Combined Use of Engineered PfAGO and LbCas12a for Nucleic Acid Detection.

The COVID-19 pandemic has underscored the urgent need for rapid, accurate, and accessible nucleic acid detection technologies. Here, we present NAEPLS (Nucleic Acid Detection Based on Engineered PfAgo and LbCas12a with Split crRNA), a novel diagnostic platform that synergistically integrates the engineered DNA-guided nuclease PfAgo (mPfAgo) from Pyrococcus furiosus with CRISPR-Cas12a for ultrasensitive and specific pathogen detection. By leveraging mPfAgo's RNA-cleaving capability and LbCas12a's compatibility with split crRNA, NAEPLS achieved detection of SARS-CoV-2 RNA. Coupling this assay with RT-RPA enabled ultrasensitive detection, achieving a limit of detection (LOD) of 10 copies/mL. The system demonstrates single-nucleotide specificity, robustly discriminating mutations in mocked samples (such as D614G) as well as in clinical isolates (such as S371P). Notably, the compatibility with lateral flow strip visualization allows this method to be implemented as a rapid POCT platform, particularly valuable in resource-limited settings. Validation with 20 clinical samples (Ct 20-39) showed 100% concordance with RT-qPCR, including reliable detection of low viral loads (Ct > 35). This study establishes a transformative paradigm for nucleic acid diagnostics, merging the precision of PfAgo with the amplification power of CRISPR for high-performance, field-deployable pathogen detection.