnostics, food safety, and environmental surveillance. We introduce a one-step Padlock DNA ligation-driven Rolling Circle Transcription-assisted CRISPR/LwCas13a detector named PROTRACTOR, tailored for the discernment and quantitative assessment of nucleic acids. The PROTRACTOR platform harnesses template-mediated padlock DNA ligation to transform target RNA/DNA into single-stranded circular DNA. Subsequent rolling circle transcription (RCT) spawns RNA transcripts replete with tandem repeats of the sequences of interest. These transcripts are then specifically targeted by CRISPR/LwCas13a, enabling their detection through fluorescent signals or lateral flow strips (LFS). Innovatively eschewing both reverse transcription and amplification, this approach allows for the direct measurement of RNA/DNA molecules. Demonstrated by the ultrahigh sensitivity (down to 10 copies/mL), rapidity (< 10 minutes), superb specificity (single nucleotide precision), simplicity, and affordability of use, PROTRACTOR's broad utility in nucleic acid assays has been validated with viral genomes, microRNAs, and genetically modified organisms (GMOs). Additionally, a novel point-of-care testing (POCT) device for SARS-CoV-2 integrates PROTRACTOR with expeditious RNA extraction and LFS readouts, producing outcomes within a mere 10 minutes, independent of advanced instrumentation. Furthermore, coupling with a chip-based digital PCR platform, PROTRACTOR facilitates absolute DNA quantification under isothermal conditions in a comparable timeframe. Cumulatively, our findings indicate PROTRACTOR's vast potential for both qualitative and quantitative nucleic acid analysis, whether in lab settings or POCT, across various domains such as biomedicine, agriculture, food verification, and environmental review, with particular benefits in resource-limited environments.