One-Tube Nested PCR Coupled with CRISPR-Cas12a for Ultrasensitive Nucleic Acid Testing

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