Ultrasensitive CRISPR Platform Enables Rapid and Extraction-Free Detection of Viral Nucleic Acid.

CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.