From PCR to CRISPR: Comparing the detection performance of different nucleic acid detection technologies

Real-time quantitative PCR (qPCR) is the gold standard for viral nucleic acid detection but requires specialized laboratories, trained personnel, and costly equipment, resulting in prolonged testing time. Isothermal amplification technologies (e.g., recombinase polymerase amplification (RPA)) have advanced rapidly, while CRISPR-based molecular diagnostics have gained widespread attention for their advantages in speed, specificity, and sensitivity. However, systematic investigations of different detection methods in practical applications remain limited. Especially for the same nucleic acid detection method, there are significant differences in detection time and detection limit in the existing literature, highlighting the urgent need to establish a unified standard to guide practical application. This study developed optimized RPA-assisted CRISPR nucleic acid detection systems (two-step and one-step methods) and systematically compared their performance with qPCR and RPA-fluorescence methods in viral nucleic acid detection. By systematically optimizing reagent concentrations and operational procedures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was used as the target to evaluate key metrics: detection time, sensitivity, and specificity. Results showed that qPCR had the highest sensitivity but required the longest detection time. The RPA-assisted CRISPR/Cas12a and Cas13a two-step method achieved a sensitivity of 1 copy/μL, demonstrating superior sensitivity and specificity within a 15-minute detection window compared to RPA-fluorescence assays. The RPA-assisted one-step method demonstrated good operational convenience with slightly reduced sensitivity and maintained specificity. • Developed optimized RPA-assisted CRISPR systems for rapid viral nucleic acid detection. • Achieved 1 cp/μL sensitivity in 15-min two-step CRISPR-Cas12a/Cas13a. • qPCR showed highest sensitivity but required longest detection time. • One-step CRISPR offered easy use with minor sensitivity loss.