One-Tube Nested\nPCR Coupled with CRISPR-Cas12a for\nUltrasensitive Nucleic Acid Testing

Nucleic acid testing with high sensitivity and specificity\nis of\ngreat importance for accurate disease diagnostics. Here, we developed\nan in situ one-tube nucleic acid testing assay. In this assay, the\ntarget nucleic acid is captured using magnetic silica beads, avoiding\nan elution step, followed directly by the polymerase chain reaction\n(PCR) and clustered regularly interspaced short palindromic repeats\n(CRISPR)-Cas12a detection. This assay achieved visual readout and\na sensitivity of 120 copies/mL for detecting SARS-CoV-2. More importantly,\nthe assay demonstrated over 95% sensitivity and 100% specificity compared\nto the gold standard real-time quantitative PCR (RT-qPCR) test by\nusing 75 SARS-CoV-2 clinical samples. By integrating nested PCR and\nCas12a, this all-in-one nucleic acid testing approach enables ultrasensitive,\nhighly specific, and cost-effective diagnosis at community clinics\nand township hospitals.